不同浓度Mg-6Zn合金浸提液对肠上皮细胞凋亡及其相关基因Caspase-3表达的影响

Effects of Different Concentration of Mg-6 Zn Alloy Extracts on Apoptosis and the Related Caspase-3 Expression in Intestinal Epithelial Cells

研究不同浓度Mg-6Zn合金浸提液对大鼠肠上皮细胞IEC-6的凋亡及凋亡相关基因Caspase-3表达的影响。将Mg-6Zn合金制成不同浓度(20%和40%)的浸提液体外培养大鼠肠上皮细胞IEC-6,并以含10%胎牛血清的高糖DMEM(Dulbecco′s modified Eagle′s medium)培养基作为阴性对照。采用噻唑蓝(MTT)法通过计算细胞相对增殖率检测不同浓度合金浸提液对IEC-6细胞的毒性作用;采用流式细胞仪检测不同浓度合金浸提液对IEC-6细胞凋亡的影响;采用逆转录聚合酶链反应(RT-PCR)法检测不同浓度合金浸提液对IEC-6细胞凋亡相关基因Caspase-3mRNA表达的影响。实验结果表明,20%和40%两种浓度的镁合金浸提液在体外培养IEC-6细胞第七天时细胞毒性分级分别为0级和1级,属于无毒性,符合医用材料细胞毒性分级的合格标准。40%和20%浓度镁合金浸提液中,IEC-6细胞的凋亡比例及Caspase-3活化度相对于对照组都增高,差异有统计学意义(P<0.05),40%浓度组中,IEC-6细胞的凋亡比例及Caspase-3活化度相对于20%浓度组增高,差异有统计学意义(P<0.05),说明Mg-6Zn合金浸提液在一定范围内处于较高浓度时易诱导IEC-6细胞的凋亡及Caspase-3的表达,且随浓度升高影响越大。

The effects of different concentration of Mg-6Zn alloy extracts on apoptosis rate and the expression of caspase-3 in intestinal epithelial cells （IEC）-6 were evaluated. IEC-6 cells were cultured in different concentration of Mg-6Zn alloy extracts （40%, 20%）, and the Dulbecco＇s modified Eagle＇s medium-high glucose cell medium contai- ning 10% fetal bovine serum was served as a control group. The cytotoxicity of Mg-6Zn alloy on IEC-6 cells was stu- died by calculating the cell relative growth rate through the MTT method. The apoptosis of IEC-6 cells was measured through flow cytometry, And the expression of caspase-3 was investigated using real-time polymerase chain reaction. The results showed that the cytotoxicity of these extracts was grade 0--1, demonstrating non-toxic to the IEC-6 cells which cultured for 7 days, indicating that the Mg-6Zn alloy meet the standard of the tests for cytotoxicity of medical materials. The level of apoptosis and the expression of caspase-3 were up-regulated in IEC-6 cells cultured in 40% and 20% Mg-6Zn alloy extract as compared to the control group. And the results were also increased in 40% alloy extract as compared to the 20% group. All the differences were significant. The significant differences （P〈0. 05） showed that the Mg-6Zn alloy extract at a level of high concentration could induce the apoptosis and the related caspase-3 expression in IEC-6 cells in vitro, and the influence was more effective as the concentration of extract increased.