Real-time Fluorescent Quantitative Polymerase Research Articles

Heterogeneity of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells in mice.

This study aimed to explore the heterogeneity and gene ontology of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells (CNCs) in mice. The embryos of Wnt1-Cre;R26RmTmG and Pax2-Cre;R26RmTmG at embryonic day (E)8.0-E9.25 were collected for histological observation. We performed immunostaining to compare green fluorescent protein (GFP)-positive CNCs in Pax2-Cre;R26RAi9 and Wnt1-Cre;R26RAi9 mice at E15.5. Single-cell RNA sequencing (scRNA-seq) was used to analyze the first branchial arch GFP-positive CNCs from Wnt1-Cre;R26RmTmG and Pax2-cre;R26RmTmGmice at E10.5. Real time fluorescence quantitative polymerase chain reaction (q-PCR) was performed to validate the differential genes. Wnt1-Cre-marked and Pax2-Cre-marked CNCs migrated from the neural plateto first and second branchial arches and to the first branchial arch, respectively, at E8.0. Although Wnt1-Cre-marked and Pax2-Cre-marked CNCs were found mostly in cranial-facial tissues, the former had higher expression in palate and tongue. The results of scRNA-seq showed that Pax2-Cre-marked CNCs specifically contributed to osteoblast differentiation and ossification, while Wnt1-Cre-marked CNCs participated in limb development, cell migration, and ossification. The q-PCR data also confirmed the results of gene ontology analysis. Pax2-Cre mice are perfect experimental animal models for research on first branchial arch CNCs and derivatives in osteoblast differentiation and ossification.

Effects of small GTP-binding protein GDP dissociation stimulator on adipocyte hypertrophy and glucose metabolism disorder in mice

Objective: To explore the effect and mechanism of small GTP-binding protein GDP dissociation stimulator (SmgGDS) on the development of obesity. Methods: (1) 8-week-old C57BL/6J mice were randomly assigned to normal diet and high fat diet group, with 6 mice in each group. They were fed regular feed and a high fat diet containing 60% fat for 4 months, respectively. The expression of SmgGDS in epididymal adipose tissue (eWAT), liver, and skeletal muscle were measured using Western-blot. (2) 6-week-old wild-type (WT) and SmgGDS knockdown (KD) mice were divided into four groups, each receiving high fat diet for 4 months (7 in each group) and 7 months (9 in each group). Glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted; The weight, adipose tissue, and liver weight of mice were recorded; HE staining examined adipose tissue structural changes; Western-blot determined extracellular signal-regulated kinase (ERK) 1/2 phosphorylation levels in eWAT; Real time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA levels of CCAAT/enhancer binding protein α (C/EBPα), C/EBPβ and peroxisome proliferator activated receptor γ (PPARγ) in eWAT. (3) Mouse embryonic fibroblasts (MEFs) extracted from WT and KD mice were induced for differentiation. Oil red O staining and Western-blot were used to detect lipid droplet and expression of SmgGDS and phospho-ERK; C/EBPα, C/EBPβ and PPARγ mRNA levels were measured using RT-qPCR. (4) 10-week-old C57BL/6J mice were randomly assigned into two groups, with 7 mice in each group. Mice were infected with SmgGDS overexpressing adeno-associated virus (AAV-SmgGDS) or empty vector intraperitoneally, then fed with high fat diet. After 4 weeks, performed GTT and ITT; Recorded the weight and adipose tissue weight of mice; HE staining was used to analyze structural changes of eWAT; Western-blot was used to detect the phosphorylation level of ERK in eWAT. Results: (1) The expression of SmgGDS was significantly upregulated in eWAT of high fat diet fed mice (normal diet group: 0.218±0.037, high fat diet group:0.439±0.072, t=2.74, P=0.034). (2) At 4 months of high fat diet intervention, the glucose tolerance (60 minutes after glucose injection, WT group: 528 mg/dl±21 mg/dl, KD group: 435 mg/dl±17 mg/dl, t=3.47, P=0.030; 90 minutes, WT group: 463 mg/dl±24 mg/dl, KD group: 366 mg/dl±18 mg/dl, t=3.23, P=0.047;120 minutes, WT group: 416 mg/dl±21 mg/dl, KD group: 297 mg/dl±16 mg/dl, t=4.49, P=0.005) and insulin sensitivity (15 minutes after insulin injection, WT group: 77.79%±3.45%, KD group: 54.30%±2.92%, t=3.49, P=0.005; 30 minutes, WT group: 62.27%±5.31%, KD group: 42.25%±1.85%, t=2.978, P=0.024; 90 minutes, WT group: 85.69%±6.63%, KD group: 64.71%±5.41%, t=3.120, P=0.016) of KD mice were significantly improved compared to the WT group, with an increase in eWAT weight ratio (WT: 4.19%±0.18%, KD: 5.12%±0.37%, t=2.28, P=0.042), but a decrease in average adipocyte area (WT group: 5221 μm²±241 μm², KD group: 4410 μm²±196 μm², t=2.61, P=0.026). After 7 months of high fat diet, the eWAT weight ratio of KD mice decreased (WT: 5.02%±0.20%, KD: 3.88%±0.21%, t=3.92, P=0.001) and adipocyte size decreased (WT group: 6 783 μm²±390 μm², KD group: 4785 μm²±303 μm², t=4.05, P=0.002). The phospho-ERK1 in eWAT increased (WT group: 0.174±0.056, KD group: 0.588±0.147, t=2.64, P=0.025), and mRNA level of PPARγ significantly decreased (WT group: 1.018±0.128, KD group: 0.029±0.015, t=7.70, P=0.015). (3) The expression of SmgGDS was significantly increased in differentiated MEF (undifferentiated: 6.789±0.511, differentiated: 10.170±0.523, t=4.63, P=0.010); SmgGDS knock-down inhibited lipid droplet formation in MEF (WT group: 1.00±0.02, KD group: 0.88±0.02, t=5.05, P=0.007) and increased ERK1 (WT group: 0.600±0.179, KD group: 1.325±0.102, t=3.52, P=0.025) and ERK2 (WT group: 2.179±0.687, KD group: 5.200±0.814, t=2.84, P=0.047) activity, which can be reversed by ERK1/2 inhibitor. (4) SmgGDS over expression resulted in weight gain, increased eWAT weight (control group: 3.29%±0.36%, AAV-SmgGDS group: 4.27%±0.26%, t=2.20, P=0.048) and adipocyte size (control group: 3525 μm²±454 μm², AAV-SmgGDS group: 5326 μm²±655 μm², t=2.26, P=0.047), impaired insulin sensitivity(30 minutes after insulin injection, control group: 44.03%±4.29%, AAV-SmgGDS group: 62.70%±2.81%, t=3.06, P=0.019), and decreased ERK1 (control group: 0.829±0.077, AAV-SmgGDS group: 0.326±0.036, t=5.96, P=0.001)and ERK2 (control group: 5.748±0.287, AAV-SmgGDS group: 2.999±0.845, t=3.08, P=0.022) activity in eWAT. Conclusion: SmgGDS knockdown improves obesity related glucose metabolism disorder by inhibiting adipogenesis and adipose tissue hypertrophy, which is associated with ERK activation.

