摘要
目的研究丙型肝炎病毒非结构蛋白4B对肝细胞细胞周期及PCNA表达的影响,探讨HCV-NS4B在肝细胞增殖机制中的可能作用。方法利用脂质体介导将空白载体PCXN2组及重组质粒PCXN2-NS4B转染入LO2肝细胞,G418筛选。RT-PCR鉴定质粒成功转染入肝细胞内。MTT法绘制生长曲线,观察NS4B对肝细胞生长的影响;流式细胞仪检测细胞周期的情况;免疫组化方法观察PCNA的表达情况。结果NS4B转染入LO2有表达,生长曲线示HCV-NS4B可促进肝细胞生长;细胞周期检测示转染PCXN2-NS4B组S期、G2/M期较PCXN2组增加,两组比较差异有统计学意义(P<0.05);转染PCXN2-NS4B组PCNA表达率较PCXN2组高,两组比较差异有统计学意义(P<0.01)。结论HCV-NS4B可能通过调节PCNA的表达,促进DNA合成,促进肝细胞增殖。
Objective To investigate the effect of hepatitis C virus non-structural protein 4B（HCV-NS4B）on cell cycle,expressions of PCNA in hepatic ceils, and to study the possible role of hepatitis C Virus non-sttuctural in the hepatic cell proliferation. Methods The experiment was divided into empty vector PCXN2 group and PCXN2-NS4B group. The recombinant plasmid （PCXN2-NS4B）and the empty vector were transfected into LO2 liver cells with liposome. Screening was performed with G418. Plasmid mRNA was detected by reverse transcription PCR. The growth curve was made by MTT. Flow cytometry was used to determine cell cycle, the protein expressions of PCNA were determined by immunohistochemical method. Results Stable expression of the recombinant plasmid was found in transfected LO2 liver ceils. HCV-NS4B could promote proliferation of hepatic cell. Flow cytometry analysis showed that the levels of G0/G1 phase in PCXN2 cells was higher than that in PCXN2-NS4B（ P 〈 0.01） ;but the rate of G2/M phase and S phase in PCXN2 cells was lower than those in PCXN2-NS4B. The expression of PCNA in PCXN2-NS4B was 36.29% ± 4.62%, in group PCXN2 were significantly lower than those in PCXN2-NS4B（ P 〈 0.01 ）. Conclusion Stable expression of the recombinant plasmid was found in transfected LO2 liver cells. HCV-NS4B could promote the expression of PCNA and disturb cell cycle, which may improve the proliferation of hepatic cells.
出处
《四川医学》
CAS
2008年第1期7-9,共3页
Sichuan Medical Journal
基金
四川省教育厅资助项目(20003531)
