摘要
目的探讨降低硫氧还蛋白5(EndoPDI)基因表达对于胃癌细胞增殖状态、细胞周期、凋亡等生物学特性及浸润转移能力的影响。方法 EndoPDI基因特异的核糖核酸干扰(RNAi)载体通过脂质体介导法转染人胃癌细胞系细胞(MKN45),筛选获得稳定转染的细胞系,经过免疫细胞化学、逆转录聚合酶链反应(RT-PCR)法及蛋白免疫印记(Western-blot)法证实。使用生长曲线法、平板克隆形成实验、细胞迁徙实验等方法分析稳定转染株相关生物学特性的变化,每种检测实验均设立RNAi载体转染细胞组、点突变对照组和空白MKN45细胞组。结果稳定转染的EndoPDI RNAi细胞系经过免疫细胞化学法、RT-PCR法及Western blot法证实,表达抑制率可达70%左右。与对照组细胞相比,EndoPDI RNAi组细胞株生长减慢,各时间点细胞计数显著低于对照组(P<0.05);流式细胞仪检测细胞周期显示,S期比例显著低于对照组,G0-G1期比例显著高于对照组(P<0.05)。平板克隆形成实验结果显示,EndoPDI RNA i组克隆形成率显著低于对照组(P<0.05);细胞迁徙实验结果提示,EndoPDI RNA i组穿膜率显著低于对照组(P>0.05)。结论 EndoPDI基因可能具有促进细胞生长增殖作用,降低EndoPDI表达可减少处于分裂周期细胞比例、提高凋亡比例,降低EndoPDI基因表达可能对胃癌细胞的恶性生物学行为有抑制作用。
Objective To investigate the influence of down-regulation of EndoPDI gene expression on proliferation, apoptosis, invasion and cell cycle of the gastric cancer line MKN 45. Methods The specific silengcing sequences of EndoPDI were designed and synthesized based on the sequence of EndoPDI mRNA. The RNAi vectors specific to EndoPDI gene were constructed and transfected into MKN45 cell by liposome. The stable expression cell clones were selected. The cell cycles and apoptosis of these clones were detected by flow cytometry. The proliferation was analyzed by cell growth curve and colony formation assay respectively. The invasion capacity was tested using cancer cell migration assay. Results Immunocytochemistry,RT-PCR and Western-blot methods detected that 70% of EndoPDI gene expression in stable transfected cell line was suppressed. The growth of EndoPDI RNAi cell line was much slower than its control groups. The cell counts on day 5,6 and 7 were significantly reduced in comparison with those in control groups （P 〈 0.05）. Cell cycle analysis showed that a significant proportion of the EndoPDI RNAi ceils were increased in G0-G1 compared with those of control groups （P 〈 0.05）. The colony formation rate of EndoPDI RNAi cell line was lower than that of control （P 〈 0.05）. Furthermore,the cell migration rate of EndoPDI RNAi cell line was significantly decreased than that of control groups （P 〈 0.05）. Conclusion EndoPDI may promote the cell proliferation and invasion of gastric cancer cells. Down-regulating the expression of EndoPDI in ~astric cancer cell lines can ameliorate malignant phenotvpe.
出处
《中华保健医学杂志》
2014年第1期20-23,共4页
Chinese Journal of Health Care and Medicine
