【作者】 杨连君；

【导师】 王文亮；

【作者基本信息】 第四军医大学 ， 病理学与病理生理学， 2001， 博士

【摘要】 研究乙醇对肝细胞和肝肿瘤细胞凋亡的诱导作用，对于探讨乙醇诱导的肝细胞凋亡与肝脏疾病的关系具有重要的意义。目前关于建立乙醇诱导原发性肝细胞癌（简称肝癌）细胞凋亡模型的报道很少。bcl-2和bax分别是抑制细胞凋亡和促进细胞凋亡基因，并且同属于bcl-2基因家族。关于bcl-2和bax在肝癌的表达情况目前尚无定论。关于bax过表达对肝癌细胞凋亡的影响，以及bcl-2过表达对乙醇诱导的肝癌细胞凋亡的调控作用尚未见报道。制备抗肝癌凋亡细胞的单克隆抗体（mAb）能够探讨细胞凋亡相关分子表达的变化和产生情况，并且可能用于细胞凋亡的检测，是目前细胞凋亡研究中较新的探索性领域。尚未见在抗原不明确的情况下制备抗肝癌调亡细胞mAb的报道。 本研究围绕着乙醇诱导肝癌细胞凋亡及其Bax和Bcl-2的表达情况、转染bcl-2和bax对肝癌细胞凋亡及其细胞周期蛋白p21^WAF1/CIP1和p27^Kip1表达的影响和抗乙醇诱导的肝癌凋亡细胞mAb的制备进行以下几个方面的工作： ①用噻唑蓝（MTT）法检测浓度分别为20～100mL/L的乙醇作用6h对肝癌细胞系HCC-9204的杀伤作用。然后用60mL/L乙醇作用6h诱导其发生凋亡，以台盼蓝染色、苏木精-伊红（HE）染色、May-Grunwald-Giemsa（MGG）染色、Hoechst 33258染色、吖啶橙/溴化乙啶（AO/EB）双染色、透射电子显微镜观察、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法检测和流式细胞仪分析不同细胞周期DNA含量等方法检测细胞凋亡的发生情况。用免疫细胞化学ABC法染色和图像定量分析检测诱导凋亡前后细胞的Bax和Bcl-2表达水平的变化。结果显示，随着乙醇浓度的增加，其对HCC-9204细胞的杀伤作用逐渐增强。60mL/L乙醇作用6h可使HCC-9204细胞发生明显的凋亡形态学变化，并且大部分细胞为台盼蓝拒染和TUNEL阳性着色。流式细胞仪分析可见明显的亚二倍体凋亡峰。诱导凋亡后的HCC-9204细胞Bax的表达水平明显增高（P＜0.05），而Bcl-2在诱导凋亡前后均未见表达。 ②用免疫组织化学ABC法检测Bcl-2和Bax在肝癌和正常肝组织的表达情况，并分析其与肝癌组织病理分级之间的关系。用脂质体介导的基因转染法分别将含有人bcl-2和bax cDNA的真核表达载体转染到HCC-9204细胞中，建立稳定高表达Bax的肝癌细胞，并用克隆化的方法建立100％稳定表达Bcl-2的肝癌细 第四军医大学博士学位论文 摘要 胞株。用免疫细胞化学ABC法染色和图像定量分析检测转染比－2和bax前后细胞 的昨”“”哑和p27“’‘表达水平。用MTT法、TU’N’EL法和流式细胞仪检测转染 bclZ和bax前后的细胞凋亡发生情况。结果显示，正常肝组织的Bcl－二阳性率只 有4．二％＊／24X肝癌组织的k上阳性率为258％口／66X二者具有显著性差 异汐＜005卜 正常肝组织的Bax阳性率为刀8％厂 门4)，肝癌组织的Bax阳性 率为439％c9“6X二者也具有显著性差异o＜005卜肝癌组织的k卜2和Bax 阳性率与其病理分级之间均未见明显的相关性。在正常肝组织中，B。12的阳性卡 率明显低于B＜ (＜001卜在肝癌组织中，BC12的阳性率也明显低于BCX(尸＜ 005卜 建立了稳定高表达Bax的肝癌细胞和100％稳定表达Bcl上的肝癌细胞株。 与未转染的HCC习204细胞相比，转染bC12的细胞的昨”“””m和p27“…表达水 平均明显下降(＜0刀5X而转染bax的细胞的p”“’“。m和p27M‘表达水均明显 升高（尸、005）。在转染b8X的细胞中可观察到有少量凋亡细胞产生。60 InL／L 乙醇作用6 h后，转染比－2和转染bax的细胞的杀伤率、TUN’－L指数和亚二倍体 凋亡峰比例均分别明显低于和高于未转染的＊＊C习204细胞(＜005卜 ①以乙醇诱导凋亡的HCC、9204细胞为抗原，用环磷酚胺消减免疫法免疫 8＊AB／叫。鼠。取其脾细胞与BALS／。小鼠骨髓瘤细胞系SP二川融合。＊NsA法筛 选。计算细胞融合率和抗体阳性率。有限稀释法连续克隆化。然后用免疫细胞 化学ABC法进行进一步筛选和分析。Western blot法分析ab相关抗原的分子 量。结果显示，消减免疫法和常规免疫法的细胞融合率未见明显差异；与诱导 凋亡的细胞反应强，而与未诱导凋亡的细胞反应较弱的抗体的产生率也未见明 显差异。消减免疫法的总抗体产生率明显低于常规免疫法（＜0刀1），与诱导凋 亡的和未诱导凋亡的细胞均呈强反应的抗体的产生率也明显低于常规免疫法（尸 ＜001）。初步获得了1株与乙醇诱导凋亡的＊＊C9204细胞反应强，而与未诱导 凋亡的HCC－9204细胞反应较弱的mAb。其相关抗原定位于HCC－9204细胞的细 胞核，分子量约为75 ku。 以上结果说明，低浓度乙醇能够诱导HCC习204细胞凋亡，其凋亡的发生与 Bax表达水平增高有关。与正常肝组织相比，肝癌组织出现Bcl－2表达低度升高， Bax表达更多还原

