摘要
目的研究RNA干扰人pttg表达对肺癌SPC-A-1细胞恶性表型的影响。方法构建针对人pttg的RNA干扰真核表达载体,运用脂质体介导转染SPC-A-1细胞并用G418筛选出阳性克隆株;半定量PCR和Western blot检测转染后PTTG mRNA及蛋白的表达水平;人工计数法检测阳性克隆株SPC-A-1细胞染色体倍性的变化;氚-胸腺嘧啶核苷掺入法观察抑制人pttg表达后SPC-A-1细胞增殖能力的改变;TUNEL法检测细胞凋亡的变化情况。结果成功构建针对人pttg基因的RNA干扰真核表达载体(命名为Si-hPTTG),转染后筛选获得人pttg下调表达的SPC-A-1细胞株;Si-hPTTG转染使SPC-A-1细胞PTTG mRNA和蛋白的表达较未转染组和阴性对照质粒组均显著下调;Si-hPTTG转染后SPC-A-1细胞染色体众数和平均数增加,3H掺入量显著降低,细胞凋亡明显增加。结论人pttg下调表达可增加SPC-A-1细胞染色体众数和平均数,伴有减轻染色体结构畸变的趋势;显著抑制SPC-A-1细胞增殖,增加细胞凋亡。人pttg可能是肺癌治疗的新靶点。
Objective To observe the effects of human pttg down-regulation on chromosome number, cell proliferation and apoptosis in SPC-A-1 cells. Methods The RNA interference （RNAi） expression vector targeting human pttg was constructed and transfected into SPC-A-1 ceils by lipofectamine, and then positive cell clones were screened by G418. The expression of PTTG mRNA and protein was confirmed by Semi-quantitation PCR and Western blot respectively. The changes of chromosomal number were counted through metaphase chromosomal preparation. Cell proliferation was determined by 3 H-TdR incorporation. Cell apoptosis was evaluated by TUNEL. Results The RNAi expression vector targeting human pttg was constructed （named Si-hPTTG） . The expression of PTTG mRNA and protein significantly decreased in SPC-A-1 cells transfected with Si-hPTTG. Both the modal and the average number of cell chromosome increased in SPC-A-1 with pttg down-expression, and the chromosomal appearance was increasingly ameliorated. 3 H-thyrnidine incorporation significantly decreased. On the other hand, cell apoptosis significantly increased. Conclusion The down-expression of human pttg in SPC-A-1 cells can remarkably restrain cell proliferation and increase cell apoptosis, decrease both the modal and average number of cell chromosomes, and ameliorate the disturbance of the chromosomal appearance. The data described here have provided a proof of the concept that vector-based RNAi targeting human pttg could be used therapeutically for lung cancer.
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2009年第3期238-243,共6页
Chinese Journal of Histochemistry and Cytochemistry
基金
中国博士后科学基金项目资助(20070411050)
江苏省博士后科研资助计划项目资助(0701023B)
