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骨形成蛋白7基因诱导人肝癌细胞的凋亡
引用本文:肖时湘,张宁.骨形成蛋白7基因诱导人肝癌细胞的凋亡[J].中国生物工程杂志,2009,29(11):7-11.
作者姓名:肖时湘  张宁
作者单位:天津市第三中心医院 天津 300170
摘    要:目的 构建表达重组人骨形成蛋白7 (bone morphogenic protein 7, BMP7)基因的重组逆转录病毒,观察其对人肝癌细胞HepG2的凋亡诱导活性,并探讨其作用机制。方法 克隆BMP7基因,以loxP同源重组法构成逆转录病毒载体pLP-LNCX-BMP7(pLLBMP7),转染包装细胞PT67进行病毒包装并测定病毒滴度;将逆转录病毒感染人成骨细胞,MTT法检测细胞生长变化,琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;Western blotting检测BMP7,caspase-3和bcl-2蛋白表达。结果 重组逆转录病毒载体pLLBMP7经鉴定连接正确,转染PT67细胞后上清液中可得到病毒,滴度达1×109pfu;MTT检测见pLLBMP7病毒组48和72h细胞抑制率高于对照组(35.1% vs. 5.3%,68.5% vs.18.3%,均p<0.05),48h可见BMP7蛋白高表达。琼脂糖凝胶电泳出现典型梯形条带;流式细胞仪检测出现凋亡峰,于转染48h后达最高峰,其凋亡百分率高达14.42%;BMP7蛋白高表达时caspase-3蛋白的表达亦有显著升高,但bcl-2蛋白未见表达差异。结论 构建了BMP7逆转录病毒,在体外能够有效地诱导人肝癌细胞HepG2的凋亡,其可能是通过激活caspase-3而发生作用。

关 键 词:骨形成蛋白7基因  逆转录病毒  肝癌细胞  凋亡  
收稿时间:2009-05-21
修稿时间:2009-09-02

Apoptosis-inducing Effect of BMP7 Gene on Human Liver Cancer Cell Line HepG2
XIAO Shi-xiang,ZHANG Ning.Apoptosis-inducing Effect of BMP7 Gene on Human Liver Cancer Cell Line HepG2[J].China Biotechnology,2009,29(11):7-11.
Authors:XIAO Shi-xiang  ZHANG Ning
Institution:Tianjin Third Central Hospital,Tianjin 300170,China
Abstract:Objective:To construct recombinant retrovirus expressing human bone morphogenetic protein-7 gene BMP7 and to discuss its apoptosis-inducing activities and the mechanism in liver cancer cell line HepG2. Methods:BMP7 gene was amplified and reconstructed with retroviral plasmid pLP-LNCX by loxP homologous recombination, and then the plasmid pLP-LNCX-BMP7 ( pLLBMP7) was transferred into packing cell line PT67 and the supernatant was collected to assay viral titer. MTT assay was adopted to observe HepG2 cells amplification. 48h after pLLBMP7 infection agarose electrophoresis and flow cytometry were used to verify apoptosis of tumor cells, and then the expression of BMP7, caspase-3 and bcl-2 proteins were detected by Western blotting. Results: Recombinant retrovirus pLLBMP7 was justified and transformed into PT67 package cell with supernatant viral titer amounted to 5 × 10~9 pfu/ml. In MTT assay retrovirus group had no evident difference from controls in cellular inhibition 72h later (35. 1% vs. 5. 3% ,68. 5% vs. 18. 3% , p < 0. 05 ). 48h after transfection, agarose electrophoresis of genomic DNA showed typical ladder-like pattern and flow cytometry analysis showed obvious apoptosis peaks with the highest percentage rate of apoptotic cells present and cellular caspase-3 expression could be seen in pLLBMP7 group without any change of bcl-2. Conclusion: BMP7 retrovirus vector was reconstructed and could express BMP7 protein correctly in vitro and it could induce apoptosis in HepG2 cell line in vivo by activating caspase-3 expression.
Keywords:BMP7 gene  Retrovirus  Liver cancer cell  Apoptosis
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