人肝癌细胞系7402、7721及癌旁肝细胞7701在重组腺病毒-胸苷激酶/更昔洛韦系统作用前后差异蛋白检测及CK18表达

DETECTION OF DIFFERENCE PROTEIN AND OBSERVATION OF PATHOMORPHISM ON HUMAN HEPATOMACARCINOMA CELL LINE 7402,7721 AND ADJACENT LIVER CELL 7701 BEFORE AND AFTER ADV-TK/GCV TREATMENT

目的检测重组腺病毒-胸苷激酶/更昔洛韦(adenovirus-thymidine kinase/gancyclovir,ADV-TK/GCV)系统作用前后人肝癌细胞株7402、7721及癌旁肝细胞7701差异蛋白表达,进一步阐明ADV-TK/GCV自杀基因系统作用机制。方法利用肽质量指纹图谱和双向电泳分析法检测ADV-TK/GCV系统对人肝癌细胞7402、7721及癌旁肝细胞7701作用前后的差异蛋白;常规病理学技术苏木精伊红染色、电子显微镜技术观察作用前后细胞病理形态学改变;免疫细胞化学方法检测细胞角蛋白18(cytokeratin 18,CK18)的表达部位、分布范围及量的变化。结果7721及7701对ADV-TK/GCV系统作用不敏感,人肝癌细胞7402经自杀基因药物作用后,多种蛋白表达呈现出差异性,差异点主要表现为与物质能量代谢相关的酶类及与细胞骨架相关的蛋白,7402细胞的CK18表达上调明显。加药组7402细胞死亡明显,残留细胞体积变大,胞浆疏松,空泡变性。电镜观察显示加药组细胞器水肿,变化明显,微丝增多,且多分布于核周围,凋亡现象明显。7402免疫细胞化学染色显示加药组CK18的表达增强且向核周聚集。结论 ADV-TK/GCV自杀基因系统作用后,敏感株7402蛋白表达具有差异性,其中CK18表达显著上调且出现分布改变,并引起细胞形态学的变化。

Objective To detecte the expression of the difference proteins of the human hepatoma carcinoma cell line 7402,7721 and adjacent liver cell 7701 before and after the treatment of the adenovirus-thymidine kinase/gancyclovir （ ADV-TK/GCV ） system, further elucidating the mechanism of the suicide gene system ADV-TK/GCV on hepatoma cells. Methods Peptide mass fingerprinting （PMF） and two-dimensional electrophoresis analysis were used to test the expression of the difference proteins of the human hepatoma cell lines 7402,7721 and the adjacent liver cell line 7701 before and after the treatment of the ADV-TK/GCV system; moreover,we observed the cell pathological morphology changes after the treatment with Hematein Eosin （ HE）, one of the routine methods of pathology. We also used the electron-microscopy to detect the expression port, distribution and quantity of cytokeratin 18 （CK18） of dosing group and control group with immunohistochemistry. Results 7721 and 7701 were sensitive to ADV-TK/GCV system. The expression of many proteins of the human hepatoma cell lines 7402,exhibited differences after the disposal method of suicide gene drugs,the expression of CK18 was upregulated obviously by comparative analysis. Cellular morphology changed significantly by HE. Cells in the control group were mostly spindle-shaped and nucleoli were obvious, while most cells in dosing group died. The volume of residual cell was small, round, and hyperchromatic. Electron microscopy observations showed that the control abundant organelles, accidentally saw cell apoptosis. Dosing group of cell organelle were oedematous obviously, microfilament increased and throughout around the nucleus, apoptosis phenomenon was obvious. The immunocytochemistry showed that the expression of CK18 of the control group was equally distributed in the cytoplasm and cell membrane, but the expression of CK18 of the treatment group increased and was uneven distribution, mainly concentrated in the membrane surrounding the cytoplasm, some of which were circular distribution. Conclusion After the suicide gene system ADV-TK/GCV played a role, protein expression of the sensitive line 7402 showed differences, which suggested the expression of CK18 upregulated memorably, the distribution presented differences and caused changes in the morphology.