Apoptosis Rate In Group Research Articles

Mechanism of Dexmedetomidine Alleviating Myocardial Ischemia-Reperfusion Injury Through Mitochondrial and ER Oxidative Stress Pathway

Objective: To analyze the mechanism of dexmedetomidine through mitochondrial and ER oxidative stress pathway in neonatal rat cardiomyocytes. Methods: SD neonatal rats were selected as the research object, and the control group (Group A), H2O2 group (Group B), dexmedetomidine group (Group C) and dexmedetomidine + H2O2 group were constructed (Group D) four groups of neonatal rat cardiomyocyte oxidative injury models. After drug treatment, the morphology, LDH, GSH, ROS, casease-3,8,9 and 12 activities of neonatal rat cardiomyocytes were detected in turn. The mitochondrial membrane potential and apoptosis rate were detected by flow cytometry, and GRP78 and IRE1 were detected by Western blot α, Expression of Bcl-2 and Bax proteins. Results: 500umol/l was selected as the best reaction concentration of H2O2. H2O2 can cause the disorder of cytoskeleton sequencing and the disappearance of myocardial striations, while dexmedetomidine can reduce the damage of H2O2 to cardiomyocytes. Compared with group A, the activity levels of LDH and ROS in cardiomyocytes in group B increased significantly, and the activity level of GSH decreased significantly. Compared with group B, the activity of GSH in group D was significantly increased, while the activity of LDH and ROS decreased significantly. Compared with group A, the activity levels of Caspase-3,8,9 and 12 in cardiomyocytes of group B increased significantly. Compared with group B, the activity of Caspase-3,8,9 and 12 in cardiomyocytes in group D decreased significantly. The apoptosis rate of cardiomyocytes in group A was (18.36 ± 5.68)%, and that in group D was (39.64 ± 9.36)%. Compared with group A, the apoptosis rate in group B increased significantly; there was no significant change in the apoptosis rate in group C. Compared with group A, the ratio of Bax to Bcl-2 in group B increased significantly; compared with group B, the ratio of Bax to Bcl-2 in group D decreased significantly. Compared with group A, GRP78 and IRE1 α Significantly improved; Compared with group B, GRP78 and IRE1 in group D α Significantly reduced. Conclusion: Dexmedetomidine can inhibit the oxidative stress response and apoptosis induced by H2O2 in neonatal rat cardiomyocytes.

Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity

To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

Improving porcine SCNT efficiency by selecting donor cells size

ABSTRACT Considerable advancements have recently been achieved in porcine somatic cell nuclear transfer (SCNT), but the efficiency remains low. Donor cell size might play an important role in SCNT, but its effects in pigs remain unclear. This study aimed to evaluate the efficiency of porcine SCNT by selecting donor cells of suitable size. Porcine fetal fibroblasts (PFFs) were divided into three groups, group S (small, d ≤ 13 μm), group M (medium, 13 μm<d ≤ 18 μm), and group L (large, d > 18 μm), and their biological characteristics were analyzed. Next, SCNT was performed using PFFs of different sizes to evaluate the developmental potential of reconstructed embryos. The data showed that PFFs in groups S, M and L accounted for 17.5%, 47.7% and 34.8% of cells, respectively. Morphologically, cells in group S exhibited clear and regular cell membranes and nuclei, whereas cells in groups M and L displayed varying degrees of cell membrane protuberance, karyo-pyknosis, autophagy and mitochondrial abnormalities. In addition, the growth status and proliferation capabilities of cells in group S were significantly better than those of group M and group L. The percentage of cells at G0/G1 in group S and M were significantly greater than group L. The senescence rate of group S was lower than group M and group L. The apoptosis rate of group S was significantly lower than that of group L but comparable to that of group M . The cleavage rate of group S was also significantly greater than that of group M but comparable to that of group L . The blastocyst rate of group S was significantly greater than that of group M and group L. The blastocyst cell numbers of group S were also significantly greater than those of group M and group L. These findings suggested that small PFFs with a diameter of less than 13 μm are more suitable donor cells for SCNT in pigs. Abbreviations: DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; PFFs: Porcine fetal fibroblast cells; SCNT: Somatic cell nuclear transfer.

Effects of Bone Powder Extracts with Different Hydroxyapatite Contents on Proliferation, Apoptosis, and Cell Phase of Human Umbilical Vein Endothelial Cells

ABSTRACTBone powder with different contents of hydroxyapatite may have different effects on angiogenesis in bone defect areas. The effects of bone powder with different content of hydroxyapatite on proliferation, apoptosis and cell cycle of vascular endothelial cells (HUVECs) were studied. In the experimental group, HUVECs were cultured in the incubator with different contents of hydroxyapatite bone powder extract. The experimental group was divided into four subgroups: group A had a low hydroxyapatite content of 10–15%; group B had a hydroxyapatite content of 25–30%; group C had a hydroxyapatite content of 80–90%; and group D had a hydroxyapatite content of more than 90%. The control group was treated with the culture of umbilical vein endothelial cells alone. Firstly, we used scanning electron microscope (SEM) to observe the morphology of bone powder with different hydroxyapatite content in normal condition. After that, we carried out a series of experiments to observe the changes of cell morphology, proliferation ability, apoptosis and cell cycle in different culture groups. The results showed that there was no significant difference in cell morphology and proliferation ability among different experimental groups. However, the apoptosis rate in group A was higher than that in group C. The underlying mechanism may be related to G1 arrest and G2 transformation in smaller and higher concentration groups, respectively. We can thus preliminarily conclude that a high concentration of hydroxyapatite in bone powder extract would be most suitable for cell cultures.