【Abstract】 Postgraduate for Doctor’s degree: Lianjun YangSupervisor: Prof. Wenliang Wang Department of Pathology, Fourth Military Medical University, Xi ’an 710032To study the inducing effect of ethanol on apoptosis in hepatocytes and liver tumor cells is important and helpful to probe into the relationship between ethanol-induced apoptosis in hepatocytes and liver diseases. Currently, there are only few such reports as establishing the model of ethanol-induced apoptosis in hepatocellular carcinoma (HCC) cells. Bcl-2 and bax are respectively anti-apoptotic and proapoptotic genes, and both of them belong to bcl-2 gene family. There is no definite conclusion about the expression of bcl-2 and bax in HCC recently. There is no such report about the regulating effects of bax overexpression on apoptosis in HCC cells and bcl-2 overexpression on ethanol-induced apoptosis in HCC cells until now. Preparing anti-apoptotic cell monoclonal antibody (mAb) is helpful to investigate the change and production of apoptosis-related molecules, it may also be used in apoptosis detection, and it is a newly explored domain in the study of apoptosis There is no such report as preparing anti-apoptotic cell mAb as its antigen is unclear until now.Centering on ethanol-induced apoptosis in HCC cells and its expression of Bax and Bcl-2, the effect of bcl-2 and bax transfection on apoptosis in HCC cells and its expression of cell cycle proteins p21WAF1/cn>1 and p2710pl, and preparation of mAb against ethanol-induced apoptotic HCC cells, this study contains the following work:?The cytotoxicity of HCC cell line HCC-9204 treated with 20 ~ 100 mL / L ethanol for 6 h was detected by Methabenzthiazuron (MTT) assay. Then the cells were induced to undergo apoptosis with 60 mL / L for 6 h. The happening of apoptosis was detected by trypan blue staining, Haematine-eosin (HE) staining, May-Grunwald-Giemsa (MGG) staining, Hoechst 33258 staining, Acridine orange / Ethidium bromide (AO / EB) double staining, Transmission electronic microscope observation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and DNA contents assay in different cell cycles by flow cytometer. The change of Bax and Bcl-2 expression level in HCC-9204 cells before and after inducing apoptosis was detected by immunocytochemical ABC staining method and image-quantitative analysis. The results showed that, as the concentration of ethanol increased, the cytotoxicity of HCC-9204 cells increased gradually. Sixty mL / L for 6-6-h could make obviously morphologically apoptotic change in HCC-9204 cells, and most of the cells could be stained with trypan blue and were TUNEL positively-stained. There was significant sub-Gl apoptotic peak detected by flow cytometer. The Bax expression level in HCC-9204 cells after inducing apoptosis increased significantly, but there is still no detectable Bcl-2 both before and after inducing apoptosis.?Expression of Bcl-2 and Bax in HCC and normal liver tissues was detected by immunohistochemical ABC method, and its relationship with the pathological grades of HCC tissues was analyzed. Eukaryon expression vectors containing human bcl-2 and bax cDNA were respectively transfected into HCC-9204 cells, HCC cells that express Bax stably and highly were established, and an HCC cell strain that express Bcl-2 at a 100 % positive rate was obtained by cloning. The p21WAF1/CIP1 and p27Kipl expression level in the cells before and after transfected with bcl-2 and bax was detected by immunocytochemical ABC staining method and image-quantitative analysis. The happening of apoptosis in the cells before and after transfected with bcl-2 and bax was detected by MTT assay, TUNEL assay and flow cytometer. The results showed that, the positive rate of Bcl-2 in normal liver tissues was only 4.2 % (1 / 24), that in HCC tissues was 25.8 % (17 / 66), and there was significant difference between them (P < 0.05). The positive rate of Bax in normal liver tissues was 70.8 % (17 / 24), that in HCC tissues was 43.9 % (29 / 66), and there was更多还原