摘要
目的构建凋亡素基因(vp3)重组真核表达质粒(pcDvp3),观察vp3基因在人乳腺癌细胞435中的表达及诱导凋亡作用,探讨其诱导肿瘤细胞凋亡的作用机制。方法(1)克隆vp3基因并与pcDNA3.1质粒连接构建pcDvp3重组真核表达载体,测序鉴定;(2)用脂质体将pcDvp3和pcDNA3.1转染人乳腺癌细胞435,48h后分别用透射电镜、琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;(3)建立人乳腺癌细胞435的裸鼠模型,分组给药后测定抑瘤率,并用原位凋亡(TUNEL法)检测肿瘤细胞的凋亡情况。结果(1)核酸序列分析表明已构建成重组质粒pcDvp3;(2)pcDvp3转染肿瘤细胞48h后,电镜下可见明显形态改变及凋亡小体形成;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,其凋亡百分率高达14.42%;(3)裸鼠实验可见pcDvp3组抑瘤率分别为65.52%和68.23%,明显高于pcDNA3.1组(t=4.06,P<0.01),TUNEL检测可见pcDvp3组肿瘤细胞凋亡。结论vp3基因在体内外均能够有效地诱导人乳腺癌435细胞凋亡。
Objective To construct recombinant plasmid pcDvp3 and observe the apoptosis- inducing effect of vp3 gene on human breast cancer cell 1ine-435. Methods （ 1 ） vp3 gene was cloned into the plasmid pcDNA3.1 to form the recombinant plasmid pcDvp3. Then the nucleotides sequencing was processed. （2） 48 h after transfection of pcDvp3 and pcDNA3. 1 into breast cancer cell lines435, optical microscopy, electric-microscopy, agarose electrophoresis and flow cytometry were used to verify apoptosis of tumor cells. （3） Nude mouse model of human breast cancer cells 435 was established to observe the tumor- inhibiting rate and TUNEL was adopted to identify apoptosis. Results （ 1 ） Sequence analysis justified the recombination of plasmid pcDvp3. （2） 48 h after transfection into breast cancer cells435, distinct morphological transformation and typical apoptosis bodies were observed, agarose electrophoresis of genomie DNA showed typical ladder-like pattern and flow cytometry analysis showed apoptosis peaks with the percentage of 14. 42%. （3） Tumor-inhibiting rates in pcDvp3 groups were 65.52% and 68.23% , much higher than that in pcDNA3. 1 group（ t =4. 06,P 〈0.01 ） , and cellular apoptosis could be seen by TUNEL assay. Conelusion vp3 gene could induce apoptosis in breast cancer ce11435 both in vitro and in vivo.
出处
《中华普通外科杂志》
CSCD
北大核心
2005年第10期660-662,共3页
Chinese Journal of General Surgery
作者简介
通信作者：于春梅，E—mail：chunmei660301@163．com