Aseptic capillary vitrification of human spermatozoa: Cryoprotectant-free vs. cryoprotectant-included technologies

The protocol of aseptic cryoprotectant-free vitrification on human spermatozoa is well documented. However, data about the effect of permeable cryoprotectants at this procedure is limited. Presented study aimed to test the aseptic capillary vitrification technologies using permeable cryoprotectant-included or cryoprotectant-free media. Thirty-two normal samples were included and analyzed after vitrification in three different media and thawing. Three treatment groups were formed: Group 1, basic medium; Group 2, basic medium with 0.25 M sucrose; Group 3, basic medium with glycerol. Before plunging into liquid nitrogen, capillaries were filled by 10 μl of spermatozoa suspension and isolated from liquid nitrogen by location in hermetically closed 0.25 ml straws. Progressive motility, plasma membrane integrity, total motility/viability after 24, 48 and 72 h in vitro culture, apoptosis and mitochondrial membrane potential (ΔΨm) were determined after thawing at 42 °C. Progressive motility of spermatozoa in groups 1, 2, 3 was 24.9 ± 1.7%, 34.5 ± 2.8% and 34.0 ± 1.4%, respectively (P1-2,3<0.05). The plasma membrane integrity of spermatozoa in groups 2 and 3 (48.4 ± 2.9% and 45.5 ± 3.9%, respectively) was higher than in Group 1 (33.3 ± 2.1%, P < 0.05). After 24 h, 48 h and 72 h in vitro culture, the total motility and viability of spermatozoa in Group 1 was significantly lower than Group 2 and Group 3. The apoptosis rate in Group 3 (44.5 ± 3.0%) and Group 2 (47.7 ± 4.1%) were lower than in Group 1 (52.5 ± 4.4%; P < 0.05). ΔΨm rates in Group 3 and Group 2 were higher than in Group 1 (P < 0.05) with no statistical differences between this parameter in Group 2 and Group 3 (P > 0.1). In conclusion, supplementation of medium for aseptic capillary technology for cryoprotectant-free vitrification of human spermatozoa by permeable cryoprotectant does not improve the quality of spermatozoa after warming.

Expression of miR-34a in cataract rats and its related mechanism.

Expression of miR-34a in cataract rats and its related mechanism were investigated. A total of 30 SD rats were selected and divided into three groups: group A: 2-month-old lucent lens, group B: 18-month-old lucent lens, and group C: 18-month-old naturally occurring cataract lens. The lens was taken and measured by LOC III to determine the degree of lens opacity of the three groups of rats. qPCR was used to detect expression of miR-34a and mRNA of SIRT1 and P53. Western blotting was used to detect the protein expression of SIRT1 and P53. Cell apoptosis was detected by flow cytometry. The lens of rats in group C was more turbid than that of groups A and B (P<0.05). The expression levels of miR-34a and P53 mRNA in the rats lens of group C were significantly higher than those in groups A and B, and the expression of SIRT1 mRNA was significantly lower than that of groups A and group B (P<0.05). Expression of miR-34a in group A was significantly higher than that in group B, the mRNA expression of SIRT1 was significantly lower than that in the lucent lens of 18-month-old rats (P<0.05). The expression of SIRT1 protein in group C was significantly lower than that in groups A and group B, while the expression level of P53 protein in group C was significantly higher than that of groups A and B. The expression of SIRT1 protein in group B was significantly higher than that in group A (P<0.05). The apoptosis rate of group C was higher than that of groups A and group B (P<0.05). In conclusion, the upregulation of expression level of miR-34a is related to cataract occurrence in rats, which may be caused by regulation of SIRT1 protein.

Effects of lncRNA HOTAIR on proliferation and apoptosis of myeloma cells through NF-κB pathway.

The aim of this study was to explore the influences of long non-coding ribonucleic acid (lncRNA) homeobox transcript antisense intergenic RNA (HOTAIR) on the proliferation and apoptosis of myeloma cells and its molecular mechanism. The myeloma cells were randomly divided into three groups, including: group A (myeloma cell group), group B [HOTAIR-small-interfering RNA (siRNA) group], and group C (HOTAIR negative control group). The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed to detect the expression of lncRNA HOTAIR in myeloma cells. The flow cytometry was adopted to determine the apoptosis of myeloma cells. Meanwhile, the protein expression of nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) was detected via Western blotting. In addition, the activity of the myeloma cells was measured using methyl thiazolyl tetrazolium (MTT) assay. The expression of HOTAIR in group A, group B, and group C was (2.19 ± 0.33), (1.37 ± 0.25), and (2.51 ± 0.27), respectively. Compared with group A and group C, the expression of HOTAIR was significantly downregulated in group B, in which the cells interfered with siRNAs. The expression of HOTAIR was significantly higher in group C than group A and group B (p<0.05). HOTAIR expression reached the highest level in group C, followed by group A and group B, respectively. The results of MTT indicated that the activity of the myeloma cells significantly increased at 24 h, 48 h, and 72 h in group C when compared with group A and group B. However, the activity of the myeloma cells was relatively low in group B, showing a slow rising trend. The activity of myeloma cells in group A remarkably increased when compared with group B, but it was lower than group C. Furthermore, the activity of the myeloma cells was higher in group C than group B (p<0.05). Western blotting indicated that the protein expression of NF-κB in group A, group B, and group C was (0.79 ± 0.22), (0.51 ± 0.17), and (0.95 ± 0.31), respectively. Compared with group A and group C, the protein expression of NF-κB was significantly downregulated in group B (the cells interfered with siRNAs) (p<0.05). Meanwhile, the protein expression of NF-κB was markedly higher in group C than in group A and group B (p<0.05). The protein expression of NF-κB was the highest in the cells of group C. Flow cytometry demonstrated that the apoptosis rate in group A, group B, and group C was (9.57 ± 1.71), (20.33 ± 1.63), and (8.74 ± 1.23), respectively. Compared with group A and group C, the apoptosis was significantly elevated in group B, in which the cells interfered with siRNAs (p<0.05). Compared with group A, the number of apoptotic myeloma cells significantly decreased in group C (p<0.05). LncRNA HOTAIR activates the expression of NF-κB in myeloma cells and promotes the proliferation of myeloma cells.