Efficacy evaluation and mechanism of Bacillus subtilis EBS03 against cotton Verticillium wilt

BackgroundIn our previous study, a strain EBS03 with good biocontrol potential was screened out of 48 strains of cotton endophyte Bacillus subtilis by evaluating the controlling effect against cotton Verticillium wilt. However, its mechanism for controlling Verticillium wilt remains unclear. The objective of this study was to further clarify its controlling effect and mechanism against cotton Verticillium wilt.ResultsThe results of confrontation culture test and double buckle culture test showed that the inhibitory effects of EBS03 volatile and nonvolatile metabolite on mycelium growth of Verticillium dahliae were 70.03% and 59.00%, respectively; the inhibitory effects of sporulation and microsclerotia germination were 47.16% and 70.06%, respectively. In the greenhouse test, the EBS03 fermentation broth root irrigation had the highest controlling effect at 87.11% on cotton Verticillium wilt, and significantly promoted the growth of cotton seedlings. In the field experiment, the controlling effect of EBS03 fermentation broth to cotton Verticillium wilt was 42.54% at 60 days after cotton sowing, and the boll number per plant and boll weight in EBS03 fermentation broth seed soaking, root irrigation, and spraying treatments significantly increased by 19.48% and 7.42%, 30.90% and 2.62%, 15.99% and 9.20%, respectively. Furthermore, EBS03 improved the resistance of cotton leaves against the infection of V. dahliae, and induced the outbreak of reactive oxygen species and accumulation of callose. In addition, the results of real time fluorescent quantitative polymerase chain reaction (RT-qPCR) detection showed that EBS03 significantly induced upregulation expression level of defense-related genes PAL, POD, PPO, and PR10 in cotton leaves, enhanced cotton plant resistance to V. dahliae, and inhibited colonization level of this fungal pathogen in cotton.ConclusionBacillus subtilis EBS03 has a good biological defense capability, which can inhibit the growth and colonization level of V. dahliae, and activate the resistance of cotton to Verticillium wilt, thus increase cotton yield.

Effects and mechanism of human umbilical vein endothelial cells-derived exosomes on wound healing in diabetic rabbits

Effects and mechanism of human umbilical vein endothelial cells-derived exosomes on wound healing in diabetic rabbits

Enhancing or inhibiting apoptosis? The effects of ucMSC-Ex in the treatment of different degrees of traumatic pancreatitis.

The animal models of traumatic pancreatitis (TP) were established to evaluate the specific mechanisms by which umbilical cord mesenchymal stem cell-derived exosomes (ucMSC-Ex) exert therapeutic effects. Sixty four rats were randomly divided into eight groups, including TP groups with three different degrees and relevant groups with ucMSC-Ex treated. The degrees of pancreatic tissue injury were evaluated by Histological Examination. Furthermore, enzyme-linked immunosorbent assay were applied to evaluate the activity of pancreatic enzymes and levels of inflammatory factors in serum. Finally, the apoptotic effects of each group were evaluated by TUNEL, western blot (WB), and real time fluorescence quantitative polymerase chain reaction (RT-qPCR). The pancreatic histopathological score and serum amylase and lipase levels gradually increased in various degrees of TP and the levels in the treatment group were all significantly decreased. The apoptosis index gradually increased in each TP group and significantly decreased in the treatment group in TUNEL results. WB and RT-qPCR showed the same trend, that bax and caspase-3 gradually increased and bcl-2 gradually decreased in TP groups. Compared with TP groups, the expression of bax and caspase-3 were lower while bcl-2 expression was higher in the treatment group. ucMSC-Ex suppressed the inflammatory response and inhibited pancreatic acinar cell apoptosis to promote repair of injured pancreatic tissue.

In vitro behaviour of human gingival fibroblasts cultured on 3D-printed titanium alloy with hydrogenated TiO2 nanotubes

