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目的:研究钙磷酸蛋白C对乳癌细胞MDA-MB-435S凋亡的影响及相关机制。方法:采用倒置显微镜、琼脂糖凝胶电泳和流式细胞术等方法观察钙磷酸蛋白C处理前后MDA-MB-435S细胞形态、基因组DNA断裂和凋亡细胞数的变化;采用RT-PCR检测钙磷酸蛋白C处理前后MDA-MB-435S细胞凋亡相关基因bcl-XL、bcl-2、IAP-1、bax及Smac mRNA表达的变化;采用Western blotting法测定Bax和Bcl-2蛋白的表达。结果:与MDA-MB-435S亲本细胞(对照组)相比,钙磷酸蛋白C处理后的MDA-MB-435S细胞(实验组)出现凋亡形态学特征(如胞膜发泡、染色质凝集、凋亡小体形成等)改变,显示特征性的DNA“梯状”断裂变化,凋亡细胞数明显增多。RT-PCR和Western blotting结果显示:与对照组相比,实验组细胞中促凋亡基因bax和Smac mRNA表达上调,抗凋亡基因bcl-2mRNA表达下调,但抗凋亡基因bcl-XL和IAP-1mRNA表达没有明显变化;实验组Bax蛋白表达上调,Bcl-2蛋白表达下调。结论:钙磷酸蛋白C可诱导MDA-MB-435S凋亡,该作用可能与上调bax和Smac基因表达及下调bcl-2基因表达有关。
Objective: To study the effect of calcium phosphate-C on the apoptosis of breast cancer cell line MDA-MB-435S and its related mechanism. Methods: The morphological changes, DNA fragmentation and apoptotic cell number of MDA-MB-435S cells were observed by inverted microscope, agarose gel electrophoresis and flow cytometry. Calcium The expressions of bcl-XL, bcl-2, IAP-1, bax and Smac mRNA in MDA-MB-435S cells before and after phosphoprotein C treatment were detected by Western blotting. RESULTS: Compared with MDA-MB-435S parental cells (control group), apoptotic morphological features of MDA-MB-435S cells treated with Calcium Phosphoprotein C (such as membrane foaming, chromatin condensation , Apoptotic body formation, etc.) changes, showing the characteristic DNA “ladder ” fracture change, a significant increase in the number of apoptotic cells. The results of RT-PCR and Western blotting showed that compared with the control group, the expression of pro-apoptotic genes bax and Smac mRNA in the experimental group was up-regulated and the expression of the anti-apoptotic gene bcl-2 mRNA was down-regulated. However, the anti-apoptotic genes bcl- -1 mRNA expression did not change significantly; the experimental group Bax protein expression increased, Bcl-2 protein expression down. CONCLUSION: Calcium phosphate C can induce the apoptosis of MDA-MB-435S, which may be related to up-regulation of bax and Smac gene expression and down-regulation of bcl-2 gene expression.