摘要
目的构建pcDvp3重组真核表达质粒,并观察凋亡素基因(vp3)在人乳腺癌细胞435中的表达及诱导凋亡作用。方法(1)扩增vp3基因,与pcDNA3.1连接构建pcDvp3重组真核表达载体,并测序;(2)将pcDvp3和pcDNA3.1转染Hela细胞,免疫荧光技术检测凋亡素蛋白表达;(3)将pcDvp3和pcDNA3.1空质粒转染人乳腺癌细胞435和正常细胞,转染48h后用透射电镜、琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡。结果(1)核酸序列分析证明克隆的vp3基因与文献报道一致,且正确插入载体中,表明真核表达载体pcDvp3构建成功;(2)转染pcDvp3的Hela细胞48h后可见明显的荧光,而pcDNA3.1组未见;(3)电镜可见细胞明显形态改变及凋亡小体形成;电泳见典型梯形条带;流式细胞仪检测出现凋亡峰,于转染48h后达最高,凋亡百分率为14.42%;而转染pcDNA3.1细胞未见上述改变。结论vp3在体内能够表达并有效地诱导人乳腺癌435细胞凋亡。
Objective To discuss the mechanism of vp3 inducing tumor cells into apoptosis by constructing recombinant expression plasmid pcDvp3 and studying its expression and apoptosis inducing effect on human breast cancer cells 435.Methods (1) Apoptin gene fragment was amplified and cloned into the plasmid pcDNA3.1 to form the recombinant plasmid pcDvp3.The nucleotides sequencing was processed.(2) Hela cells were transfected with pcDvp3 and pcDNA3.1 separately.The vp3 expression was detected by immunofluorescent method.(3) The human breast cancer cell line 435 was transfected with pcDvp3 by liposome.Optical microscope,electron microscope,agarose electrophoresis and flow cytometry were used to detect the apoptosis of the tumor cells.[WT5”HZ]Results (1) The sequence analysis demonstrated that the cloned vp3 was the same as that published in literature and inserted into the vector accurately,which suggested recombinant plasmid pcDvp3 had been constructed successfully.(2) Two days fter the COS cells were transfected with vp3,the obvious fluorescence was seen,while in the pcDNA 3.1 group,no fluorescence was found.(3) Distinct cellular morphological transformation and typical apoptosis bodies could be observed in the tumor cells.Agarose electrophoresis of the DNA extracted from the transfected tumor cells showed typical ladder bands.Flow cytometry analysis showed obvious apoptosis peaks and the highest percentage rate of apoptotic cells was present at the time of 48 h after transfection with the apoptotic rate being 14.42%.Conclusion vp3 could be expressed in vivo and in vitro and effectively induced apoptosis of human breast cancer cell line 435.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2005年第7期801-803,共3页
Chinese Journal of Experimental Surgery
