猪MNSFβ稳定表达细胞系的建立及促凋亡功能研究

发布时间：2018-03-26 07:31

本文选题：猪MNSFβ基因　切入点：猪BCL-G基因　出处：《西北农林科技大学》2015年硕士论文

【摘要】：单克隆非特异性免疫抑制因子-β(Monoclonal Nonspecific Suppressor Factorβ,MNSFβ)是一种在机体内广泛分布的非抗原特异性免疫抑制因子,又是一种新的细胞凋亡调节因子和潜在的肿瘤抑制因子,可与不同的靶蛋白结合调控信号通路,在免疫调节和细胞凋亡过程中发挥重要作用。近年来在小鼠上被广泛研究,但其功能在猪中鲜有报道,猪是一种比小鼠更理想的医学实验动物模型,对猪MNSFβ(p MNSFβ)基因的研究将为人类MNSFβ基因及相关疾病的研究提供重要的理论支持。本论文主要针对p MNSFβ基因的促凋亡功能展开研究,探究p MNSFβ是否与p BCL-G发生相互作用以及p MNSFβ与p BCL-G结合对细胞凋亡的影响。本研究获得的研究结果如下:1.通过瞬时转染和新霉素(G418)筛选稳定表达GFP-p MNSFβ猪脐静脉血管内皮细胞株(SUVEC),经倒置荧光显微镜观察和western blot鉴定,稳定表达GFP-p MNSFβ的SUVEC细胞株构建成功。2.免疫共沉淀实验表明,pMNSFβ与pBCL-G蛋白能够发生相互作用。3.通过形态学观察和细胞核染色发现,随着星孢菌素(STS)浓度的提高,皱缩、变圆、脱落、体积缩小,Hoechst 33342染色的细胞核内呈现亮蓝色荧光等典型细胞凋亡特征越来越明显,细胞凋亡的数量也相应增多。而且与对照组(GFP)相比,实验组(GFP-pMNSFβ)的细胞凋亡特征更明显。4.用Annexin V-PE与PI进行双重染色结合流式细胞术分析表明,稳定表达的pMNSFβ蛋白对STS诱导的细胞凋亡具有显著的促进作用;p BCL-G与p MNSFβ蛋白共表达协同促进STS诱导的细胞凋亡。综上所述,本论文首次通过实验获得稳定表达GFP-p MNSFβ的SUVECs细胞株,证明了p MNSFβ能够促进STS诱导的细胞凋亡,阐明了p MNSFβ与p BCL-G蛋白能够发生相互作用,且p MNSFβ与p BCL-G蛋白对STS诱导的细胞凋亡有协同促进作用。
[Abstract]:Monoclonal Nonspecific Suppressor Factor 尾 (MNSF 尾) is a widely distributed non-antigen-specific immunosuppressive factor, and it is also a new apoptosis regulator and a potential tumor suppressor. It plays an important role in immunomodulation and apoptosis. It has been widely studied in mice in recent years, but its function is rarely reported in pigs. Pig is a more ideal animal model than mice. The study of porcine MNSF 尾 p MNSF 尾 gene will provide important theoretical support for the study of human MNSF 尾 gene and related diseases. This paper mainly focuses on the apoptotic function of p MNSF 尾 gene. To explore whether p MNSF 尾 interacts with p BCL-G and the effect of p MNSF 尾 binding with p BCL-G on apoptosis. The results of this study are as follows: 1. The stable expression of GFP-p MNSF 尾 in porcine umbilical vein blood was screened by transient transfection and neomycin G418). The tube endothelial cell line was identified by inverted fluorescence microscope and western blot. The stable expression of GFP-p MNSF 尾 in SUVEC cell line was successfully constructed. 2. The immunoprecipitation assay showed that pMNSF 尾 could interact with pBCL-G protein. By morphological observation and nuclear staining, it was found that with the increase of the concentration of GFP-p MNSF 尾, it shrinks, becomes round and falls off. The typical apoptotic characteristics such as bright blue fluorescence and the number of apoptotic cells in the nuclei stained by Hoechst 33342 were more and more obvious, and compared with those in the control group, the number of apoptotic cells in the cells with Hoechst 33342 was higher than that in the control group. The apoptotic characteristics of GFP-pMNSF 尾 in experimental group were more obvious. 4. Double staining with Annexin V-PE and Pi combined with flow cytometry showed that, The stable expression of pMNSF 尾 protein significantly promoted the apoptosis induced by STS. The coexpression of p BCL-G and p MNSF 尾 protein co-promoted the apoptosis induced by STS. In conclusion, SUVECs cell lines expressing GFP-p MNSF 尾 stably were obtained for the first time in this paper. It was proved that p MNSF 尾 could promote apoptosis induced by STS, and that p MNSF 尾 and p BCL-G protein could interact with each other, and p MNSF 尾 and p BCL-G protein had synergistic effect on STS induced apoptosis.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.2

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