家蚕Bm30Kc6蛋白对过氧化氢诱导的家蚕细胞凋亡的抑制作用  

The Protective Effect of Silkworm(Bombyx mori) Bm30Kc6 on BmN Cells Apoptosis Induced by Hydrogen Peroxide

作　　者：应慧慧[1,2] 余海鹏[1,2] 寿鑫[1,2] 全滟平[1,2] 张耀洲[1,2] 童富淡[1,2] 于威[1,2] 

机构地区：[1]浙江理工大学生物化学研究所,杭州310018 [2]浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《农业生物技术学报》2013年第4期441-447,共7页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No.2011AA100603);浙江省自然科学基金项目(No.Y3090339;No.Y3110187)

摘　　要：30K蛋白是一类家蚕(Bombyx mori)血淋巴中具有最强抗细胞凋亡活性的蛋白,家蚕Bm30Kc6蛋白作为30K蛋白家族的成员之一,也具有较强的抗细胞凋亡作用,但其抗细胞凋亡的作用机制还不十分清楚。本研究将本实验室成功构建的重组病毒Bacmid-Bm30Kc6接种家蚕BmN细胞大量表达后,利用Ni-NTA蛋白亲和层析柱纯化重组蛋白Bm30Kc6。SDS-PAGE和Westernblot检测结果显示纯化蛋白的纯度较高。然后利用不同浓度(0.1、1、5和10mmol/L)的H2O2处理家蚕BmN细胞,诱导细胞发生凋亡,结果表明,在培养基中加入终浓度为5mmol/LH2O2孵育4h后即可成功诱导BmN细胞凋亡。在上述建立的H2O2诱导的家蚕BmN细胞凋亡体外损伤模型的基础上,分析了Bm30Kc6蛋白的抗细胞凋亡的活性及作用机制。首先利用CelldeathDetection ELISA试剂盒检测了细胞内的DNA片段化程度,检测结果表明,Bm30Kc6蛋白(终浓度5μg/mL)可明显降低H2O2诱导的家蚕BmN细胞内的DNA片段化程度。然后利用Cellproliferation ELISA试剂盒检测了细胞的增殖情况,结果表明,Bm30Kc6蛋白可以显著增强家蚕BmN细胞的活力。进一步利用8-isoprostane EIA试剂盒检测细胞内氧化应激标志物8-isoprostane的浓度变化,结果显示,家蚕Bm30Kc6蛋白可以显著降低胞内8-isoprostane的浓度。此外,Western blot分析结果显示,Bm30Kc6蛋白可显著抑制家蚕BmN细胞中细胞色素c从线粒体中释放进入细胞质。Bm30Kc6蛋白一方面可以通过降低BmN细胞内的氧化应激水平从而起到抑制细胞凋亡的作用;另一方面,Bm30Kc6蛋白也可以通过阻断H2O2诱导激活的线粒体通路来抑制细胞发生凋亡。本研究将为进一步深入研究家蚕Bm30Kc6蛋白的抗凋亡机制提供基础资料。It has been clear that silkworm （Bombyx mori） 30K protein in larval hemolymph has the highest apoptosis inhibition activities. As one of the member of 30K apolipoproteins family, Bm30Kc6 has been reported to have obvious anti-apoptotic activity, but the anti-apoptotic mechanism was still not very clear. In this study, the silkworm BmN cells were infected with recombinant virus Bacmid-Bm30Kc6 constructed by our lab and the expressed proteins were purified by a Ni-NTA agarose affinity filler. SDS-PAGE and Western blot results showed that the Bm30Kc6 proteins were highly purified. Then BmN cells were treated with different concentrations of hydrogen peroxide （0.1, 1, 5 and 10 mmol/L） to induce cell apoptosis, the results showed that a final concentration of 5 mmol/L H20~ incubated with BmN cells for 4 h could be successfully induced cell apoptosis. Based on this cell damage model, the anti-apoptotic mechanism of Bm30Kc6 was studied. Firstly, cell death detection ELISA kit was used to evaluate the cell apoptosis, the results showed that 5 ixg/mL Bm30Kc6 could remarkably slow down DNA fragmentation in Bran cells. Then cell proliferation ELISA kit was used to test the cell viability, the results showed that Bm30Kc6 could also enhance the viability of BmN ceils obviously. Moreover, 8-isoprostane EIA kit was used to test the concentration of 8-isoprostane inside BraN cells, the results showed that H：O2 treatment could enhance the formation of 8-isoprostane obviously, however, Bm30Kc6 protein could decrease the concentration of 8-isoprostane obviously. Finally, Western blot results showed that Bm30Kc6 could inhibited the H20^-induced cytochrome c released from mitochondria to cytoplasm inside BmN cells. In conclusion, Bm30Kc6 may inhibit BraN cell apoptosis by decreasing the level of oxidative stress inside cells and inhibit cell apoptosis by blocking the mitochondrial signaling pathway. This study will provide basic information for further study on the anti-apototic mechanism of Bm30Kc6 portein.

关 键 词：家蚕 Bm30Kc6 细胞凋亡 抑制作用 

分 类 号：S512.1[农业科学—作物学]