Selective laser melting (SLM), as one of the most common 3D-printed technologies, can form personalized implants, which after further surface modification can obtain excellent osseointegration. To study the surface properties of SLM titanium alloy (Ti6Al4V) with hydrogenated titanium dioxide (TiO2)nanotubes (TNTs) and its influence on the biological behaviour of human gingival fibroblasts (HGFs), we used SLM to prepare 3D-printed titanium alloy samples (3D-Ti), which were electrochemically anodizing to fabricate 3D-TNTs and then further hydrogenated at high temperature to obtain 3D-H2-TNTs. Polished cast titanium alloy (MP-Ti) was used as the control group. The surface morphology, hydrophilicity and roughness of MP-Ti, 3D-Ti, 3D-TNTs and 3D-H2-TNTs were measured and analysed by scanning electron microscopy (SEM), contact angle metre, surface roughness measuring instrument and atomic force microscope, respectively. HGFs were cultured on the four groups of samples, and the cell morphology was observed by SEM. Fluorescence staining (DAPI) was used to observe the number of adhered cell nuclei, while a cell counting kit (CCK-8) was used to detect the early adhesion and proliferation of HGFs. Fluorescence quantitative real time polymerase chain reaction (RT–qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of adhesion-related genes and fibronectin (FN), respectively. The results of this in vitro comparison study indicated that electrochemical anodic oxidation and high-temperature hydrogenation can form a superhydrophilic micro-nano composite morphology on the surface of SLM titanium alloy, which can promote both the early adhesion and proliferation of human gingival fibroblasts and improve the expression of cell adhesion-related genes and fibronectin.Graphical abstract

Differential gene expression and bioinformatics analysis in sepsis secondary to pneumonia

To analyze and screen the key genes of sepsis secondary to pulmonary infection by bioinformatics, and to provide theoretical basis for the basic research of the disease and find an ideal animal model program. Experiment 1 (bioinformatics analysis): gene expression data sets of pulmonary infection secondary sepsis patients and multiple sepsis animal models were screened by Gene Expression Omnibus (GEO) Database, and gene differences were analyzed by R software. Differential genes were analyzed by gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Correlation analysis was conducted between differential genes and clinical symptoms in the data set of pulmonary infection secondary sepsis, and the correlation heat map between differential genes and clinical symptoms was drawn. Key genes were screened by weighted gene co-expression network analysis (WGCNA) and protein-protein interaction network analysis (PPIN) clustering. Experiment 2 (sepsis animal model preparation): male mice weighing 21-25 g were randomly divided into the key genes group and the control (Sham) group. And cecal ligation and puncture (CLP) was used to establish mouse sepsis model, while the mice in sham group were performed by exposure of cecum. And all the mice were scarified 24 hours after surgery to extract the total RNA from lung tissue, real time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression of key genes. Experiment 1 (bioinformatics analysis): 319 differential genes were showed by GSE 134364 and GSE 65682 data set analysis of pulmonary infection secondary sepsis. And there was no genetic difference between community acquired pneumonia (CAP) and hospital acquired pneumonia (HAP) in patients with pulmonary infection secondary to sepsis. Obvious differences existed between differential genes in animal models, and there was no common differential gene. Differential genes in patients and animal models were similarly enriched in GO function, mainly in cell differentiation, regulation of cell process, and regulation of cellular response to stimuli, there were significant differences in pathway enrichment, among which, CLP animal models showed higher consistency with patients. The key genes obtained by WGCNA and PPIN analysis were MAPK14, NLRC4 and LCN2. Experiment 2 (sepsis animal model preparation): animal experiment results showed that the mRNA expressions of MAPK14, NLRC4 and LCN2 in lung tissue of CLP model mice were significantly up-regulated compared with the sham group. MAPK14, NLRC4 and LCN2 are key genes involved in the regulation of biological processes of pulmonary sepsis secondary to infection, and are potential research directions of this disease. What's more, CLP animal model can better reflect the biological characteristics of patients with pulmonary infection secondary sepsis, and is one of the ideal animal model schemes for pulmonary infection secondary sepsis.

Dual-target one-step nested PCR for sensitive detection of SARS-CoV-2 nucleic acids

The Corona Virus Disease reported in 2019 (COVID-19) poses a significant threat to human and public health. Its early and accurate detection can reduce the spread and recurrence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Real-time reverse transcription fluorescent quantitative polymerase chain reaction (RT-qPCR) is the “gold standard” for detecting the nucleic acid of SARS-CoV-2. This study developed and tested a dual-target (ORF1ab and N gene) one-step nested RT-qPCR (DTO-N-PCR) to detect SARS-CoV-2. Ten-fold serial dilutions of mixed synthetic DNA from SARS-CoV-2 ORF1ab and N gene were used as templates to test the sensitivity of DTO-N-PCR. Its specificity was subsequently tested using throat swab specimens from 10 COVID-19 patients and 35 healthy participants. DTO-N-PCR was more sensitive and specific than conventional RT-qPCR. It has unique features, including a dual-target (ORF1ab and N gene), rapid one-step operation of reverse transcription and PCR, four pairs of inner and outer primers, and specific probes. These features aid in its rapid, accurate, and efficient detection of SARS-CoV-2 RNA.

Effect of acupuncture on microglia activation in prefrontal cortex of chronic stress-induced depression rats

To observe the effect of acupuncture on the expression of proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), microglia marker ion calcium adaptor protein (Iba-1) and triggering receptor expressed on myeloid cells 2 (TREM2) in the prefrontal cortex of chronic stress-induced depression rats, so as to explore its antidepressant mechanism. Thirty-two SD rats were randomly divided into normal control, model, acupuncture and fluoxetine groups, with 8 rats in each group. The depression model was established by using chronic mild unpredictable stress methods for 6 weeks. Manual acupuncture stimulation was applied to "Baihui" (GV20) and "Yintang" (GV29) for 10 min before modeling for 6 weeks. Fluoxetine (10 mg/kg, 1 mg/mL) was given to rats of the fluoxetine group by gavage before stress stimulation, once every day for 6 weeks. The open field test was used to evaluate the behavioral changes of rats. The contents of IL-1β, IL-6 in the prefrontal cortex were determined by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to determine the expression of Iba-1 in the prefrontal cortex. The TREM2 gene expression in the prefrontal cortex was determined by real time fluorescent quantitative polymerase chain reaction (RT-PCR). After modeling, the crossing numbers and rearing times were significantly decreased in the model group relevant to the control group (P<0.05). After the treatment, the crossing numbers were significantly increased in the acupuncture and fluoxetine groups (P<0.05), while the rearing times in the acupuncture group were significantly increased (P<0.05). Compared with the control group, the contents of IL-1β, IL-6 and the expression of Iba-1 positive cells in the prefrontal cortex were significantly increased in the model group (P<0.05), while the expression of TREM2 gene in the prefrontal cortex was significantly decreased (P<0.05). After the treatment, the increased levels of IL-1β, IL-6 and Iba-1 positive cells and the decreased TREM2 gene expression were considerably reversed in both acupuncture and fluoxetine groups compared with the model group (P<0.05). Acupuncture intervention plays a positive role in anti-depression in rats, which may be related to its effects in inhibiting the activation of microglia, reducing the expression of proinflammatory cytokines, and increasing TREM2 expression in the prefrontal cortex.