Vitrification freezing of large ovarian tissue in the human body

BackgroundThis study aims to carry out the vitrification freezing of a large ovarian tissue in the human body, and evaluate its feasibility.ResultsA total of 18 ovarian tissues in the human body were selected, and each tissue was cut into three large ovarian cortex slices. These tissues were randomly divided into three groups: vitrification freezing group (group A), programmed freezing group (group B), and fresh control group (group C). Then, the morphological analysis and apoptosis detection of each ovarian tissue was carried out, followed by the recycling of ovarian tissues at three weeks after the heterotransplantation of nude mice, in order to detect the follicle preservation conditions. The immunohistochemistory method was applied to detect the follicle activity. In comparing the proportion of primordial follicle with normal morphology after unfreezing between group A and group B, the difference was not statistically significant (P > 0.05). Furthermore, the incidence of follicle apoptosis in group A and group B was higher than that in the group C (P < 0.05). However, when comparing between group A and group B, the difference was not statistically significant (P > 0.05). The interstitial cell apoptosis rate in group A was lower than that of the group B, showing that the difference was statistically significant (P < 0.05).ConclusionsCompared with programmed freezing, the vitrification freezing of large ovarian tissues in the human body was feasible to a certain extent. This can be used as an alternative scheme to realize the freeze preservation of ovarian tissues in the human body.

A study on the protective effects of protein transduction domain 4-Cu, Zn superoxide dismutase fusion protein aganist global cerebral ischemia/reperfusion injury in rats

Objective To investigate the protective effects of protein transduction domain 4-Cu, Zn superoxide dismutase fusion protein (PTD4-Cu, Zn-SOD) against cerebral ischemia/reperfusion injury (CI/RI) in rats. Methods A total of 120 adult male rats were randomly divided into four groups (n=30): sham group (group SH), ischemia/reperfusion group(group I/R), superoxide dismutase group (group SOD), PTD4-Cu, Zn-SOD group (group PTD4-SOD). For group I/R, SH and PTD4-SOD, their bilateral carotid arteries were temporarily occluded over 6 min after electrocoagulation of bilateral vertebral arteries for 24 h to establish a CI/RI model. Before reperfusion, rats in group SOD and PTD4-SOD were injected with 6 mg/kg SOD or 6 mg/kg PTD4-SOD via the tail vein, respectively, while those in group SH and group I/R received the same volume of phosphate buffer saline(PBS) via the tail vein. Then the brain tissue samples were harvested 2, 6, 12, 24, 72 h after reperfusion(n=6). The pathological changes of the hippocampus were observed by hematoxylin-eosin (H-E) staining. The activity of SOD in the hippocampus was detected by xanthine oxidase method. The apoptosis rate of hippocampal cells was examined by flow cytomrtry. Results H-E stain: the neurons in hippocampal CA1 region of group SH were tightly arranged, where the cytoplasm was reddish and the nuclear was stained blue, with the clear nucleus. The neurons of group I/R were arranged in a disordered manner and idioblast counts were increased. The cytoplasm and nucleolus obviously shrunk, where the nucleolus disappeared or dissolved. Compared with group I/R, group PTD4-SOD presented tightly arranged neurons in hippocampal CA1 region, with little sparse idioblasts. The changes in group SOD was between group PTD4-SOD and group I/R. SOD activity test: compare with group SH, remarkable difference was found in groups I/R, SOD and PTD4-SPD 12, 24 and 72 h after perfusion (P<0.05). Compared with group PTD4-SOD, remarkable difference were found in group I/R and group SOD at each time point (P<0.05). The cell apoptosis rate of hippocampus: compared with group SH, there were marked differences in the apoptosis rate in group I/R, group SOD, group PTD4-SOD at each time point (P<0.05). Compared with group PTD4-SOD, remarkable difference were found in group I/R and group SOD at each time point (P<0.05). Conclusions PTD4-Cu, Zn-SOD can relieve CI/RI, which may be associated with enhanced SOD activity and free radical scavenging. Key words: Ischemia-reperfusion injury; Free radicals; Protein transduction domain-superoxide dismutase fusion protein

Expression of miR-490-5p, miR-148a-3p and miR-608 in bladder cancer and their effects on the biological characteristics of bladder cancer cells.

