乙醇诱导肝癌细胞凋亡及其单克隆抗体的制备

Ethanol induced apoptosis in hepatoma cells and preparation of its monoclonal antibody

目的 :探讨原发性肝细胞癌 (简称肝癌 )细胞凋亡过程中相关分子的表达情况 ,制备抗肝癌凋亡细胞相关抗原的单克隆抗体 (mAb)。方法 :用含 6%乙醇的培养液培养肝癌细胞系HCC 92 0 4细胞 6h诱导其凋亡 ,透射电镜观察细胞形态变化 ,TUNEL法检测细胞DNA片段化发生情况 ,流式细胞仪分析细胞DNA含量变化。环磷酰胺消减免疫法免疫小鼠。杂交瘤法制备抗凋亡细胞的mAb。ELISA法筛选在凋亡细胞高表达 ,而在非凋亡细胞低表达的mAb。有限稀释法连续克隆化。用免疫细胞化学ABC法进一步筛选和分析 ,并进行杂交瘤细胞染色体和免疫球蛋白 (Ig)类型分析。 结果 :6%乙醇作用HCC 92 0 4细胞 6h后 ,可见明显的凋亡形态学变化 ,大部分细胞为TUNEL染色阳性 ,并且流式细胞仪检测可见亚二倍体凋亡峰。经连续克隆化筛选出 1株在乙醇诱导凋亡的HCC 92 0 4细胞的胞核高表达 ,而在未经诱导凋亡的HCC 92 0 4细胞低表达的mAb。染色体分析结果符合mAb杂交瘤细胞特征 ,其Ig类型为IgG1。 结论 :低浓度乙醇诱导凋亡的HCC 92 0 4肝癌细胞中有某些相关分子的表达升高。初步获得了 1株可能是抗乙醇诱导的肝癌细胞凋亡相关抗原的mAb。

Purpose To investigate the expression in apoptosis related molecules in hepatocellular carcinoma (HCC), and prepare monoclonal antibody (mAb) against HCC apoptosis related antigens. Methods HCC cell line HCC 9204 cells were treated with 6% ethanol for 6 h. The morphological changes of the cells were observed with transmission electron microscope. The DNA fragmentation was detected by TUNEL assay. The DNA content of the cells was analyzed with flow cytometer. Mice were immunized by cyclophosphamide subtractive immunization method. MAb against apoptotic cells was prepared by hybridoma method. The mAbs, which reacted strongly with apoptotic cells, but weakly with non apoptotic cells, were selected by ELISA method. Clone by limited dilution method continually. The mAb was selected and analyzed more with immunocytochemical ABC method. The chromosomes of the hybridoma cells and the type of the immunoglobulin (Ig) were analyzed. Results After HCC 9204 cells were treated with 6% ethanol for 6 h, significantly apoptotic morphological changes were observed, most cells were TUNEL positively stained, and sub G1 apoptotic peak was found. A mAb was selected out after cloning continually, and it reacted with the nuclei of the ethanol induced apoptotic HCC 9204 cells strongly, but weakly with non apoptotic HCC 9204 cells. The result of chromosome analysis coincided with the characteristic of hybridoma cells, and its Ig type is IgG1. Conclusion There is increase of some associated molecules expression during apoptosis in HCC 9204 cells induced with low concentration ethanol. A mAb, which may be against ethanol induced apoptosis related antigens of HCC, was obtained.