Real-time Fluorescence Quantitative Method Research Articles

The Effect of Rootstock on the Activity of Key Enzymes in Acid Metabolism and the Expression of Related Genes in ‘Cabernet Sauvignon’ Grapes

The types and contents of organic acids in wine grapes determine wine quality. To explore the effects of different rootstocks on the acid metabolism of ‘Cabernet Sauvignon’ grapes, various perennial rootstock–scion combinations were used as experimental materials. High-performance liquid chromatography (HPLC) was used to determine citric acid, tartaric acid, and malic acid contents during fruit development. Succinic acid and oxalic acid contents and the activity of related enzymes were measured using spectrophotometry. The expression levels of related genes were measured using a real-time fluorescence quantitative method. The results showed that all four rootstock types significantly reduced oxalic acid and citric acid contents in the grapes, while increasing succinic acid content to varying degrees. Employing 110R, SO4, and Kangzhen3 rootstocks increased tartaric acid and malic acid contents. Enzyme activity analysis revealed that 110R, SO4, and Kangzhen3 rootstocks increased the NAD-MDH enzyme activity, which positively correlated with malic acid content. Simultaneously, these rootstocks reduced the NADP-ME enzyme activity level. NAD-MDH and PEPC gene expression levels were higher in ‘Cabernet Sauvignon’ grapes grafted with 110R, SO4, and Kangan3 rootstocks compared to control self-rooted seedlings. Grafting these three rootstocks enhanced malic acid accumulation in ‘Cabernet Sauvignon’ grapes.

The bio-derived material acacetin ameliorated hyperlipidemia and intestinal barrier damage in mice by modulating gut microbiota

The increasing incidence of hyperlipidemia has been linked to disruptions in gut flora. The bio-derived material, acacetin, is a naturally flavonoid compound extracted from various plants that has been shown to protect the liver and lowers blood lipid levels, its potential to mitigate gut barrier damage caused by a high-fat diet (HFD) has not been fully explored. This work aimed to investigate the effects of acacetin on HFD-induced intestinal barrier disruption and its potential interaction with gut microbiota regulation. Forty-eight female ICR mice were divided into three groups: control group (standard diet containing 6% daily energy from fat), HFD group (45% of daily energy from fat), and the acacetin (AC) group (HFD with acacetin 30 mg/kg body weight). The test period lasted for eight weeks. In addition to lipid metabolism parameters, serum lactate dehydrogenase (LDH) activity, diamine oxidase (DAO) activity, and lipid metabolism, we used the real-time fluorescence quantitative method to measure the expression of zonula occludens 1 (ZO-1) and occludin genes. The microorganisms were analyzed by 16 s RNA and functional gene prediction analysis. The results indicated that acacetin treatment could alter serum biochemical parameters and reduce body weight, liver weight gain, and abdominal fat accumulation. Furthermore, acacetin increased the expression levels of ZO-1 and occludin in HFD mice. In addition, acacetin altered the structure, diversity, and function of intestinal flora, characterized by the restoration of the Firmicutes/Bacteroidetes ratio. Additionally, the species abundance were significant correlation with lipid factors, DAO, and LDH. Alistipes and Acetatifactor were the prevalent genus in the AC group. Acacetin downregulated HFD-induced Facultative_anaerobic phenotypes related to Clostridium according to the BugBase analysis. The KEGG study revealed that acacetin altered the functional composition of microorganisms, as evidenced primarily by variations in the abundance of metabolic pathways involved in lipid metabolism and intestinal epithelial injury. The COG category showed acacetin increased the abundance of Cytoskeleton associated with the intestinal barrier. Overall, acacetin ameliorated HFD-induced hyperlipidemia and intestinal barrier damage in mice by modulating intestinal bacteria, exhibited a good clinical application prospect.

The Therapeutic Effects of Oral Intake of Hydrogen Rich Water on Cutaneous Wound Healing in Dogs.