Changes in the expression of miR-490-5p, miR-148a-3p and miR-608 in bladder cancer tissues were studied. A total of 30 patients with bladder cancer who had surgical resection in the Hunan Provincial People's Hospital (Changsha, China) from April 2015 to August 2016 were selected. RT-qPCR was used to detect the expression levels of miR-490-5p, miR-148a-3p and miR-608. The expression vectors of miR-490-5p, miR-148a-3p and miR-608 were respectively transfected and divided into three groups: blank cell group, gene transfection group (groups A-C) and negative transfection group (NC group). CCK8 was used to detect cell proliferation and flow cytometry was used to detect the condition of apoptosis of each group, and the Transwell chamber was used to detect the invasion ability of the cells. After the transfection, the expression level of miR-490-5p in group A was significantly higher than that in the NC and blank groups, and the expression level of miR-148a-3p in group B was significantly higher than that in the NC and blank groups. The expression level of miR-608 in group C was significantly higher than that in the NC and blank groups (P<0.001). The survival rates of the cells in groups A-C were significantly lower than those in the NC and blank groups at 48 and 72 h (P<0.001). After the transfection, the number of invasive cells and the apoptosis rates in groups A-C were significantly higher than those in the NC and blank groups (P<0.05). miR-490-5p, miR-148a-3p and miR-608 promoted proliferation of bladder cancer T24 cells and inhibited apoptosis of the cells and showed potential to become a new target for the future treatment of bladder cancer.

Expression and significance of annexin A3 in osteosarcoma HOS cells

Objective To investigate the expression and significance of annexin A3 (ANXA3) in osteosarcoma HOS cells. Methods The expression levels of ANXA3 in osteoblasts cells, osteosarcoma HOS cells and osteosarcoma U2OS cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively. The expression of ANXA3 in HOS cells was down-regulated by transfection of Small interfering RNA (siRNA) into HOS cells by liposome-mediated method. Flow cytometry was used to detect the apoptosis of cells before and after transfection. Results Compared with the osteoblast control group, the expression of RNA and protein in ANXA3 cells was significantly increased in HOS and U2OS cells. At the level of RNA, HOS cells was 2.99 times higher than that of osteoblasts cells, and U2OS cells was 1.25 times higher than that of osteoblasts cells. At the protein level, HOS was 3.25 times higher than that of osteoblasts, and U2OS was 2.33 times higher than that of osteoblasts. siRNA down-regulated ANXA3 expression 48 hours and Western blotting detection transfection efficiency, Compared with the control group, the down-regulation of the si-651 group was 0.26, the down-regulation of the si-732 group was 0.31, the down-regulation of the si-1094 group was 0.31, the positive control group was 0.37, the negative control group was 0.87. The apoptotic rate was 8.1% in the control group and 21.5% in the si-651 group, which was 15.2% higher than that in the control group. The apoptosis rate of si-732 group was 13.3%, which was 7.5% higher than that of control group. Compared with the control group increased 7.5%; si-1094 group apoptosis rate was 13.4%, compared with the control group increased 6.7%. Conclusion The expression of ANXA3 in osteosarcoma cells is improved; Down-regulation of ANXA3 expression in HOS cells can increase the apoptotic capacity. Key words: Osteosarcoma; Annexin A3; Gene silencing; Targeted therapy

Effect of attenuated expression of neuraminidase 3 via RNA interference on the proliferation and apoptosis of osteosarcoma MG-63 cells

To investigate the effect of attenuated expression of neuraminidase 3 (NEU3) via RNA interference on the proliferation and apoptosis in human osteosarcoma MG-63 cells. MG-63 cells were immunostained to observe the expression of NEU3. The cells were then divided into 5 groups: MG-63 cells in normal control group (group A) were not treated; MG-63 cells in 30, 50, and 100 nmol/L NEU3 RNA interference groups (groups B, C, and D) were transfected with 30, 50, and 100 nmol/L of NEU3 small interfering RNA (siRNA); negative control group (group E), MG-63 cells were transfected with different species negative siRNA (actin siRNA of mice, 50 nmol/L). The expression level of NEU3 mRNA was measured with real-time fluorescence quantitative PCR (qPCR). The proliferation of the cells was measured by cell counting kit 8 (CCK-8). The cell apoptosis rate was detected by flowcytometry (FCM). The expressions of cell apoptosis related proteins (Ras and Bcl-2) were detected by Western blot assay. NEU3 expressed in the cytoplasm of MG-63 cells under fluorescence microscope. The qPCR results showed that NEU3 mRNA levels were significantly lower in groups B, C, D than that in groups A and E ( P<0.05) after 24 hours of transfection; meanwhile, with the increase of siRNA concentration, NEU3 mRNA levels were significantly decreased ( P<0.05). The CCK-8 results showed that with the increase of siRNA concentration, the survival rate of MG-63 cells was significantly suppressed ( P<0.05) and the apoptosis rate of MG-63 cells was significantly accelerated ( P<0.05) after 48 hours of transfection. FCM results showed that after 24 hours of transfection, the number of live MG-63 cells decreased as that of the dead cells increased in groups B, C, D, and showing significant differences between 3 groups ( P<0.05). While the apoptosis rate in groups B, C, and D showed significant difference when compared with that of group A ( P<0.05); and when compared with group E, the apoptosis rate in groups C and D were significantly reduced ( P<0.05), but there was no significant difference between groups B and E ( P>0.05). The results of Western bolt assay showed that the protein levels of Ras and Bcl-2 in groups B and C were not significantly different from groups A and E ( P>0.05), while the protein levels of Ras and Bcl-2 were significantly decreased in group D ( P<0.05). Attenuated expression of NEU3 could inhibit the survival of MG-63 cells and accelerate its apoptosis. The results suggest that NEU3 could be a possible target for treating osteosarcoma.

