HEPN1过表达慢病毒的构建及对人泌乳素型垂体瘤细胞的影响-东南大学学报医学版

HEPN1过表达慢病毒的构建及对人泌乳素型垂体瘤细胞的影响

单位：西南医科大学附属医院神经外科, 四川泸州 646000

分类号：R736.4

出版年·卷·期（页码）：2017·36·第三期（409-416）

摘要：

目的：构建肝细胞癌下调蛋白-1（HEPN1）过表达慢病毒，观察其对人泌乳素型垂体瘤细胞HPAs HEPN1、caspase-3、Bax和Bcl-2表达以及细胞增殖、凋亡、侵袭和迁移的影响。方法：构建pCHD-HEPN1重组质粒，转染HEK293T细胞进行病毒包装，再感染HPAs细胞，实验分HEPN1组、空质粒组和空白组3组，采用荧光显微镜观察绿色荧光蛋白表达，逆转录聚合酶链反应和蛋白印迹法检测HEPN1、caspase-3、Bax和Bcl-2表达，噻唑蓝比色法、流式细胞术、细胞侵袭和划痕实验检测细胞增殖、凋亡、侵袭和迁移。结果：HEPN1组和空质粒组感染效率分别为90.3%和92.5%。与空质粒组及空白组比较，HEPN1组HEPN1、caspase-3和Bax的mRNA表达上调（P<0.05），Bcl-2的mRNA表达下调（P<0.05）；HEPN1、caspase-3和Bax蛋白表达上调（P<0.05），Bcl-2蛋白表达下调（P<0.05）；细胞总凋亡率增加（P<0.05），穿膜细胞数目减少（P<0.05），细胞迁移距离缩短（P<0.05）。结论：HEPN1过表达慢病毒可上调HPAs细胞HEPN1、Bax和caspase-3表达，下调Bcl-2表达，抑制细胞增殖，促进其凋亡，降低其侵袭和迁移能力。

Objective:To construct overexpression lentivirus targeting HEPN1 gene and observe its effect on biological behavior in pituitary tumor cells HPAs. Methods:The overexpression lentivirus targeting HEPN1 gene was constructed and transfected into HPAs cells. The experiment was assigned into 3 groups:HEPN1 group, empty plasmid group and blank group. The expression of green fluorescent protein was observed by fluorescence microscope. The expression of HEPN1, caspase-3, Bax and Bcl-2 were detected by reverse transcription PCR and Western Blot. The proliferation and apoptosis of cells were detected by MTT assay and flow cytometry, and the invasion and migration of the cells were detected by Transwell and cell scratch test. Results:The infection efficiency of HEPN1 group and empty plasmid group was 90.3% and 92.5%, respectively. Compared with empty plasmid group and blank group, the mRNA expression of HEPN1, caspase-3, Bax was up-regulated, while that of Bcl-2 was down-regulated. The protein expression of HEPN1, caspase-3, Bax was up-regulated,while that of Bcl-2 was down-regulated. The total apoptosis rate in HEPN1 group was higher than that in empty plasmid group and blank group(P<0.05). The number of crossed cells and migration distance of HEPN1 group was less than that in empty plasmid group and blank group(P<0.05). Conclusion:Overexpression lentivirus targeting HEPN1 gene can significantly up-regulate the expression of HEPN1, caspase-3 and Bax and down-regulate the expression of Bcl-2, which can inhibit the proliferation of HPAs cells, reduce their invasion and migration ability and promote their apoptosis.

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