Expression and effect of microRNA-205 in hypertrophic scar

Objective: To investigate the expression and effect of microRNA-205 (miR-205) in hypertrophic scar. Methods: The experimental research method were applied. From October 2019 to January 2020, hypertrophic scar tissue from 6 patients with hypertrophic scar [1 male and 5 females, aged (36±7) years], and remaining normal skin tissue from 6 trauma patients [2 males and 4 females, aged (38±9) years] after flap transplantation operation were collected. The above-mentioned 12 patients were admitted to the General Hospital of Northern Theater Command and met the inclusion criteria. Real time fluorescent quantitative polymerase chain reaction was used to detect the mRNA expressions of miR-205 and thrombospondin-1 (TSP-1). The hypertrophic scar tissue was taken to culture the 3rd to 5th passage of fibroblasts (Fbs) for the follow-up experiments. Fbs of hypertrophic scar was divided into TSP-1+miR-205 control group, TSP-1+miR-205 mimic group, TSP-1 mutant+miR-205 control group, TSP-1 mutant +miR-205 mimic group, which were transfected with the corresponding sequences. At 48 h after transfection, the expressions of luciferase and renal luciferase were detected by luciferase reporter gene detection kit, and the luciferase/renal luciferase ratio was calculated to indicate the activity of TSP-1. Two batches of hypertrophic scar Fbs were collected and divided into miR-205 control group, miR-205 mimic group, and miR-205 inhibitor group and miR-205 control group, miR-205 mimic group, and miR-205 mimic+TSP-1 group, respectively, which were transfected with the corresponding sequences. At 0 (immediately), 12, 24, 36, and 48 h after transfection, the cell viability was detected by microplate reader. Two batches of hypertrophic scar Fbs were collected, grouped, and treated as the cell viability detecting experiment. At 24 h after transfection, Hoechst 33258 staining was performed to observe the nuclear shrinkage, so as to reflect the apoptosis of Fbs. The number of samples in cell experiment was 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and t test. Results: The mRNA expression of miR-205 in hypertrophic scar tissue was 0.54±0.05, which was significantly lower than 1.26±0.07 in normal skin tissue (t=8.213, P<0.01). The expression of TSP-1 mRNA in hypertrophic scar tissue was 1.46±0.07, which was significantly higher than 0.68±0.11 in normal skin tissue (t=6.031, P<0.01). At 48 h after transfection, the luciferase/renal luciferase ratio reflecting the TSP-1 activity of cells in TSP-1+miR-205 mimic group was 0.532±0.028, which was significantly lower than 0.998±0.012 in TSP-1+miR-205 control group (t=26.500, P<0.01), and the luciferase/renal luciferase ratio of cells in TSP-1 mutant+miR-205 mimic group was 0.963±0.012, which was close to 0.976±0.010 in TSP-1 mutant+miR-205 control group (t=0.816, P>0.05). At 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 mimic group was significantly lower than that in miR-205 control group (t=6.169, 12.670, 27.130, 12.670, P<0.05 or P<0.01). At 0, 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 inhibitor group was significantly higher than that in miR-205 control group (t=6.169, 7.221, 7.787, 7.835, 13.030, P<0.05 or P<0.01). At 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 mimic group was significantly lower than that in miR-205 control group and miR-205 mimic+TSP-1 group (t=8.118, 26.970, 39.550, 42.490, 14.570, 12.240, 36.830, 45.220, P<0.05 or P<0.01). At 24 h after transfection, compared with miR-205 control group, the cell apoptosis in miR-205 mimic group was increased, and the cell apoptosis in miR-205 inhibitor group was decreased. At 24 h after transfection, compared with miR-205 mimic group, the cell apoptosis in miR-205 control group miR-205 mimic+TSP-1 group were decreased. Conclusions: miR-205 can inhibit the proliferation and promote the apoptosis of Fbs in hypertrophic scar by inhibiting the expression of TSP-1, which has the potential to be the therapeutic target for hypertrophic scar.

Study of thermal injury effects on human HaCaT cells under simulated microgravity environment