Effects of hypoxia-inducible factor 1α on hypoxic tolerance of human amniotic mesenchymal stem cells

Under hypoxic conditions, the survival and apoptosis of human amniotic mesenchymal stem cells (hAMSCs) were observed by transient transfection of hypoxia-inducible factor 1α (HIF-1α) gene, to investigate the effect of HIF-1α on hypoxic tolerance of hAMSCs. The hAMSCs were isolated and cultured from amniotic membrane tissue from voluntary donors who were treated with cesarean section. And the morphological observation by inverted phase contrast microscope and immunofluorescence detection of the expressions of stem cell markers OCT-4 and NANOG were performed to identify the cultured cells. The third generation hAMSCs were treated with 200 μmol/L CoCl 2, and transient transfection of plasmids were added according to the following grouping: group A was hAMSCs blank group; group B was pcDNA3.1 negative control group; group C was short hairpin RNA (shRNA) negative control group; group D was shRNA-HIF-1α interference group; group E was pcDNA3.1-HIF-1α over expression group. Cell survival rate of each group was measured by cell counting kit 8 (CCK-8) at 12, 24, 48 hours after hypoxia treatment. Flow cytometry was used to detect apoptosis rate of each group at 24 hours after hypoxia treatment. The expression levels of HIF-1α, vascular endothelial growth factor (VEGF), B-cell lymphoma 2 (Bcl-2), Bax, and cleaved Caspase-3 (C-Caspase-3) proteins were detected by Western blot at 24 hours after hypoxia treatment. CCK-8 assay showed that the cell survival rate of group D was significantly lower than those of groups A and C at all time points after hypoxia treatment; while the cell survival rate in group E was significantly increased than those in groups A and B, and the diffrences at 24 hours were significant ( P<0.05). In group E, the cell survival rate at 24 hours was significantly higher than those at 12 and 48 hours ( P<0.05). The results of flow cytometry showed that the apoptosis rate in group D was significantly higher than those in groups A and C ( P<0.05), and the apoptosis rate in group E was significantly lower than those in groups A and B ( P<0.05). Western blot showed that the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group D were significantly decreased when compared with those in groups A and C, and the expressions of Bax and C-Caspase-3 proteins were significantly increased ( P<0.05). On the contrary, the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group E were significantly higher than those in groups A and B, and the expressions of Bax and C-Caspase-3 proteins were significantly decreased ( P<0.05). Overexpression of HIF-1α gene can significantly improve hAMSCs tolerance to hypoxia, the mechanism may be related to up-regulation of VEGF and Bcl-2 expressions, and down-regulation of Bax and C-Caspase-3 expressions.

Effect of long non-coding RNA TapSAKI on the renal tubular epithelial cells induced by hypoxia

Objective To study the effect of long non-coding(LncRNA) TapSAKI on HK-2 cells of renal tubular epithelial cells induced by hypoxia. Methods The cultured HK-2 cells were divided into the normal oxygen group (group A), hypoxia injury group (group B), hypoxia injury+ control siRNA group (group C), and hypoxia injury+ TapSAKI siRNA group (group D). The real-time quantitative polymerase chain reaction (qPCR) was used to detect the change in TapSAKI expression; proliferation was measured by CCK-8 assay; cell cycle and cell apoptosis were measured by flow cytometry; Western blot was used to detect cycle related CyclinD1 protein, CDK2 and apoptosis related protein Bcl-2 and Bax. Results From the result of qPCR, compared with group A, the expression of TapSAKI gene in group B increased significantly (48.92±0.31 vs.1.00±0.01, P<0.05). Compared with group C, the expression of TapSAKI gene in group D decreased significantly(4.70±0.60 vs.48.31±0.29, P<0.05). The result of CCK-8 showed that the proliferation in group B significantly decreased compared with group A (P<0.05). Compared with group C, the proliferation in group D significantly increased (P<0.05). The flow cytometer test results showed that the apoptosis rate in group B was higher than that in group A [(26.38±1.21)% vs.(6.45±0.46)%, P<0.05]. Comparing with group C, the apoptosis rate in group D decreased significantly [(10.98±0.88)% vs.(21.59±1.30)%, P<0.05]. Compared with group A, Bax expression in group B increased, and Bcl-2 expression decreased (1.304±0.082 vs.0.411±0.002, 0.390±0.007 vs.1.027±0.022, all P<0.05). In group D, the expression of Bax was lower than that in group C, and the expression of Bcl-2 increased, both of which were statistically significant(0.655±0.819 vs.1.419±0.087, 0.819±0.034 vs.0.437±0.014, all P<0.05). Compared with group A, the proportion of G0/G1 phase cells in group B significantly increased (69.82±1.14 vs.34.46±0.82, P<0.05), and the proportion of S phase cells decreased significantly (11.6±0.60 vs.42.23±1.46, P<0.05). Compared with group A, CyclinD1 and CDK2 protein expressions in group B decreased (0.659±0.062 vs.1.723±0.084, 0.414±0.015 vs.0.87±0.031, all P<0.05). Compared with group C, group D G0/G1 phase cells significantly decreased (30.77±0.33 vs.61.81±1.50, P<0.05), the proportion of S phase cells significantly increased(40.32±0.72 vs.17.92±0.71, P<0.05), CyclinD1 and CDK2 protein expressions increased (2.049±0.027 vs.0.626±0.024, 0.89±0.104 vs.0.424±0.012, all P<0.05). Conclusions Under hypoxic conditions, LncRNA TapSAKI in renal tubular epithelial cells was abundant, which may inhibit renal tubular epithelial cell proliferation and accelerate apoptosis.It is suggested that LncRNA TapSAKI may play a role in the deterioration of cell proliferation and apoptosis. Key words: Long non-coding RNA; TapSAKI; Renal tubular epithelial cell; Hypoxia