This study explored the effects of drinking Hydrogen-rich water (HRW) on skin wound healing in dogs. Eight circular wounds were analyzed in each dog. The experimental group was treated with HRW thrice daily, while the control group was provided with distilled water (DW). The wound tissues of dogs were examined histopathologically. The fibroblasts, inflammatory cell infiltration, the average number of new blood vessels, and the level of malondialdehyde (MDA) and superoxide dismutase (SOD) activity in the skin homogenate of the wound was measured using the corresponding kits. The expressions of Nrf-2, HO-1, NQO-1, VEGF, and PDGF were measured using the real-time fluorescence quantitative method. We observed that HRW wounds showed an increased rate of wound healing, and a faster average healing time compared with DW. Histopathology showed that in the HRW group, the average thickness of the epidermis was significantly lower than the DW group. The average number of blood vessels in the HRW group was higher than the DW group. The MDA levels were higher in the DW group than in the HRW group, but the SOD levels were higher in the HRW group than in the DW group. The results of qRT-PCR showed that the expression of each gene was significantly different between the two groups. HRW treatment promoted skin wound healing in dogs, accelerated wound epithelization, reduced inflammatory reaction, stimulated the expression of cytokines related to wound healing, and shortened wound healing time.

Study on protective mechanism of Tibetan medicine Ershiwuwei Songshi Pills on cholestatic liver injury in rats based on FXR signaling pathway

This paper aimed to investigate the protective effect and mechanism of the Tibetan medicine Ershiwuwei Songshi Pills on α-naphthalene isothiocyanate(ANIT)-induced cholestatic liver injury in rats based on the farnesol X receptor(FXR) signaling pathway. SD rats were randomly divided into blank group, model group, ursodeoxycholic acid(UDCA) group, Tibetan medicine Ershiwuwei Songshi Pills low, medium and high dose groups(0.09, 0.18, 0.36 g·kg~(-1)). A prophylactic dosing regimen was used in the experiment. From the 1 st to 4 th days, the UDCA group and the Tibetan medicine Ershiwuwei Songshi Pills suspension groups received prophylactic gavage administration; on the 5 th day, the blank control group was given an equal volume of olive oil blank reagent, and the remaining groups were given ANIT modeling reagent. Administration was continued on day 5 to 6 in each administration group. Forty-eight hours after modeling on the 7 th day, blood was collected from the femoral artery of rats. Serum alkaline phosphatase(ALP), alanine aminotransferase(ALT), aspartate aminotransferase(AST), direct bilirubin(DBIL), total bilirubin(TBIL), and total bile acid(TBA) levels were detected, and liver histopathological changes were observed. The relative expression changes of FXR, SHP, CYP7 A1, MRP2, MRP3, NTCP, BSEP mRNA in liver tissues were detected by Real-time fluorescence quantitative method, and the expression changes of FXR, SHP, UGT2 B4 protein in liver tissues were detected by Western blot. The results showed that the Tibetan medicine Ershiwuwei Songshi Pills significantly reduced the levels of ALT, ALP, DBIL, TBIL and TBA in the serum of the ANIT mo-del rats(P<0.01, P<0.05), significantly up-regulated the mRNA expressions of SHP and NTCP(P<0.01, P<0.05), significantly down-regulated the mRNA expression of CYP7 A1 and MRP3(P<0.01, P<0.05); and significantly up-regulated the protein expressions of FXR and SHP(P<0.01, P<0.05). The Tibetan medicine Ershiwuwei Songshi Pills have an obvious protective effect on ANIT-induced cholestatic liver injury in rats, and its mechanism may be related to the bile acid metabolism mediated by the FXR signaling pathway.