Objective: To investigate the thermal injury effects on human HaCaT cells under simulated microgravity environment. Methods: The human HaCaT cells were collected and divided into simulated microgravity thermal injury (SMGTI) group, normal gravity thermal injury (NGTI) group, and normal gravity false injury (NGFI) group according to the random number table. Cells in NGTI and NGFI groups were cultured routinely in culture bottle, and cells in SMGTI group were cultured in the rotary cell culture system to simulate microgravity environment. Cells in SMGTI and NGTI groups were bathed in hot water of 45 ℃ for 10 minutes to make thermal injury model, and cells in NGFI group were bathed in warm water of 37 ℃ for 10 minutes to simulate thermal injury. At post injury hour (PIH) 12, cell morphology of 3 groups was observed under inverted phase contrast electron microscope. At PIH 2, 6, and 12, single cell suspension in the 3 groups was collected to detect the cell cycle by flow cytometer and the mRNA expressions of heat shock protein 70 (HSP70), matrix metalloproteinase 9 (MMP-9), and cysteine-aspartic protease 3 (caspase-3) by real time fluorescence quantitative reverse transcription polymerase chain reaction, and the experiments were repeated for 3 times. At PIH 2, 6, and 12, cell culture supernatant in the 3 groups was collected to detect the concentration of heparin-binding epidermal growth factor (HB-EGF) by enzyme linked immunosorbent assay method, the experiment was repeated for 3 times. The sample in each group and each time point was 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference test, Kruskal-Wallis H test, and Mann-Whitney U test. Results: (1) At PIH 12, cells in NGFI group showed regular shape and regular arrangement, with no cell debris. The cell shape in NGTI group was generally regular, with fewer cell debris and closer arrangement than that in NGFI group. The cells in SMGTI group showed more irregular shapes, different sizes, and dead cell debris. (2) The percentage of G1 phase cells in NGTI group was significantly higher than that in NGFI group and SMGTI group at PIH 2, respectively (P<0.05), and the percentage of G1 phase cells in NGTI group was significantly lower than that in NGFI group and SMGTI group at PIH 6 and 12, respectively (P<0.05). The percentage of G2/M phase cells in NGTI group was significantly lower than that in SMGTI group at PIH 2 (P<0.05), and the percentage of G2/M phase cells in NGTI group was significantly higher than that in NGFI group and SMGTI group at PIH 6 and 12, respectively (P<0.05). The percentage of S phase cells in NGTI group at PIH 2, 6, and 12 was significantly higher than that in SMGTI group (P<0.05), and the percentage of S phase cells in NGTI group at PIH 2 and 6 was significantly lower than that in NGFI group (P<0.05). (3) The HSP70 mRNA expressions of cells in NGTI group were 2.50±0.30 and 3.99±0.35 at PIH 2 and 6, which were significantly higher than 1.14±0.15 and 0.82±0.27 in NGFI group (P<0.05), and 1.17±0.53 and 1.65±0.59 in SMGTI group (P<0.05). The MMP-9 mRNA expression of cells in SMGTI group was significantly higher than that in NGTI group at PIH 2, 6, and 12, respectively (Z=-2.319, -2.882, -2.908, P<0.05). At each time point after injury, the mRNA expression of caspase-3 of cells in NGTI group was similar to that in NGFI group and SMGTI group, respectively (P>0.05). (4) The concentration of HB-EGF in cell culture supernatant of NGTI group was significantly lower than that in NGFI group at PIH 2, 6 and 12 (P<0.05), and the concentration of HB-EGF in cell culture supernatant of SMGTI group was significantly higher than that in NGTI group at PIH 2 and 6 (P<0.05). Conclusions: The proliferation and secretion functions and expression of wound repair related protein of human HaCaT cells inflicted with thermal injury in simulated microgravity environment showed complex and diversified changes, which provide theoretical basis for further research on damage repair under weightlessness.

Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects

Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects

The function and molecular mechanism of CEP55 in anaplastic thyroid cancer.

The purpose of this study was to determine the role of centrosomal protein of 55 kDa (CEP55) in anaplastic thyroid cancer (ATC) and to further explore the mechanism, which might provide a new molecular marker for treatment of ATC. The expression level of CEP55 in clinical cases was tested by fluorescence quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Also, qRT-PCR assay was performed in different TC cell lines. The relationship between CEP55 expression and clinicopathological characteristics was statistically analyzed. Kaplan-Meier curve and Cox's proportional hazards regression model were performed in survival analysis. Further, Western blot assay was used to analyze the protein expression changes in PI3K/Akt pathway. The expression level of CEP55 in TC tissues showed a noticeable upgrade, especially in ATC. In vitro, CEP55 expression was also increased in four kinds of TC cells, in which, the highest expression was found in ATC (TA-K) cells. The clinicopathological features, including lymph node metastasis, distant metastasis, and prognostic index were found to be correlated with the expression level of CEP55. Besides, the ATC patients with higher expression of CEP55 had a statistically worse overall survival (OS) time. In univariate analyses and multivariate analyses, the CEP55 level was an independent prognosis index of patients with ATC. In vitro study, CEP55 protein expression level was significantly reduced in si-CEP55-transfected TA-K cells. Notably, the downregulation of CEP55 could suppress the phosphorylation of PI3K and AKT. This study found that CEP55 could promote ATC progression, and PI3K/AKT pathway might be the downstream target of its action. These results provided a new therapeutic direction for the treatment of ATC.

The effect of miRNA-34a antisense oligonucleotide on non-small cell lung cancer cell line HCC827

Objective To investigate the effect of antisense oligonucleotides of miRNA-34a on non-small cell lung cancer (NSCLC) and its molecular mechanism. Methods The expression of miRNA-34a in human non-small cell lung cancer cell line HCC827 and human normal lung cell MRC-5 was detected by real time fluorescence quantitative polymerase chain reaction (qRT-PCR). HCC827 cells were divided into three groups: blank control group, negative control group, anti-sense oligonucleotide group(liposome 2000 transfected anti-sense oligonucleotide miRNA-34a); cell counting kit-8 (CCK-8) method was used to detect cell proliferation, Jimsa staining was used to detect cell cloning ability, Transwell test was used to detect cell migration and invasion ability; RT-PCR and Western blot were used to detect phosphatase and tensin homolog (PTEN), phosphorylation-protein kinase B (p-Akt), phosphatidylinositol-3-kinase (PI3K) mRNA and protein expression. Results The relative expression of miRNA34a in HCC827 cells was significantly higher than that in human normal lung cells (P 0.05). At 48 h, 72 h and 96 h, the proliferation level of HCC827 cells in anti-sense oligonucleotide miRNA-34a group was significantly lower than that in negative control group and blank control group (P<0.05). The cell cloning rate of antisense oligonucleotide miRNA-34a group was significantly lower than that of negative control group and blank control group (P<0.01). The number of migration and invasion of HCC827 cells in antisense oligonucleotide RNA-34a group was significantly lower than that in negative control group and blank control group (P<0.01). The relative expression of PTEN mRNA and protein in antisense oligonucleotide miRNA-34a group was significantly higher than that in negative control group and blank control group (P<0.05); the relative expression of p-Akt, PI3K mRNA and protein in antisense oligonucleotide miRNA-34a group were significantly lower than that in negative control group and blank control group (P<0.05). Conclusions The expression level of miRNA-34a in human non-small cell lung cancer cells is significantly higher than that in human normal lung cells. Antisense oligonucleotides of miRNA-34a can inhibit the proliferation, cloning, migration and invasion of human non-small cell lung cancer cells. The mechanism may be related to the negative regulation of PTEN/p-Akt/PI3K signaling pathway. Key words: Oligonucleotides, antisense; miRNA-34a; Carcinoma, non-small-cell lung; Cell line, tumor

