摘要
目的研究雷替曲塞对微卫星高度不稳定性(MSI-H)结肠癌细胞株LOVO增殖、凋亡的作用及分子机制。方法取对数生长期的MSI-H结肠癌细胞株LOVO细胞,经10%FBS的DMEM完全培养基培养后,分别加至60μM雷替曲塞(雷替曲塞组)和2μg/ml 5-氟尿嘧啶(5-FU)(5-FU组)的完全培养基培养,同时设置不加药物的空白对照组。采用CCK-8法检测雷替曲塞及5-FU对LOVO细胞增殖的影响;采用FITC-Annexin V-PI染色法和流式细胞仪检测雷替曲塞及5-FU作用于LOVO细胞24 h后对细胞凋亡的影响;Western Blot法检测雷替曲塞及5-FU作用24 h后凋亡相关蛋白-B细胞淋巴瘤/白血病-2蛋白(Bcl-2)和Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶3前体(Pre-caspase 3)、半胱氨酸天冬氨酸蛋白酶3(cleaved-caspase 3)等蛋白在LOVO细胞中的表达水平。结果CCK-8法细胞增殖实验检测结果显示,与对照组比较,雷替曲塞作用后细胞的增殖明显受到抑制,其中在72 h最明显(P均<0.05),而5-FU组无变化(P>0.05)。FITC-Annexin V-PI染色结果显示,与对照组及5-FU组相比,雷替曲塞作用于LOVO细胞24 h后,细胞早期凋亡数目和晚期凋亡数目均增加,差异有统计学意义(P均<0.05)。流式细胞术检测显示,与对照组比较,雷替曲塞组的细胞凋亡率明显增高(P<0.05),5-FU组无变化(P>0.05)。Western Blot检测结果显示雷替曲塞组的促凋亡蛋白cleaved-caspase3、Bax蛋白的表达水平明显增加,而抑制凋亡蛋白Bcl-2、Pre-caspase3蛋白表达水平明显降低(P均<0.05)。结论雷替曲塞可促进MSI-H结肠癌细胞的凋亡,为雷替曲塞对结肠癌的治疗提供了参考依据。
Objective To investigate the effect of raltitrexed on the proliferation and apoptosis of mierosatellite high insta- bility（MSI-H） colon cancer cell line LOVO cells and its molecular mechanisms. Methods After being cultured with 10% FBS DMEM complete medium, MSI-H colon cancer cell line LOVO cells in the logarithmic growth phase were added into 60 μM rahitrexed complete medium （ rahitrexed group） and 2μg/ml 5-fluorouracil （5-FU） complete medium （ 5-FU group）, respectively, and blank control group without drugs was set up. The Cell Count Kit （CCK）-8 method was used to detect the influence of rahitrexed and 5-FU on cell proliferation. FITC-AnnexinV-PI staining method and flow cytometry were used to respectively detect the influence of raltitrexed and 5-FU on cell apoptosis 24 hours after treatment. Western blot method was used to detect the relative expression levels of apoptosis-related proteins cleaved-caspase3, Bax, Bcl-2 and pre-caspase3. Results CCK-8 cell proliferation test showed that cell proliferation was inhibited significantly after rahitrexed treatment compared with control group, especially in 72 hours （ all P 〈 0. 05 ）, while it remained unchanged in 5-FU group （ P 〉 0. 05 ）. FITC-Annexin V-PI stainning results showed that the number of LOVO cells both early apoptosis and late apoptosis increased significantly 24 hours after rahitrexed treatment compared with control group and 5-FU group（ all P 〈 0. 05 ）. Flow cytometry results showed that the cells apoptosis rate in rahitrexed group increased significantly compared with control group （ all P 〈 0.05 ） , while it remained unchanged in 5-FU group （ P 〉 0.05 ）. Western Blot showed that the relative expression levels of pro-apoptotic proteins cleaved-caspase3 and Bax proteins increased significantly ,while the relative expression lev- els of apoptosis-inhibifing proteins Bcl-2 and pre-caspase3 proteins decreased significantly in raltitrexed group （ all P 〈 0. 05）. Conclusion Raltitrexed can promote the apoptosis of MSI-H cancer cells, and this provides an experimental refer- ence for its treatment of colon cancer.
出处
《中国临床研究》
CAS
2016年第12期1610-1613,共4页
Chinese Journal of Clinical Research
基金
南京市医学科技发展项目(YKK13185)