Correlation between miR-564, TGF-β1, and radiation-induced lung injury*

Abstract Objective Our study aimed to analyze the expression of miR-564 and TGF- β1 in cancer tissues and the serum of patients with radiation-induced lung injury, and to investigate the relationship between them and radiation-induced lung injury. Methods In situ hybridization and real-time fluorescence quantitative method were used to detect the expression of miR-564. Additionally, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were performed to detect the expression of TGF- β1. Results The overall incidence of acute radiation pneumonia was 55.9% (100/179). The incidence of ≥ grade 2 radioactive pneumonia was 24.0% (43/179) and that of grade 1 was 31.8% (57/179). The expression of miR-564 in grade ≥ 2 was slightly higher than that in patients without or with grade 1, but there was no statistical difference (P = 0.86). The serum level and ratio of miR-564 in patients with grade ≥ 2 were significantly higher than those without or with grade 1 (P = 0.005, P = 0.025, respectively). The expression of TGF- β1 in grade ≥ 2 was significantly higher than that of patients without or with grade 1 (P = 0.017). The serum levels of TGF- β1 in grade ≥ 2 were significantly higher than those in patients without or with grade 1 (P = 0.038). Although the ratio of TGF- β1 in radiation pneumonia of grade ≥ 2 was significantly higher than that of without or with grade 1, there was no significant difference (P = 0.24). Moreover, patients with higher expression of miR-564 and lower expression of TGF-β1 had better prognosis. Conclusion MiR-564 and TGF-β1 are predictors of radiation-induced lung injury. Monitoring its changing trend can improve the accuracy of predicting radiation-induced lung injury. The levels and ratio of serum miR-564 and TGF- β1 in patients with radiation-induced lung injury are related to the severity of radiation-induced lung injury.

The influence of overexpressions of microRNA-375 on the expression of thymic stromal lymphopoietin and IL-4, IL-13 in allergic rhinitis mice

Objective: To detect the expression of thymic stromal lymphopoietin (TSLP) and IL-4, IL- 13 (interleukin-4, interleukin-13) through overexpression of microRNA-375 in allergic rhinitis (AR) mice. Methods: AR mice models with overexpression of miRNA375 were induced by ovalbumin (OVA) in Lentivirus overexpression vector AR group (Overexpression vector+OVA), lentivirus control vector AR group (Control vector+OVA) as well as AR (OVA) group. While saline was administered in control (Saline) group, nasal mucosa of each group were collected for further use. Relative expressions of TSLP and IL-4, IL-13 mRNA and proteins were detected by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analysis. Results: Real-time fluorescence quantitative method verified the overexpression of miRNA375 in allergic rhinitis group, and the same method has been adopted to verify the expressions of IL-4 and IL-13 genes in the overexpression group. The relative expressions of TSLP and IL-4, IL-13 mRNA and protein decreased in overexpression of microRNA-375 nasal mucosa compared with AR group (P

Effect of Yiqihuoxue prescription on myocardial energy metabolism after myocardial infarction via cross talk of liver kinase B1-dependent Notch1 and adenosine 5′-monophosphate-activated protein kinase

ObjectiveTo investigate the effect of Yiqihuoxue prescription (YQHX) from Traditional Chinese Medicine (TCM) on myocardial glucose and lipid metabolism after myocardial infarction via the cross talk between the liver kinase B1 (LKB1)-dependent Notch1 and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK). YQHX was prepared with substances with properties that benefit, to activate blood circulation based on the TCM theory. MethodsAnimal models of myocardial infarction were established by ligating Sprague Dawley rats' left anterior descending coronary arteries. The animals were randomly divided into a myocardial infarction (MI) group, a YQHX group, a perindopril group, a γ-secretase inhibitor, Notch signal inhibitor (DAPT) group, a DAPT+YQHX group and a sham group. The related drugs were administered on the second day after operation, and changes in the relevant indexes were examined on weeks 1 and 4. Changes in cardiac structure and function were examined by echocardiography. The glucose and free fatty acids (FFA) were examined by ELISA. The expression of Notch, LKB1 and AMPK mRNA was examined by a real-time fluorescence quantitative method. The expression of glucose transporter 4 (GLUT4), and the expression of total acetyl-CoA carboxylase (ACC) and its phosphorylation were examined by western blotting. ResultsCompared with the sham group, the expression of Notch, LKB1 and AMPK mRNA in the MI group was lower. Compared with the MI group, the expression of these mRNAs in the YQHX and perindopril groups was higher, and their expression in the DAPT group was lower. At all time points, the protein expression of GLUT4 and pACC decreased in the MI group. On week 1, the expression of pACC protein was higher. In the DAPT group, the expression of pACC protein decreased. Compared with the YQHX group, the expression of pACC protein in the DAPT + YQHX group was lower. On week 4, compared with the MI group, the expression of GLUT4 protein in the YQHX group and the perindopril group was higher. The expression of GLUT4 protein in the DAPT group decreased. Compared with the YQHX group, the expression of GLUT4 protein in the DAPT+YQHX group was lower. There was no significant difference in the expression of ACC protein between the groups. ConclusionYQHX promoted cross talk between the LKB1-dependent Notch1 and AMPK in myocardial tissue after myocardial infarction. Furthermore, it regulated the glucose and lipid metabolism of cardiomyocytes at different time points, thereby ameliorating the cardiac energy metabolism via different mechanisms and protecting the heart.