Expression and significance of microRNA-125b in tongue squamous cell carcinoma

The expression of microRNA-125b in tongue squamous cell carcinoma (TSCC) was detected and analyzed for its relationship with the clinicopathological features of TSCC. Real time fluorescence-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of microRNA-125b in 35 TSCC tissues and adjacent normal tissues from 35 TSCC cases. The relationship between the expression of microRNA-125b in TSCC tissues and the clinicopathological features of patients with TSCC was analyzed. In situ hybridization (ISH) was used to detect the expression level of microRNA-125b gene in the TSCC tissues and adjacent normal tissues. RT-qPCR results showed that the relative expression levels of microRNA-125b in the TSCC issues was 2.32±0.69, and that of normal tissues was 0.87±0.32. The statistical results showed that the expression level of microRNA-125b was significantly higher in the TSCC tissues than in the normal tissues (P<0.001). The expression level of microRNA-125b in the TSCC tissues was not significantly correlated with age, gender, pathological grade, and lymph node metastasis but was positively correlated with TNM stage. Patients with high TNM stage had high microRNA-125b expression levels (P<0.05). The ISH results showed that the expression levels of microRNA-125b in the TSCC tissues were 0.010±0.003, and that of normal tissues was 0.004±0.001. The expression levels of microRNA-125b in the 35 TSCC tissues were significantly higher than those in the normal tissues (P<0.05). MicroRNA-125b is highly expressed in TSCC and associated with TNM stage, suggesting that high microRNA-125b expression may be involved in the development of TSCC.

Role of intestinal barrier in the pathogenesis of autoimmune hepatitis

Objective To observe and analyze the role of intestinal barrier in the pathognesis of autoimmune hepatitis (AIH), to explain the pathogenesis of AIH and to explore the intestinal based new treatment strategies. Methods A total of 14 AIH patients from January to December 2017 at Tianjin Medical University General Hospital (six patients without liver cirrhosis, and eight patients with liver cirrhosis) and 10 healthy controls were enrolled. The serum levels of D-lactic acid (D-Lac) and diamine oxidase (DAO) were detected by enzyme-linked immunosorbent assay. Real time fluorescence quantitative polymerase chain reaction was used to detect the relative expression levels of connexin (zonula occluden-1 (ZO-1), occludin), cytokines (interleukin(IL)-2, interferon(IFN)-γ, IL-4, IL-10) and Toll-like receptor 4 (TLR4) in terminal ileal tissues of each group. The relative expression of secretory immunoglobulin A (sIgA) in the terminal ileum was determined by Western blotting. Thirty BALB/c mice were selected and divided into blank control group, dextran sulfate sodium (DSS) group, concanavalin A (ConA) group, DSS+ ConA group, and DSS+ bacterium+ ConA group, with six mice in each group. The relative expression levels of ZO-1, occludin in mouse colonic tissues, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and inflammatory activity degree of liver tissues (Knodell score) of each group were measured. T-test and one-way analysis of variance were performed for statistical analysis. Results The serum D-Lac and DAO levels of AIH with liver cirrhosis group and AIH without liver cirrhosis group were both higher than those of healthy control group ((1 768.2±147.1) μg/L, (436.2±197.0) μg/L vs. (100.2±10.9) μg/L, and (11.5±2.5) U/L, (5.4±0.9) U/mL vs. (3.5±0.9) U/mL), and the levels of D-Lac and DAO of AIH with liver cirrhosis group were the highest; and the differences were statistically significant (t=5.512, 36.010, 4.088 and 9.443, F=396.958 and 46.640, all P<0.01). The relative expression levels of ZO-1 and occludin in the terminal ileal mucosa of AIH with liver cirrhosis group were lower than those of healthy control group (0.20±0.14 vs. 1.67±0.51, 0.12±0.09 vs. 0.90±0.21), and the relative expression of ZO-1 in AIH without liver cirrhosis group was lower than that in healthy control group (0.99±0.37 vs. 1.67±0.51); and the differences were statistically significant (t=8.641, 7.407 and 2.295, all P<0.05). The relative expression levels of IL-2 and IFN-γ in terminal ileal tissues of AIH with liver cirrhosis group were higher than those of healthy control group (1.11±0.43 vs. 0.24 ±0.16, and 3.50 ± 1.90 vs. 0.32±0.30), however the relative expression of sIgA in terminal ileal tissues was lower than that of healthy control group (0.506±0.024 vs. 1.081±0.102); and the differences were statistically significant (t=4.679, 3.981 and 5.493, all P<0.05). While the relative expression levels of IL-10 in AIH with liver cirrhosis group and AIH without liver cirrhosis group were lower than that in healthy control group (0.30±0.20, 0.42±0.24 vs. 0.84± 0.23), and the relative expression levels of TLR4 in ileum mucosa of the both groups were higher than that of healthy control group (8.74 ±5.13, 6.74 ±3.65 vs. 0.89 ± 0.70); and the differences were statistically significant (t=3.095, 4.816, 3.856 and 3.685, all P<0.05). The relative expression levels of ZO-1 and occludin of DSS+ ConA group were lower than those of ConA group (0.14±0.08 vs. 0.98±0.13, and 0.09±0.02 vs. 0.98±0.16), however serum ALT, AST levels and the Knodell score were all higher than those of ConA group ((5 496.67±618.83) U/L vs. (3 325.00±1 030.06) U/L, (8 825.00±1 165.35) U/L vs. (5 433.33±1 691.14) U/L, and 18.00±2.00 vs. 9.33±3.01); and the differences were statistically significant (t=13.480, 13.520, 4.227, 4.045 and -2.892, all P<0.05). The relative expression levels of ZO-1 and occludin in DSS+ bacterium+ ConA group were higher than those in DSS+ ConA group (0.46±0.08 vs. 0.14±0.08, and 0.53±0.15 vs. 0.09±0.02), while serum ALT and AST levels were lower than those of DSS+ ConA group ((4 343.33±252.16) U/L vs. (5 496.67±618.83) U/L, and (6 123.33±1 086.60) U/L vs. (8 825.00±1 165.35) U/L); and the differences were statistically significant (t=6.928, 7.122, 4.228 and 4.153, all P<0.01). Conclusions AIH patients have increased intestinal permeability and impaired intestinal barrier which is more serious in patients with liver cirrhosis than in patients without cirrhosis. The intestinal barrier injury can aggravate ConA-induced immune-mediated liver injury. While the protection and repair of intestinal barrier can alleviate immune-mediated liver injury induced by ConA. Key words: Hepatitis, autoimmune; Concanavalin A; Intestinal mucosal barrier; Dextran sulfate sodium; Bifidobacterium