Effects of microRNA-140 gene transfection with nucleus localization signal linked nucleic kinase substrate short peptide conjugated chitosan on rabbit articular chondrocytes

To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) ( NNSCS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes in vitro. Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with NNSCS to form NNSCS/pDNA complexes, respectively. Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits. The second generation chondrocytes were divided into 3 intervention groups: normal cell control group (group A), NNSCS/GV268 empty plasmid transfection group (group B), and NNSCS/GV268-miR-140 transfection group (group C). NNSCS/GV268 and NNSCS/GV268-miR- 140 complexes were transiently transfected into cells of groups B and C. After transfection, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140; Annexin Ⅴ-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes; the expressions of Sox9, Aggrecan, and histone deacetylase 4 (Hdac4) were detected by RT-qPCR. RT-qPCR showed that the expression of miR-140 in group C was significantly higher than that in groups A and B ( P<0.05). Compared with groups A and B, the apoptosis rate in group C was decreased and the proliferation activity was improved, Sox9 and Aggrecan gene expressions were significantly up-regulated, and Hdac4 gene expression was significantly down-regulated ( P<0.05). There was no significant difference in above indexes between groups A and B ( P>0.05). Exogenous gene can be carried into the chondrocytes by NNSCS and expressed efficiently, the high expression of miR-140 can improve the biological activity of chondrocytes cultured in vitro, which provides important experimental basis for the treatment of cartilage damage diseases.

Expression and significance of hypoxia-inducible factor 1α in endplate chondrocytes of rats

To explore the expression and significance of hypoxia-inducible factor 1α (HIF-1α) in endplate chondrocytes, and to study the relations between HIF-1α expression and endplate chondrocytes apoptosis. Eight Sprague Dawley rats were selected to obtain the L 1-5 intervertebral disc endplate; the endplate chondrocytes were isolated by enzyme digestion method, and the endplate chondrocytes at passage 3 were cultured under 20% O 2 condition (group A), and under 0.5% O 2 condition (group B). Cell morphology was observed by inverted phase contrast microscope and cell apoptosis was detected using flow cytometry after cultured for 24 hours; the mRNA expression of HIF-1α was detected by real-time fluorescent quantitative PCR, the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1α mRNA. HIF-1α specific RNAi sequence compound was constructed and transfected into cells. The transfected endplate chondrocytes at passage 3 were cultured under 0.5% O 2 condition in group C and group D (HIF-1α gene was silenced). After cultured for 24 hours, cells were observed via immunofluorescence staining of HIF-1α, and cell apoptosis was detected using flow cytometry. Meanwhile, the mRNA expressions of HIF-1α, collagen type II (COL II), Aggrecan, and SOX9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. At 24 hours after culture, small amount of vacuoles necrotic cells could be observed in group A and group B; there was no significant difference in apoptosis rate between groups A and B ( t=1.026, P=0.471), and HIF-1α mRNA and protein expressions in group B were significantly higher than those in group A ( t=22.672, P=0.015; t=18.396, P=0.013), but, there was no significant difference in protein expressions of Bax and Bcl-2 between groups A and B ( t=0.594, P=0.781; t=1.251, P=0.342). The number of vacuolar necrosis cells in group D was significantly higher than that in group C, and HIF-1α positive cells were observed in group D. The apoptosis rate of group D was significantly higher than that of group C ( t=27.143, P=0.002). The mRNA expressions of HIF-1α, COL II, Aggrecan, and SOX9 in group D were significantly lower than those in group C ( t=21.097, P=0.015; t=34.829, P=0.002; t=18.673, P=0.022; t=31.949, P=0.007). The protein expressions of HIF-1α and Bcl-2 in group D were significantly lower than those in group C ( t=37.648, P=0.006; t=16.729, P=0.036), but the protein expression of Bax in group D was significantly higher than that in group C ( t=25.583, P=0.011). HIF-1α mRNA expression is up-regulated under hypoxia condition, which will increase the hypoxia tolerance of endplate chondrocytes. Cell apoptosis is suppressed by the activation of HIF-1α in endplate chondrocytes under hypoxia condition.

