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Objective To construct the small interfering RNA (siRNA) targeting heparanase gene and its expressing vector,and to observe its interference effect on the expression of heparanase gene and inhibitory effect on the invasive potential of human malignant melanoma A375 cells.Methods Three siRNAs were designed.The recombinant plasmid pRNATU6.1/heparanase-siRNA was designed and constructed. A375 cells were cultured,and transfected with pRNATU6.1/heparanase-siRNA.The cells treated with lipo- fectamine or Opti-MEM served as the controls.Real-time fluorescence quantitative PCR and Western blot were performed to evaluate the expression of heparanase RNA and protein in these treated A375 cells.The in vitro invasive potential of treated A375 cells was assessed by Matrigel gel assay.Results The siRNA targeting heparanase gene was successfully cloned to the eukaryotic expressing vector pRNATU6.1.The expression levels of both heparanase RNA and protein decreased significantly in siRNA-transfected A375 cells than those in the control cells.The in vitro invasive potential of siRNA-transfected cells was also signifi- cantly inhibited as compared with that of the control cells (P
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Aim To construct prokaryotic expression vector for endogenetic angiogenesis inhibitor Arresten gene and to express recombinant.Pharmacology experiments found that the expression product had the function of restrain blood vessel growing on the chorioallantoic membrane(CAM).Methods The total RNA was extracted from the placenta organize of the normal puerpera,and Arresten gene was amplified by RT-PCR to construct recombinant vector pBV220-Arr.Then,it was transformed into E.coli JM109 for yielding recombinant human Arresten protein.The method to detect inhibiting angiogenesis activity was CAM study.Results The plasmid pBV220-Arr.could be expressed in E.coli JM109 from two hours to eight hours,and the expression yield reached the highest in two hours.The Arresten protein could restrain blood vessel growth of CAM evidently than that of angiostatin.Conclusion The recombinant plasmid pBV220-Arr was construted successfully and the target protein Arresten can express in E.coli JM109,the protein of Arresten had the evident effect of inhibite the activity of angiogenesis.
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Objective To study the inhibitory effect of human antisense high mobility group box 1 (HMGB1) gene on the growth of human pancreatic adenocarcinoma cell line (PANC-1).Methods HMGB1 was cloned and reversely inserted into eukaryotic expression vector pcDNA3.1 to get pcDNA3.1/anti-HMGB1, which was then transfected by liposome method into PANC-1 cells.After G418 selection, HMGB1 expression of PANC-1 cells before and after transfection was detected by RT-PCR and Western blot, the cell proliferating activity in vitro was examined by MTT analysis, and cell cycle and apoptosis were assessed by flow cytometry.Results pcDNA3.1/anti-HMGB1 vector was obtained.After transfection with pcDNA3.1/anti-HMGB1, HMGB1 gene expression of PANC-1 cells was inhibited in both mRNA and protein level, and the proliferation of cultured pancreatic adenocarcinoma cells was suppressed with a suppression rate higher than 40 % on the sixth day.The cells of G1 phase increased obviously while the cell number of S phase decreased, the number of apoptosic cell increased.Conclusion Antisense HMGB1 gene can inhibit the proliferation of human ancreatic adenocarcinoma cells and increase the cell apoptosis rate.
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Objective To understand the expression of heat shock protein(HSP70) mRNA induced by avermectins in the hepatic tissue of rats.Methods Wistar rats aged 6 weeks were randomly divided into 4 groups,12 in each including 6 male and 6 female.The rats in the experimental groups were treated with avermectins by gavage once a day at the doses of 2.5,1.25 and 0.625 mg/kg respectively for 14 consecutive days,one group was the negative control.The general state was concerned and after the exposure,the content of total protein(TP) in the serum was determined,the hepatic tissue was sampled to examine the expression of HSP70 mRNA by polymerase chain reaction(PCR).Results The content of TP in the serum in the 2.5 mg/kg group was lower than that in the control group(P
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This paper briefly describes the mechanism of neurochemical changes in AD,the roles of the epigenetics,the regulation of gene expression and the mechanism of epigenetics in SAD.On the basis of the basic study on the AD during the decades,researchers initially revealed the mystery of the unfathomable neurochemical changes attack on AD.Especially in recentyears,researchers summarized the abnormal protein expression on SAD into epigenetics disease.This achievement helps researchers point out the new and strategic direction on AD pathogenesis,prevention and control mechanism.With the understanding of biological characteristics of epigenetics,AD needs to be studied in depth.