香菇菌C91-3凋亡相关蛋白24414对小鼠肝癌细胞Hca生物学功能的影响

目的]通过诱导表达香菇菌C91-3凋亡相关蛋白24414,研究其对小鼠肝癌细胞Hca的生物学功能影响,探讨其诱导肿瘤细胞凋亡的机制。方法]构建真核重组表达质粒pPIC9K-24414,用毕赤酵母GS115体系进行诱导表达,将已鉴定的诱导表达蛋白作用于小鼠肝癌细胞Hca,用MTT法测定不同浓度目的蛋白组、顺铂(DDP)组及空白对照组的抑瘤率,用流式细胞术检测目的蛋白对肿瘤细胞的凋亡作用,同时用MTT法检测目的蛋白对正常鸡胚成纤维细胞的毒性作用。结果]Western-blot结果显示目的蛋白成功表达;MTT法检测结果显示,不同浓度此蛋白对Hca细胞的生长抑制作用(OD595)与对照组比较差异均有显著性意义(P<0.05),7.5μg/mL、15μg/mL和30μg/mL目的蛋白的最大抑瘤率分别为70.03%、82.02%和74.66%,均超过5μg/mL DDP的作用;流式细胞术结果显示,此蛋白具有诱导Hca细胞凋亡的能力,早晚双期细胞凋亡诱导率与对照组比较差异均有显著性意义(P<0.05),浓度为30μg/mL时诱导凋亡的作用最强,早晚两期凋亡率分别为(9.28±0.04)%和(9.37±0.03)%;同时此蛋白对正常细胞无毒性作用。结论]香菇菌C91-3凋亡相关蛋白24414在毕赤酵母GS115中成功诱导表达,此蛋白对小鼠肝癌细胞Hca生长具有抑制作用,能够诱导其产生凋亡,为进一步深入研究香菇菌C91-3的抗肿瘤机制奠定了基础。

Study of biological function of Lentinus edodes C91-3 apoptosis protein 24414 on mouse hepatoma Hca cell line

Objective]To express and characterize the apoptosis protein 24414 of Lentinus edodes C91-3 in Pichia pastoris GS115.Study the biological function of the characterized protein on mouse hepatoma Hca cell line.Methods]Construct recombinant plasmid pPIC9K-24414.Induce the interest protein to be expressed in Pichia pastoris GS115.Co-cultured the characterized protein with Hca cell line and detected it with flow cytometry to study the apoptosis function.At the same time,detected the toxic effect of the characterized protein with MTT method by co-culturing the characterized protein with chick embryo fibroblast.Results]Westen-blot showed that the target protein was expressed successfully.The MTT assay showed that there was a great difference(OD595) between the interested protein group and control group according to Hca cell growth inhibition(P<0.05),the maximum inhibition rate of 7.5 μg/mL,15 μg/mL and 30 μg/mL interested protein was 70.03%,82.02% and 74.66% respectively,which was stronger than 5 μg/mL DDP group.The flow cytometry showed that there was a significantly difference between the interest protein groups and control group according to the apoptosis function(P<0.05).The strongest apoptosis function was in 30 μg/mL protein group.The apoptosis rates(%) of early and late stage were 9.28±0.04 and 9.37±0.03.While,there was no toxic effect of the characterized protein on chick embryo fibroblast.Conclusion]Lentinus edodes C91-3 apoptosis protein 24414 were expressed successfully in pichia pastoris GS115.The protein can induce the apoptosis of mouse hepatoma Hca cell,which placed a foundation for further anti-tumor mechanism studies of Lentinus edodes C91-3.