Effects of transient exposure to high glucose on biological behaviors of human dermal microvascular endothelial cells

Objective: To observe the effects of transient exposure to high glucose on biological behaviors of human dermal microvascular endothelial cells cultured in vitro. Methods: The dividing method and treatment of cells for the detection of all indexes in this study were as follows. Human dermal microvascular endothelial cells of the 4th passage were divided into 3 groups according to the random number table, with 12 wells in each group. Cells in control group (C) were cultured with complete culture solution containing 5 mmol/L D-glucose for 7 d. Cells in transient high glucose group (THG) were cultured with complete culture solution containing 30 mmol/L D-glucose for 2 d and complete culture solution containing 5 mmol/L D-glucose for 5 d. Cells in prolonged high glucose group (PHG) were cultured with complete culture solution containing 30 mmol/L D-glucose for 7 d. (1) The cell morphology in groups C and PHG on culture day 7 and that in group THG on culture day 2 and 7 was observed by inverted optical microscope. (2) On culture day 0, 2, 4, and 7, cell proliferation rate was determined by cell viability analyzing counter. (3) After culture day 2, the scratch experiment was performed, and the cells were further cultured. At post scratch hour (PSH) 0, 24, 48, 72, 96, and 120, the scratch area was measured, and the cell migration rates of the latter 5 time points were calculated. (4) On culture day 0, 2, 4, and 7, the cell apoptosis rate was determined by cell analyzer. (5) Cells were seeded into Matrigel to culture for 24 h after culture day 7. The formation of vessel-like structure was observed by inverted optical microscope. The length and number of branch point of vessel-like structure were calculated. (6) On culture day 2, 4, and 7, mRNA expression of vascularization-related gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) was determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, one-way analysis of variance, and LSD test. Results: (1) Cells in group C exhibited ovary shape in cobble stone order on culture day 7. Cells in group THG exhibited long ovary shape and lost cobble stone order on culture day 2 and kept the same changes on culture day 7. Cells in group PHG exhibited long ovary shape and lost cobble stone order on culture day 7. (2) On culture day 0, there was no significant difference in cell proliferation rate among the 3 groups (F=0.23, P>0.05). On culture day 2, cell proliferation rates in groups THG and PHG were similar (P>0.05), which were significantly lower than the cell proliferation rate in group C (with P values below 0.01). On culture day 4 and 7, the cell proliferation rates in groups THG and C were similar (with P values above 0.05), which were significantly higher than those in group PHG (with P values below 0.01). (3) At PSH 24-120, the cell migration rates in groups THG and PHG were similar (with P values above 0.05), which were significantly lower than those in group C (with P values below 0.01). (4) On culture day 0, there was no statistically significant difference in cell apoptosis rate among the 3 groups (F=0.78, P>0.05). On culture day 2, cell apoptosis rates in groups THG and PHG were similar (P>0.05), which were significantly higher than the cell apoptosis rate in group C (with P values below 0.01). On culture day 4 and 7, the cell apoptosis rates in groups THG and C were similar (with P values above 0.05), which were significantly lower than those in group PHG (with P values below 0.01). (5) The length of vessel-like structure of cells in group THG was (1.84±0.10)×10(5) μm, close to (1.82±0.11)×10(5) μm in group PHG (P>0.05), both significantly shorter than (2.75±0.23)×10(5) μm in group C (with P values below 0.01). The numbers of branch point of vessel-like structure of cells in groups THG and PHG were 43±5 and 46±8 respectively, which were close to each other (P>0.05) and both significantly less than 103±21 in group C (with P values below 0.01). (6) On culture day 2, 4, and 7, mRNA expressions of TIMP-3 of cells in groups THG and PHG were similar (with P values above 0.05), which were significantly lower than those in group C (with P values below 0.05). Conclusions: Transient exposure to high glucose can cause metabolic memory of morphology, migration, and angiogenesis in human dermal microvascular endothelial cells cultured in vitro, resulting in sustained changes in biological behaviors. The mechanism may be related to the changes of vascularization-related genes.

Salubrinal protects human lens epithelial cells against endoplasmic reticulum stress-associated apoptosis

