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目的 :研究外源性基因PTEN对人类大肠癌LS174 7细胞周期及凋亡的影响。方法 :以携带有PTEN基因的真核表达载体体外转染人类大肠腺癌细胞LS174 7,筛选阳性克隆的细胞并扩增培养 ,鉴定转染成功后采用流式细胞仪、透射电镜、DNA琼脂糖电泳 ,检测转染PTEN基因后LS174 7细胞的凋亡以及观察三种细胞生长曲线。结果 :流式细胞仪显示 ,细胞周期从G1期到S期发生抑制 ,并在G1期峰前出现 1个明显的凋亡峰。透射电镜下可见转染PTEN基因后细胞核染色质浓缩边集、胞浆浓缩、核碎裂及凋亡小体形成等典型的凋亡表现。细胞核DNA琼脂糖凝胶电泳显示凋亡细胞特有的“梯状”条带。转染空载体及未转染的LS174 7细胞未见凋亡表现。转染PTEN的LS174 7细胞与空白质粒载体转染的LS174 7细胞和LS174 7细胞的生长速度相比 ,后两者生长速度较快。结论 :将外源性PTEN基因导入LS174 7细胞后 ,可以抑制细胞周期 ,并可诱导细胞凋亡 ,这可能是PTEN基因抑制大肠癌细胞增殖的重要机制之一。
Objective: To study the effect of PTEN on the cell cycle and apoptosis of human colorectal cancer LS174 7 cells. METHODS: The human colon adenocarcinoma LS174 cells were transfected with the eukaryotic expression vector carrying PTEN gene in vitro. The positive clones were screened and expanded. The cells were identified by flow cytometry, transmission electron microscopy, DNA agarose The apoptosis of LS174 7 cells transfected with PTEN gene was detected by electrophoresis and the growth curves of three kinds of cells were observed. Results: Flow cytometry showed that the cell cycle was inhibited from G1 phase to S phase, and a significant peak of apoptosis appeared before G1 peak. Transmission electron microscopy showed the typical apoptotic features of PTEN gene, such as chromatin condensation edge set, cytoplasm concentration, nuclear fragmentation and apoptotic body formation. Nuclei DNA agarose gel electrophoresis revealed apoptotic cells unique “ladder” bands. Transfection of empty vector and untransfected LS174 7 cells showed no apoptosis. The growth rate of LS174 7 cells transfected with PTEN was faster than that of LS174 7 cells and LS174 7 cells transfected with blank plasmid vector. Conclusion: The introduction of exogenous PTEN gene into LS174 7 cells can inhibit the cell cycle and induce apoptosis, which may be one of the important mechanisms by which PTEN gene inhibits the proliferation of colorectal cancer cells.