摘要
目的探讨抗肿瘤药物奥沙利铂(Oxaliplatin,L-OHP)联合沉默REV7基因对人结肠癌细胞THC8307细胞凋亡的影响。方法体外培养人结肠癌细胞株THC8307,将REV7基因的特异性siRNA片段转染THC8307细胞,运用实时荧光定量PCR(Real-time PCR)和Western blot法检测转染后细胞中REV7 mRNA和蛋白表达;运用MTT方法确定L-OHP对THC8307细胞的半数抑制浓度,实验分为不作任何处理的THC8307组、加药处理的L-OHP组,药物联合REV7 siRNA处理的L-OHP+REV7 siRNA组,采用细胞免疫荧光法检测THC8307细胞Bax、BCL-2的表达定位;采用流式细胞术(FCM)测定各组细胞凋亡率;Western blot法检测各组细胞Bax、BCL-2蛋白表达水平。结果与空白对照组相比,流式细胞术结果显示L-OHP、L-OHP联合REV7 siRNA组均能促进THC8307细胞凋亡,Western blot结果显示,L-OHP、L-OHP联合REV7 siRNA可明显下调凋亡因子BCL-2、上调Bax的蛋白表达水平、BCL-2/Bax灰度比值显著性下降(P<0.05),同时L-OHP、L-OHP联合REV7 siRNA两组相比较,后者较单独L-OHP组对THC8307细胞促进凋亡作用更加显著(P均<0.05)。结论 L-OHP联合沉默REV7基因可显著性地诱导人结肠癌细胞THC8307凋亡,发挥抗肿瘤作用。
Objective To explore effect of anti-cancer drug Oxaliplatin combination REV7 tract on apoptosis of colon cancer THC8307 ceils. Methods Human colon cancer cell line THC8307 was cultured in vitro. We experimentally suppressed the REV7 expression in human colon cancer cells by the interference RNA technology （RNAi）. The expression of REV7 mRNA and protein was determined by Real-time qRT-PCR and Western blot. Half inhibition concentration of the human colon cancer cells THC8307 was determined by MTT assay. The experimental groups including THC8307 group,L-OHP group,L-OHP＋REV7 siRNA group. The cellular localization of target gene Bax, BCL-2 in THC8307 cell was examined by immunofluoresce stain. Flow cytometry was used to detect the cell apoptosis rate of THC8307 cells in different groups. Western blot was performed to measure the protein expression of Bax and BCL-2 in L-OHP group and Oxaliplatin combination REV7 siRNA group. Results Compared to the control group, anti-cancer drug L-OHP and L-OHP combined with REV7 siRNA could promote the apoptosis of THC8307 cells . Compared with the drug group , Western blot analyses revealed the protein expression of BCL-2 was significantly down-regulated while of Bax was up-regulated in L-OHP combined with REV7 siRNA group （P〈0.05）. The BCL-2/Bax grey value ratio was significantly decreased（P〈0.05 ）. L-OHP compared with L-OHP combined with REV7 siRNA, L-OHP combined with REV7 siRNA on the promotion of the apoptosis of THC8307 cells was significantly higher than a single factor did （P〈0.05）. Conclusion L-OHP combined with REV7 siRNA could significantly promote the apoptosis of THC8307 cells and played the role of anti-tumor.
作者
王蒙蒙
李昕
马璐
李元杰
隋御
马会明
徐方
WANG Mengmeng LI Xin MA Lu LI Yuanjie SUI Yu MA Huiming XU Fang(Ningxia Medical University,Department of Cell Biology and Medical Genetics,Key Laboratory of Fertility Preservation and Maintenance, Ministry of Education/Key Laboratory of Reproduction in Ningxia, Yinchuan 75000)
出处
《宁夏医科大学学报》
2017年第2期121-125,130,F0004,共7页
Journal of Ningxia Medical University
基金
国家自然科学基金(31360082)