LncRNA CADM1-AS1 serves as a new prognostic biomarker for gastric cancer.

To investigate the expression of the long non-coding RNA CADM1-AS1 (lncRNA CADM1-AS1) in gastric cancer and its clinical significance. The real time fluorescence quantitative polymerase chain reaction (qRT-PCR) was performed to detect the expression difference of lncRNA CADM1-AS1 between gastric cancer and its adjacent normal tissues. Then, the correlation between the expression of CADM1-AS1 in gastric cancer and the clinicopathological characteristics was analyzed by the Chi-square test. Moreover, the potential of lncRNA CADM1-AS1 in predicting the prognosis of patients with GC after the operation was assessed by the Log-rank test and the Cox's proportional hazards regression model. The expression of lncRNA CADM1-AS1 was significantly decreased in tumor tissues. According to the mean expression of lncRNA CADM1-AS1, the patients were divided into a high expression group and a low expression group, and the expression of lncRNA CADM1-AS1 in gastric cancer was significantly correlated with tumor differentiation, N stage, M stage, and TNM stage. Moreover, the gastric cancer patients with higher expression of lncRNA CADM1-AS1 had a statistically better overall survival (OS) time and progression-free survival (PFS) time. In univariate analyses and multivariate analyses, the expression of lncRNA CADM1-AS1 was an independent prognosis index of patients with gastric cancer. LncRNA CADM1-AS1 might be a new prognostic biomarker for gastric cancer.

Effects of Yiqi Jiedu prescription drug serum on the apoptosis of human renal cell carcinoma ACHN cells and Notch1 gene

Objective To observe the effects of Yiqi Jiedu prescription drug serum on the apoptosis of human renal cell carcinoma ACHN cells and Notch1 gene in Notch signal pathway. Methods The healthy SD female rats were selected, which were divided into 4 groups, 8 rats in each group: the rats were fed with the normal saline 10 ml·kg-1·d-1 (the blank serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 50 g·kg-1·d-1 (the high-concentration of Yiqi Jiedu prescription serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 25 g·kg-1·d-1 (the medium-concentration of Yiqi Jiedu prescription serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 12.5 g·kg-1·d-1 (the low-concentration medicated of Yiqi Jiedu prescription serum group); then the serum would be extracted 10 days later in each group. ACHN cells at exponential phase were cultured in the above 4 groups. The proliferation of ACHN cells in each group was observed by using CCK-8 method. The apoptosis of ACHN cells was detected by using flow cytometry (FCM). The expression levels of Notch1 mRNA of ACHN cells in each groups were detected by using real time fluorescence quantitative polymerase chain reaction (RT-PCR). Results The inhibition rates of ACHN cells in the high-concentration group, the medium-concentration group, the low-concentration group of Yiqi Jiedu prescription and the blank serum group 24 h later were (12.34±4.25)%, (7.47±1.40)%, (2.52±0.62)%, (1.05±0.31)%, respectively (F=15.04, P < 0.01); after 48 h, the inhibition rates were (24.20±2.41)%, (14.23±1.56)%, (5.08±1.34)%, (1.16±0.14)%, respectively (F=227.36, P < 0.01); after 72 h, the inhibition rates were (32.16±2.45)%, (25.05±3.69)%, (10.29±2.76)%, (1.07±0.71)%, respectively (F=110.51, P < 0.01). The results showed that Yiqi Jiedu prescription drug serum could significantly inhibit the proliferation of ACHN cells in a dose-dependent manner. Besides, the inhibition rate differences at all time points of the high-concentration serum group (F=31.44, P < 0.01), the medium-concentration serum group (F=49.61, P < 0.01) and the low-concentration serum group (F=68.78, P < 0.01) were all statistically significant, and they were in a time-dependent manner. The apoptosis rate of cells in the high-concentration group, the medium-concentration group, the low-concentration group of Yiqi Jiedu prescription and the control serum group was (34.5±1.5)%, (24.4±1.5)% and (13.1±0.5)%, (5.2±0.3)%, respectively, and the differences were statistically significant (F=1 153.36, P < 0.01). The relative expression level of Notch1 mRNA of cells in the high-concentration group, the medium-concentration group, the low-concentration serum group of Yiqi Jiedu prescription and the control serum group was 0.213±0.032, 0.432±0.049, 0.781±0.018, 1.013±0.047, respectively, and the differences were statistically significant (F=270.60, P < 0.01). Conclusion Yiqi Jiedu prescription drug serum can induce apoptosis of ACHN cells, and its mechanism may be related to the down-regulation of the expression level of Notch1 receptors. Key words: Kidney neoplasms; Yiqi Jiedu prescription; Apoptosis; Drug serum; Notch1 receptors

Effects of miR-21 on hypertensive rats through PTEN/PI3K/Akt/mTOR signaling pathway.

