【作者】 孙会卿；

【导师】 贺兴鄂；

【作者基本信息】 中南大学 ， 内科学， 2008， 硕士

【摘要】 背景及目的:目前大量实验研究证实HBx片断可在多数并发乙肝病毒感染的肝癌组织中检测到,它是肝细胞发生恶性转化的主要因素之一,并可提高肝癌细胞的增殖活性,促进肝癌细胞快速增长。因此,高效特异的基因治疗新技术RNA干扰靶向沉默HBx基因的应用是肝癌治疗的一种新方法。另一方面,化疗仍是目前肝癌治疗重要手段之一,但多种化疗药物耐药性是肝癌治疗失败的主要原因。而相关研究表明,HBx片断可通过特异MAPK途径上调多药耐药基因的表达,并使抗凋亡信号占主导地位,抑制化疗药物诱导的肝癌细胞凋亡。因此我们拟采用针对HBx的RNA干扰技术与常用的肝癌化疗药物或抗肿瘤药协同应用,探讨RNA干扰技术和传统的治疗方法联合应用对肝癌细胞的生长抑制作用的影响,并选择高效的治疗组合,研究其凋亡机制。方法:1.鉴定转染无关的对照序列(HK3细胞)和针对X基因shRNA(21543细胞)的稳定肝癌细胞株:复苏三种肝癌细胞(MHCC97-H,HK3,21543细胞),在含有一定浓度G418的高糖培养基中培养一段时间后,倒置荧光显微镜观察GFP的表达,初步估计转染效率并拍照。2.RT-PCR检测RNA干扰对HBx基因mRNA沉寂的程度:收集细胞提取总RNA,加用两对不同引物参照说明进行RT-PCR,根据凝胶电泳中内源基因片段条带的强弱,来判定模板的量,进而判断HBX基因的mRNA被siRNA沉寂的程度。3.观察RNA干扰后对细胞生长的影响:利用CCK8(Cell countingkit-8)测定光密度值,绘制三种不同细胞6天的不同增殖曲线。4.流式细胞仪检测细胞周期的变化。5.绘制加用三种不同浓度的5-氟脲嘧啶,顺铂,α-干扰素的肝癌细胞的生长曲线,选择最佳组合。6.TUNEL细胞凋亡检测试剂盒检测细胞凋亡。结果:1.复苏后细胞形态和GFP表达强度差别不大。2.RT-PCR结果显示相对于MHCC-97H细胞,21543细胞的HBxmRNA水平的下调显著,HK3细胞的HBx mRNA水平的下调不明显。3.靶向HBX的RNA干扰后的肝癌细胞(21543细胞)较原肝癌细胞(MHCC97-H细胞)和转染无关对照序列的肝癌细胞(HK3细胞)增殖明显减慢,后两种细胞差别不显著。4.21543细胞的细胞周期显示RNA干扰后的肝癌细胞延缓进入S期,增殖活性明显降低。5.三种不同细胞加用三种不同浓度的5-氟脲嘧啶,顺铂后细胞生长明显减慢,并呈浓度依赖性,以RNA干扰HBx后的肝癌细胞(21543细胞)生长抑制最明显,加用干扰素后细胞抑制不明显。6.相同浓度的化疗药物5-氟脲嘧啶作用上述三种不同肝癌细胞均可引起细胞凋亡,以RNA干扰后的肝癌细胞(21543细胞)最为明显。结论:1.RNA干扰HBx可明显抑制肝癌细胞的增长,细胞周期发生改变。2.RNA干扰HBx后的肝癌细胞可明显增强化疗敏感性。3.RNA干扰HBx和化疗药物二者联合应用肝癌细胞凋亡更为明显,细胞增殖明显减慢。更多还原

【Abstract】 Background and Objective:There are plenty of experiments which have proved that HBx can be detected in most of the Hepatocellular carcinoma (HCC) tissue infected by hepatitis B virus. HBx is one of the principal factors of hepatocyte malignant transformation,it can increase proliferation activity of Hepatocellular carcinoma cell and promote its growth quickly.RNA interference targeting to HBx is a new approach with high performance and specificity in the gene therapy of liver cancer .Chemotherapy is still one of important methods to treat the Hepatocellular carcinoma now.Neverthless, drug resistance in chemotherapeutics is a primary cause of treatment failure in liver cancer. Related study make clear that HBx can upregurate the expression of multidrug resistance gene through special MAPK pathway . Then anti-apoptosis signal ocuppy the manage position and supress the apoptosis of Hepatocellular carcinoma cell induced by the chemotheraputics.For this reason ,we plan to adopt the RNA interference to aim directly at HBx and traditionary antinelplastic agent of liver cancer.We shoud investigate the influence of association RNA interference with traditional therapy.We should select the therapy combination of high performance and study the apoptosis mechanism.Methods:1.Identity the stable Hepatocellular carcinoma cells transfected by shRNA aimming at HBx together with independent control series:we should reanimate three kinds of Hepatocellular carcinoma cell(MHCC97-H,HK3,21543),cultivate them in the high glucose DMEM with G418 for some days,then observe the expresion of GFP in cells through inversion fluorescent microscope and initial to estimate the transfection efficiency ,take photographs at last.2.Detect the extent of HBx gene by RNA interference by semipuanitative RT-PCR : collect three kinds of cell and extract total RNA,add two different pairs of primer and carry out RT-PCR in accordance with illustration,On the basis of endogenous gene strap in the gel electrophoresis,we assessment the tempate quantity and judge the extent of HBx gene by RNA interference.3.0bserve the influence of cell growth through RNA interference:we should utilize the Cell counting kit-8 and evaluate optical density value ,then we draw the growth curve of three kinds of cell in six days.4.Detect the diversity of cell cycle by flow cytometry5.Draw the growth curve of three kinds of cell after add different density of flurouracil ,cisplatin andα-interferon and select the best combination.6.Detect the cell apoptosis by TUNEL apoptosis detection kitResult:1 .There are no difference in the shape and GFP expression intensity in two transfected cell after resuscitation.2.The result of RT-PCR demonstrate that the HBX mRNA level of 21543 cell down regulate, the HBx mRNA level of HK3 cell is not different with MHCC97-H cell.3.The growth of 21543 cell are slower than MHCC97-H cell and HK3 cell obviously. The latter two cells are not different notably.4.The cell cycle of 21543 cell showed that Hepatocellular carcinoma cell through RNA interference targeting to HBx delay to goto S stage,then proliferation activity degrade obviously.5.Three kinds of cell adding different density of flurouracil ,cisplatin grows slower than the original cell. The growth inhibiting is dependent on the density of drug and the growth inhibiting of 21543 cell is the most obvious.That of three kind of cell adding toα-interferon is not obvious.6. Flurouracil induce apoptosis in all kinds of cell . The extent of apoptosis in 21543 cell is most obvious.Conclusion: 1. RNA interference targeting to HBx can supress the growth of hepatocellular carcinoma cell.2. Hepatocellular carcinoma cell through RNA interference targeting to HBx can intensify chemo-sensitivity.3. Combination RNA interference targeting to HBx with chemotherapeutics can induced apoptosis in more hepatocellular carcinoma cell.Cell proliferation step down accordingly.更多还原