钝顶螺旋藻藻蓝蛋白诱导HeLa细胞凋亡的分子机制研究

Study on the molecular mechanism of C-phycocyanin from Spirulina platensis induced apoptosis in HeLa cells

目的探讨钝顶螺旋藻（Spirulina platensis）藻蓝蛋白（C-phycocyanin,CPC）对体外人宫颈癌细胞（HeLa细胞）凋亡的影响及分子机制。方法首先通过MTT法检测藻蓝蛋白对HeLa细胞增殖的影响,然后通过电镜观察藻蓝蛋白处理后的细胞的凋亡形态,琼脂糖凝胶电泳观察凋亡细胞的DNA结构变化,流式细胞仪显示不同浓度藻蓝蛋白对HeLa细胞的细胞周期变化的影响,免疫组化法检测藻蓝蛋白对细胞表面凋亡相关基因表达的影响,检测凋亡相关的caspase的活性及细胞色素C的释放,以揭示藻蓝蛋白诱导HeLa细胞凋亡的作用机制。结果与未用藻蓝蛋白作用的对照组相比,藻蓝蛋白组的HeLa细胞存活率明显降低,并存在浓度剂量效应。藻蓝蛋白处理后的细胞呈现典型的凋亡形态,如细胞皱缩、细胞膜微绒毛消失、起泡,染色体密集,形成凋亡小体。凋亡细胞的DNA出现断裂,呈梯状的电泳带（DNAladder）,分子量为180~200 bp及其倍体。并且发现藻蓝蛋白处理后的处于亚二倍体峰HeLa细胞数蛋白浓度随藻蓝蛋白浓度的增高而明显增多。另外藻蓝蛋白能促进HeLa细胞内促凋亡基因（Fas、ICAM-1）的表达并抑制抗凋亡基因（Bcl-2）的表达,从而促进细胞内凋亡信号的传导。藻蓝蛋白可以激活caspase酶并可促进凋亡蛋白——细胞色素C从线粒体到细胞质释放。结论钝顶螺旋藻藻蓝蛋白具有诱导体外HeLa细胞凋亡的作用,通过促进凋亡信号在HeLa细胞内的传导从而达到抗肿瘤的效果。

Aim To investigate the influence and molecular mechanism of C-phycocyanin （ CPC ） from Spirulina platensis on apoptosis of HeLa cells in vitro. Methods Firstly, the effect of purified CPC on proliferation of HeLa cells in vitro was determined by MTT assay, and then electron microscope was exploited to observe the characteristic apoptotic features of cells treated with CPC. Subsequently, genomic DNA changes of HeLa cells were observed by agarose electrophoresis. Flow cytometric analysis revealed the influence of different concentrations of CPC on cell cycle of HeLa ceils. The expressions of apoptosis related genes of CPC treated HeLa cells were determined by immunohistochemistry analysis. In addition, the activities of caspases and the release of cytochrome c from mitochondria into the cytosol were detected. Results Compared with control cells untreated with CPC, a significant decreased in the numbers of HeLa cells in survival treated with CPC and concentration dose effects existed. CPC could induce characteristic apoptotic features including cell shrinkage, membrane blebbing, microvilli loss, chromatin margination and condensation into dense granules or blocks. DNA of HeLa cells treated with CPC showed fragmentation pattern （DNA ladder of oligomers of 180 - 200 bp） typical for apoptotic cells. HeLa cells treated with different concentrations of CPC demonstrated an increasing percentage of cells in sub-G0/G1 phase. In addition, CPC could promote the expression of pro-apoptotic gene （ Fas and ICAM- 1 ）;meanwhile, held back the anti-apoptotic gene （Bcl-2） expression, and then facilitated the transduction of tumoral apoptosis signals that resulted in the apoptosis of HeLa cells in vitro. In CPC treated HeLa cells, CPC treatment of HeLa cells also resulted in activation of caspases and release of cytochrome C from mitochondria into the cytosol. Conclusion C-phycocyanin from Spirulina platensis can induce the apoptosis of HeLa cells in vitro. By virtue of the promotion of the apoptosis signals transduction in HeLa, CPC realizes its antitumor activities.