Role of measuring the expression levels of a proliferation-inducing ligand and its receptors by real-time fluorescence quantitative method in children with lymphoma

to investigate the role of a real-time fluorescence quantitative polymerase chain reaction (PCR) in measuring the expression levels of a proliferation-inducing ligand and its receptors expression levels in peripheral blood of children with lymphoma. real-time fluorescence quantitative PCR was used to detect the expression levels of a proliferation-inducing ligand and its receptors in patients with Hodgkin disease (n = 10) or non-Hodgkin lymphoma (n = 37) and healthy control (n = 40). The correlation between the mRNA levels of a proliferation-inducing ligand and its receptors and differential stage of malignant lymphoma was analyzed. the results of 47 samples showed that the levels of proliferation-inducing ligand, B cell maturation antigen, transmembrane activator and CAML interactor in the peripheral blood of lymphoma in children were significantly higher than those in normal children (1.13 ± 0.09 vs 0.41 ± 0.09, 1.22 ± 0.11 vs 0.43 ± 0.10, 0.89 ± 0.12 vs 0.35 ± 0.08, all P < 0.05). The level of a proliferation-inducing ligand and its receptors had no significant difference between Hodgkin disease and non-Hodgkin lymphoma (P > 0.05). The level of a proliferation-inducing ligand in I-II stage (0.88 ± 0.06, 0.90 ± 0.08) of malignant lymphoma in children was significantly lower than that in III-IV stage (1.21 ± 0.09, 1.23 ± 0.09, P < 0.05) while the level of its receptors between various stages showed no difference (P > 0.05). the real-time fluorescence quantitative PCR has a high sensitivity and reproducibility. A proliferation-inducing ligand may play a great role in the development and progression of malignant lymphoma in children through its receptors of B cell maturation antigen, transmembrane activator and CAML interactor. A proliferation-inducing ligand and its receptors can be established as target molecules for early diagnosis and anti-cancer therapy.

Effect of blood samples rich in triglyceride on the result of quantitative teat of hepatitis C virus RNA

Objective To investigate the effect of blood samples rich in triglyceride on the results of quantitative test of hepatitis C virus RNA.Methods Under the action of reverse-transcriptase,DNA was reversely transcribed into HCV-cDNA.Using four types of deoxyribonucleotide as a substrate,HCV-RNA was detected by real-time fluorescent quantitative method with TaqMan probe to evaluate the effects of different concentrations of triglyceride on the test results of low- and high-load of HCV-RNA.Results In the sample containing viral load of 104 IU/ml,the viral load was declined from 104 IU/ml to 103 IU/ ml with an increase in triglyceride concentration to 4mmol/L,the test result had a statistical significance; while the viral load increased to 107,106 or 105 IU/ml,there was no marked relationship between the test result of HCV-RNA and the concentration of triglyceride.Conclusions Blood samples rich in triglyceride has an interfering effect on tests for low-load of HCV-RNA but not remarkably on those for high-load of HCV-RNA. Key words: Polymerase chain reaction; Triglyceride; HCV-RNA

Detection of TNF-related apoptosis inducing ligand gene expression by real-time fluorescent quantitative method