The aim of this study was to investigate the effects of micro-ribonucleic acid (miR)-21 on hypertensive rats through the phosphatase and tensin homolog deleted on chromosome ten (PTEN)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. A total of 10 spontaneously hypertensive rats (SHRs) were selected as the model group. Meanwhile, 10 rats with the same age were enrolled in the normal control group. Real Time-fluorescence quantitative Polymerase Chain Reaction (qRT-PCR) was performed to detect the mRNA level of miR-21 in rats of the SHR model group and control group. The tail arterial diastolic pressure of rats in the awake and resting state was measured in both groups, respectively. Pathological sections were prepared to evaluate pathological changes in myocardial tissues. Subsequently, myocardial cells were isolated, cultured and transfected with miR-21 mimics and miR-21 inhibitor, respectively. Transfection efficiency was verified using fluorescence quantitative PCR. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was utilized to determine the apoptosis level of myocardial cells. Furthermore, the expression levels of the signaling pathway-related proteins were detected via Western blotting assay. Fluorescence quantitative PCR results revealed that the expression level of miR-21 was significantly higher in the SHR model group (p<0.05). The diastolic pressure increased markedly in the SHR model group when compared with that in the control group (p<0.05). Subsequent hematoxylin and eosin (HE) staining indicated apparent myocardial tissue injury in the SHR model group (p<0.05). After transfection, the results showed that miR-21 inhibitor could effectively down-regulate the expression level of miR-21 in myocardial cells (p<0.05). Meanwhile, TUNEL staining revealed that the number of apoptotic cells in the miR-21 inhibitor group was remarkably higher than that of the other two groups (p<0.05). In addition, Western blotting results manifested that the protein expression levels of PTEN, PI3K, Akt and mTOR were significantly lower in the miR-21 mimics group (p<0.05), whereas was remarkably higher in the miR-21 inhibitor group (p<0.05). MiR-21 is involved in regulating the pathological symptoms and myocardial cell apoptosis in hypertensive rats through the PTEN/PI3K/Akt/mTOR signaling pathway.

Relationship between the expression of NOD-like receptor protein 3 inflammasome and the activity of ulcerative colitis

Objective To investigate the relationship between the expression of NOD-like receptor protein 3 (NLRP3) inflammasome in colonic mucosal tissues of ulcerative colitis (UC) patients and UC mice model and the activity of UC. Methods From December 2016 to January 2018, at Department of Gastroenterology, the First Affiliated Hospital of Zhejiang Traditional Chinese Medicine University, 60 patients with UC were recruited, of which 15 cases at remission phase, 15 cases at mild activity phase, and 15 cases at moderate activity phase, and 15 cases at severe activity phase; and 15 healthy subjects were selected as healthy control group. UC mice models were established by dextran sulfate sodium (DSS). Forty-eight BALB/c mice were divided into 2.5% DSS group, 5.0% DSS group and 7.5% DSS group and control group. The colon tissues of UC patients and UC mice models were pathologically scored. The expression of NLRP3, cysteinyl aspartate specific proteinase 1(caspase-1) and apoptosis-associated speck-like protein containing CARD (ASC) at mRNA level in colon tissues of UC patients and UC mice models were determined by real time fluorescence quantitative polymerase chain reaction (PCR). The expression of NLRP3, caspase-1 and ASC at protein level in colon tissues of UC patients and UC mice models were detected by Western blotting. One-way analysis of variance and SNK-t test were performed for statistical analysis. Results The histopathological scores of colon tissues of UC patients at remission phase, at mild activity phase, at moderate activity phase, at severe activity phase and healthy controls were 2.37±0.46, 4.84±1.29, 6.82±0.96, 9.42±1.13 and 1.23±0.55, respectively; the differences were statistically significant (F=67.68, P<0.01). The higher the degree of inflammation, the higher the pathological score, and the differences were statistically significant (all P<0.05). The pathological scores of colon tissues of mice in the 2.5% DSS group, 5.0% DSS group and 7.5% DSS group were 4.54±0.74, 6.02±1.00 and 8.43±1.46, respectively; the higher the dose of DSS, the higher the pathological score, and the differences were statistically significant (all P<0.05). The expression of NLRP3 at mRNA level of UC at remission phase, mild activity phase, moderate activity phase, severe activity phase and healthy controls were 1.15±0.10, 1.49±0.13, 2.00±0.25, 2.05±0.33 and 0.61±0.09, respectively; the expression of caspase-1 at mRNA level were 1.13±0.08, 1.51±0.19, 2.10±0.23, 2.88±0.33 and 0.61±0.11, respectively; the expression of ASC at mRNA level were 1.12±0.08, 1.88±0.33, 2.53±0.22, 3.20±0.24 and 0.59±0.12, respectively; the differences between groups were statistically significant (F=108.43, 63.25 and 105.25, all P<0.01). The higher the degree of inflammation, the higher the mRNA expression levels of NLRP3, caspase-1 and ASC, and the differences were statistically significant (all P<0.01). The higher the dose of DSS, the higher the protein expression levels of NLRP3, caspase-1 and ASC at mRNA level. The higher the degree of inflammation, the higher expression of NLRP3, caspase-1 and ASC in colon tissues of UC patients. The higher dose of DSS, the higher the protein expression levels of NLRP3, caspase-1 and ASC in colon tissues of mice. Conclusions The expression level of NLRP3 inflammasome is different in different stages of UC, the higher degree of inflammatory activity, the higher the expressie level. It is helpful to evaluate the activity of UC by detecting the expression level of NLRP3 inflammasome. Key words: Ulcerative colitis; NLRP3 inflammasome; Activity of inflammation