Smac过表达对人胰腺癌MiaPaCa-2细胞凋亡及生长的影响

摘要

目的 探讨Smac基因过表达对人胰腺癌细胞MiaPaCa-2凋亡及细胞生长的影响.方法 从人白血病K562细胞中扩增Smac cDNA,构建含Smac cDNA的真核表达载体pEGFP-C1-Smac,并转染人胰腺癌细胞MiaPaCa-2.流式细胞仪检测分析细胞凋亡百分率,凋亡相关蛋白Survivin、Bcl-2的表达及细胞周期;四甲基偶氮唑蓝检测癌细胞生长状况.结果 荧光显微镜下可见,转染pEGFP-C1-Smac的细胞胞浆自发绿色荧光.瞬时转染24h,转染pEGFP-C1-Smac的MiaPaCa-2癌细胞凋亡率(7.01±3.88)%明显高于未转染组(2.17±0.87)%和转染空载体组(4.40±1.49)%（P<0.05）.转染pEGFP-C1-Smac 24、36、48h时对MiaPaCa-2细胞生长抑制率分别为62.87%、63.58%、65.55%,均明显高于对应的转染空载体组细胞（31.63%、34.68%、59.20%，P<0.05）.Bcl-2蛋白表达水平在转染Smac基因组也有下降趋势,但组间差异无统计学意义.转染Smac基因组对MiaPaCa-2细胞Survivin、Bcl-2蛋白表达水平均较未转染组和转染空载体组细胞明显降低（P<0.05）.转染Smac基因组对MiaPaCa-2细胞周期影响,G0/G1期细胞比例明显高于未转染组及转染空载体组,S期比例减少（P<0.05）.结论 Smac过表达可促进人胰腺癌MiaPaCa-2细胞凋亡、抑制癌细胞生长,这一作用可能与Samc影响MiaPaCa-2细胞周期,下调Survivin、Bcl-2蛋白表达有关.%Objective To investigate the effect of overexpression of second mitochondria derived activator of caspases ( Smac/DIABLO) on cell apoptosis and growth of pancreatic carcinoma MiaPaca - 2 cells. Methods The cDNA fragment of human Smac gene was amplified by reverse transcription - PCR from total RNA of the human myeloid leukaemic K562 cell line. The PCR product was ligated with a plasmid vector named pEGFP - C1. The pEGFP - C1 Smac plasmid was confirmed using restriction enzyme digestion, PCR and DNA sequencing. The recombinant plasmid was transfected into MiaPaCa - 2 cell line by lipofectamine reagent. The expression of Smac was determined by fluorescence microscopy. Cell proliferation was assayed by tetrazolium bromide colorimetry. The expressions of Survivin, Bcl - 2 protein and cell cycling were detected by flow cytometry. Results After the transfection of pEGFP - C1 - Smac to MiaPaCa - 2 cells, the cytosolic expression of green fluorescence protein was identified by fluorescence microscopy. Smac transfectants for 24 hours . MiaPaCa -2 cells showed increased apoptosis with a percentage of (7. 01 ±3. 8) , which was higher than that of non transfected group (2. 17 ±0. 87 ) and free vector transfected group (4. 40 ± 1. 49 , P ＜0. 05 ) . The expression of Survivin and Bcl - 2 was significantly reduced ( P ＜ 0. 05 ) . Bcl - 2 expression had a trend of reduction,but the difference in groups was not significant. The percentages of G0/G1 cells were higher in Smac group than that of control, cytosolic Smac arrest MiaPaCa -2 cells in G0/G1 Cell. Conclusion Cytosolic overexpression of Smac in MiaPaCa - 2 cells could promote apoptosis and inhibit proliferation. The mechanism may be associated with the reduction of Survivin and Bcl - 2 expression and the effect of inhibiting cell cycling of Smac gene.