Objective To establish a real time fluorescent quantitative revers transcripatase PCR(FQ-RT-PCR) method to detect the expression level of TNF-related apoptosis inducing ligand (TRAIL) mRNA in peripheral blood mononuclear ceils (PBMC) and determine its expression level in healthy donors, HBV-caused cirrhosis patients and PBC ones. Methods Specific primers and Taqman-MGB probe were designed and β-actin was used as endogenous control. The amplified fragment was obtained by RT-PCR. The quantitative template was constructed and then the fluorescent intensity was documented on the ABI Prism7000 analyzer. The standard curve was established, according to which, the TRAIl. mRNA levels in 30 healthy individuals, 30 patients with primary biliary cirrhosis (PBC) and 25 ones with HBV-caused cirrhosis were calculated automatically by software after the values of cycle threshold (Ct) were detected continuously during amplification. Results The linear detection range of the assay for TRAIL gene was 103 - 109 copies/ ug RNA ( r=-0.997). The coefficients of variation of both intra-and inter-assay reproducibility for high concentration samples were 5.6% and 6. 3% , respectively, and those for low concentration samples were12.5% and 14. 6%. The TRAIL mRNA expression level in PBC patients was [ (3.3±2.5)×105copies/ugRNA] significantly higher than that of healthy control [ (0.5±0.2)×105 copies/ug RNA ] (t=5.994,P <0.01). TRAIl. mRNA level of HBV-caused cirrhosis patients[ (2.1±0.9)×105 copies/ug RNA] wasalso significantly elevated (t=8.536, P<0.01). However, the difference between these two diseased groups had no significance. Conclusion We have successfully set up a FQ-RT-PCR method for detecting TRAIL gene expression and found that its expression levels of peripheral blood mononuelear cells in PBC and HBV caused cirrhosis patients are elevated, which provides a new insight into mechanism study of liver injury caused by cirrhosis. Key words: Tumor necrosis factors; Leukocytes, mononuclear; Gene expression; Reverse transcriptase polymerase chain reaction; Membrane glycoproteins

Detection of the expression level of Toll-like receptor3 by establishing real-time fluorescence quantitative method

目的 建立检测人Toll样受体3(TLR3)mRNA表达水平的实时荧光定量逆转录PCR(FQ-RT-PCR)法,及探讨其在外周血单个核细胞(PBMC)中的表达水平.方法 用Beacon Designer2.1软件设计TLR3基因特异性引物和荧光探针,逆转录扩增目的片段,构建重组体pMD18-T-TLR3作为定量标准品.在ABI Prism 7000型荧光定量分析仪上,通过荧光强度达到一定阈值时的循环数来定量起始模板,建立标准曲线;并检测30名正常人及20例原发性胆汁性肝硬化(PBC)患者,20例慢性乙型肝炎肝硬化患者外周血PBMC TLR3基因的荧光强度,根据标准曲线及循环阈值,由软件自动计算样本中TLR3 mRNA的起始拷贝数.结果 FQ-RT-PCR检测TLR3基因的线性范围为102～108拷贝数/μl RNA(r=-0.9974),内参β-actin的线性范围为103～108拷贝数/μl RNA(r=-0.9984);高浓度样本批内和批间变异系数(CV)分别为6.7%和8.7%,低浓度样本批内及批间CV分别为12.3%和14.0%,30名健康人,20例PBC及20例慢性乙型肝炎肝硬化患者PBMC中均有TLR3 mRNA的表达,分别为3.46×102～4.51×103拷贝/μg RNA、4.92×102～1.42×104拷贝/μgRNA和2.58×102～7.17×103拷贝/μg RNA.结论 成功建立检测TLR3 mRNA的FQ-RT-PCR方法,可作为临床和基础研究TLR3基因表达的工具。

Measurement of the expression level of APRIL and its receptors in liver cancer tissue by real-time fluorescence quantitative method

目的 采用实时荧光定量聚合酶链反应(RFQ-PCR) 技术分析肝癌组织中增殖诱导配体(APRIL)及其受体mRNA的表达水平.方法在APRIL及其受体基因的高保守区设计相应引物和荧光探针,实时检测PCR产物的荧光强度,根据标准品建立的标准曲线,由软件自动计算出待测样本中靶基因mRNA准确含量,并以靶基因和内参β2M mRNA含量的比值作为评价靶基因表达水平的指标.结果 RFQ-PCR检测靶基因mRNA含量的线性范围为10～109 pg/ml,批内和批间重复性测定的变异系数v分别为3.87%～10.12%和6.86%～12.29%.20例肝癌组织样本的检测结果显示肝癌及癌旁组织中APRIL和BCMA的水平均显著高于远端正常组织(P 0.05).结论成功建立RFQ-PCR检测APRIL及其受体mRNA含量的方法,具有较好的检测灵敏度和重复性;而且发现APRIL在肝癌发生、发展过程中可能起了重要作用,有可能还参与了肿瘤细胞的转移,并主要是通过受体BCMA而不是TACI发挥作用。