To study the protective effect of Salubrinal on human lens epithelial cells and its mechanism in endoplasmic reticulum stress (ERS). Hydrogen peroxide (H2O2 200 μmol/L) was used to intervene in the cultured human lens epithelial cells B3 (HLE-B3) so as to create an oxidative stress model and induce ERS in the model. Different concentration of Salubrinal (10, 15, 20, 25, 30 and 35 μmol/L) were added to the cultured HLE-B3 with or without H2O2 intervention. Then the cells were cultured for 24 hours. The cell counting kit (CCK-8) assay was used to test the viability of HLE-B3. The HLE-B3 cells were divided into three groups: Group A (normal control group), Group B (H2O2 200 μmol/L group), and Group C (H2O2 200 μmol/L+ Salubrinal 25 μmol/L group). After 48 h, TUNEL and flow cytometry assay (FCM) were used to examine the effect of Salubrinal on HLE-B3 apoptosis. The expression of glucose-regulated protein 78(GRP78), C/EBP homologous protein (CHOP), cysteinyl aspartate specific proteinase 12 (Caspase-12) and phosphorylation eukaryotic translation initiation factor 2α (p-elF2α) were tested by western blot at different points in time. Data from different groups was analyzed by one-way analysis of variance (ANOVA) while Dunnett t test was used under an equal condition, Dunnett's T3 for the unequal variances. CCK-8 results showed that without the intervention of H2O2, different concentrations of Salubrinal had no inhibitive effect on HLE-B3 viability, and that survival rates were (98.6±3.3) %, (98.7±2.6) %, (99.4±3.2) %, (98.6±1.9) %, (98.8±2.5) %, (99.3±3.2) % and (99.5±2.4) %. There was no statistically significant difference between them (F=0.09, P=0.10). With the increasing of Salubrinal concentration, the survival rates of HLE-B3 in the presence of H2O2 intervention were (52.9±4.7) %, (65.0±3.6) %, (72.9±3.8) %, (84.5±3.6) %, (91.6±2.1) %, (93.1±2.9) %, (92.0±3.3) %. There was statistically significant difference from the control group (all P<0.01). However, the survival rates no longer increased (P=0.56, 0.88) if the Salubrinal concentration was greater than 25 μmol/L. FCM results indicated that apoptosis rates of Group A, B and C were (1.9±0.7) %, (8.8±0.5) %, (4.3±0.3) %, respectively and the differences were statistically significant (F=396.26, P<0.01, comparing with Group A, all P<0.01). TUNEL results showed that apoptosis indexes of Group A, B, and C were (7.7±1.0) %, (36.9±1.0) %, (16.7±2.2) %, respectively and the differences were statistically significant. (F=618.39, P<0.01, comparing with Group A, all P<0.01). RESULTS of western blotting in group B at different points in time (0, 12, 24, 36, 48 h) showed that p-elF2α had increased by (2.16±0.38) times at 6 h; GRP78 had increased by (2.56±0.15) times at 12 h; CHOP started to rise after 12 h until it dropped after 24 h, and its amount had increased by (2.49±0.23) times at 48 h; Caspase-12 had increased significantly by (3.53±0.30) times at 48 h. The expression of GRP78, CHOP and p-elF2α in group C was greater than that in Group B, but the expression of Caspase-12 in Group C was lower than that in Group B (GRP78: F=37.85, P<0.01; CHOP: F=61.09, P<0.01; Caspse-12: F=22.27, P<0.01; p-eIF2α: F=15.11, P<0.01). Salubrinal might protect HLE-B3 against H2O2-induced apoptosis by inhibiting ERS related apoptosis pathways.(Chin J Ophthalmol, 2016, 52: 437-443).

Effect of metastasis suppressor 1 gene expression on migration and apoptosis of human gastric cancer cell BIU87 by regulating the hepatocyte growth factor/c-Met signaling pathway

Objective To investigate the effect of metastasis suppressor 1 (MTSS1) gene on the biological function of bladder cancer BIU87 cells by regulating the hepatocyte growth factor (HGF)/c-Met signaling pathway. Methods Cultured gastric cancer BIU87 cells and passaged cells were divided into three groups: group A (PEF MTSS1 and PSV2neo cotransfection group); group B: (shNC and PSV2neo co transfection group); group C (non transfection group). Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR) were used to detect MTSS1, c-Met, E-cadherin and N-cadherin protein and mRNA expression levels. Flow cytometry and scratch test were performed to examine the migration, proliferation and apoptosis of BIU87 cells transfected with PEF MTSS1. Results (1) Western blotting and Real-time PCR results showed that as compared with groups B and C, MTSS1 and c-Met protein and mRNA expression levels were significantly down-regulated in group A (P 0.05). (4) Flow cytometry showed that cell apoptosis rate in groups A, B and C was respectively (26.73±3.72)%, (4.28±0.68)% and (2.73±0.41)%. The apoptosis rate in group A was significantly higher than in group C (P<0.01), and there was no significant difference between groups B and C. Conclusion The transfection of MTSS1 gene can promote the proliferation and inhibit the apoptosis of gastric cancer cell line BIU87 cells probably by regulating the HGF/c-Met signal transduction pathway. Key words: Bladder cancer; Metastasis suppressor 1 gene; Hepatocyte growth factor/c-Met signaling pathway

Effect of mitochondrial apoptosis on pulmonary fibrosis in rats with severe scald injury

To explore the role of mitochondrial apoptosis on pulmonary fibrosis in rats with severe scald injury. According to the random digital table, a total number of 32 Wistar rats were divided into 4 groups: sham burn (group A), burn group (group B), 12-week post burn recovery group (group C), and 12-week post burn recovery plus a second burn injury group (group D). In group A and group B, lung tissues were harvested on post burn day 4. After received first burn injury 12 weeks, the group C and group D received separately a second sham burn injury and burn injury. Lung tissues were harvested on post burn day 4 after the second burn injury. All tissues were examined for cells apoptosis by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL). Pulmonary fibrosis was assessed by Masson trichrome staining and Sirius red staining. The protein expression levels of cleaved Caspase-3, Bax and Bcl-2 were assessed by Western blot. Both Masson trichrome staining and Sirius red staining showed obvious pulmonary fibrosis in group C and group D. The apoptosis rates of group B, C and D were significantly higher than that in group A ((15.50±3.30)%, (7.88±3.10)%, (15.88±3.23)% vs (2.10±1.07)%, all P<0.05). Compared to group A, cleaved Caspase-3 levels were significantly higher in group B, C and D ((0.59±0.11), (0.33±0.08), (0.73±0.13) vs (0.16±0.05), all P<0.05). The ratio of Bax/Bcl-2 in group B, C and D also increased significantly ((2.08±0.30), (0.83±0.09), (1.54±0.12) vs (0.64±0.05), all P<0.05). Severe burn injury can induce pulmonary fibrosis and mitochondrial apoptosis may play an important role in the development of pulmonary fibrosis.