ANKRD49通过NF-κB信号通路抑制UV诱导小鼠精原细胞凋亡的初步研究

发布时间：2018-05-14 12:24

  本文选题：ANKRD49 + 精子发生　； 参考：《山西医科大学》2017年硕士论文

【摘要】：目的:运用分子克隆技术构建ankrd49真核表达重组质粒并建立ankrd49稳定过表达的小鼠精原细胞系(GC-1)细胞模型;分析ankrd49过表达对UV诱导GC-1细胞凋亡的影响,探讨NF-κB信号通路是否参与ANKRD49对UV诱导GC-1细胞凋亡的调控,并进一步探讨ANKRD49蛋白激活NF-κB信号通路的具体机制,为深入研究该基因的作用及其在精子发生中的调控机制提供思路。方法:1.利用分子克隆技术构建ankrd49真核表达重组质粒pMSCVpuro-ankrd49-flag;2.建立ankrd49稳定过表达的GC-1细胞模型,并采用RT-PCR和Western blotting检测ankrd49 mRNA和蛋白质的表达水平;3.通过Hoechst33258染色对紫外线(UV)诱导的GC-1细胞凋亡状况进行分析;4.运用流式细胞术检测过表达ankrd49基因对UV诱导的GC-1细胞凋亡率和线粒体膜电位下降率的影响;5.运用Caspase-3酶活性检测试剂盒检测过表达ankrd49基因对GC-1细胞Caspase-3酶活性的影响;6.利用Western blotting检测过表达ankrd49基因后GC-1细胞内凋亡相关蛋白多聚ADP核糖聚合酶—PARP(poly ADP-ribose polymerase)、Cleaved-Caspase-3、Bcl-xL和Bax的表达变化;7.利用流式细胞术、caspase-3酶活性检测试剂盒和免疫印迹技术分别检测过表达ankrd49的gc-1细胞中加入nf-κb信号通路抑制剂(pdtc)后,gc-1细胞的凋亡率、caspase-3酶活性和凋亡相关蛋白parp、cleaved-caspase-3、bcl-xl和bax的表达变化情况;8.分别提取过表达组和空载体组细胞胞浆总蛋白和核蛋白,并采用westernblotting检测p65蛋白的亚细胞定位。结果:1.pcr扩增、双酶切分析及dna序列测定结果均表明真核表达重组质粒pmscvpuro-ankrd49-flag构建成功;2.rt-pcr和westernblotting检测结果显示ankrd49基因在gc-1细胞中实现了稳定过表达;3.hoechst33258染色结果显示,ankrd49稳定过表达组细胞的凋亡百分比为(0.03±0.01)×100%,明显低于空载体组(0.23±0.05)×100%和裸细胞组(0.25±0.04)×100%,(p0.01);4.流式细胞术结果显示过表达ankrd49组细胞的早期凋亡率为2.71%±0.50%,明显低于空载体组11.66%±1.01%和裸细胞组11.31%±1.74%,过表达ankrd49组细胞的线粒体膜电位下降率为11.51%±1.53%,明显低于空载体组和裸细胞组(30.54%±2.42%和28.99%±1.61%),(p0.01);5.caspase-3酶活性实验结果显示过表达ankrd49组细胞caspase-3酶活性为1.09±0.15,明显低于空载体组(3.01±0.32)和裸细胞组(3.12±0.23),(p0.01);6.westernblotting检测结果显示ankrd49稳定过表达组cleaved-parp、cleaved-caspase-3蛋白的表达水平明显低于对照组,而bcl-xl蛋白表达水平显著高于对照组;7.过表达ankrd49的gc-1细胞中加入nf-κb信号通路抑制剂(pdtc)后,经过流式细胞术检测表明pdtc处理组细胞早期凋亡率明显高于pdtc未处理组(p0.01),caspase-3酶活性实验结果显示pdtc处理组caspase-3酶活性明显升高(p0.01),westernblotting检测结果显示pdtc处理组细胞内cleaved-parp、Cleaved-Caspase-3蛋白的表达水平明显增高,而Bcl-x L蛋白表达水平明显降低(P0.05);8.提取空载体组和过表达组细胞胞浆和核蛋白进行Western blotting检测,结果表明ANKRD49可以促进p65蛋白入核(P0.01)。结论:1.成功构建了ankrd49的真核表达重组质粒,并建立ankrd49稳定过表达GC-1细胞模型。2.ANKRD49蛋白对UV诱导GC-1细胞凋亡具有抑制作用。3.ANKRD49蛋白通过激活NF-κB信号通路抑制GC-1细胞凋亡。
[Abstract]:Objective: to construct ankrd49 eukaryotic expression recombinant plasmid with molecular cloning technique and to establish a mouse spermatogonial cell line (GC-1) cell model with stable ankrd49 expression, analyze the effect of ankrd49 overexpression on UV induced apoptosis of GC-1 cells, and explore whether NF- kappa B signaling pathway participates in the regulation of ANKRD49 on UV induced GC-1 cell apoptosis, and further explores the regulation of UV induced GC-1 cell apoptosis. The specific mechanism of ANKRD49 protein activation of NF- kappa B signaling pathway provides ideas for in-depth study of the role of the gene and its regulation mechanism in spermatogenesis. Method: 1. the recombinant plasmid pMSCVpuro-ankrd49-flag of ankrd49 is constructed by molecular cloning technology; and 2. to establish a GC-1 cell model for the stable expression of ankrd49, and to use RT-PCR and. Western blotting was used to detect the expression level of ankrd49 mRNA and protein; 3. the apoptosis of GC-1 cells induced by ultraviolet (UV) was analyzed by Hoechst33258 staining. 4. the effect of ankrd49 gene expression on the apoptosis rate of UV induced GC-1 cells and the decrease of mitochondrial membrane potential was detected by flow cytometry; 5. using Caspase-3 enzyme activity. The detection kit detected the effect of ankrd49 gene on the activity of Caspase-3 enzyme in GC-1 cells; 6. Western blotting was used to detect the apoptosis related protein polypolymerized ADP ribose polymerase and PARP (poly ADP-ribose polymerase) in GC-1 cells after ankrd49 gene expression, and 7. by flow cytometry. Caspase-3 enzyme activity detection kit and immunoblotting technique were used to detect the apoptosis rate, caspase-3 enzyme activity and apoptosis related protein PARP, cleaved-caspase-3, Bcl-xL and Bax, respectively, after the nf- kappa B signaling inhibitor (PDTC) was added to the GC-1 cells expressing ankrd49, respectively. 8. the expression group and the empty carrier were extracted, respectively. The cytoplasmic total protein and nucleoprotein were detected and the subcellular localization of p65 protein was detected by westernblotting. Results: 1.pcr amplification, double enzyme digestion analysis and DNA sequencing results showed that eukaryotic expression recombinant plasmid pmscvpuro-ankrd49-flag was constructed successfully; 2.rt-pcr and westernblotting detection results showed that ankrd49 gene was real in GC-1 cells. 3.hoechst33258 staining showed that the percentage of apoptotic cells in the ankrd49 stable overexpression group was (0.03 + 0.01) x 100%, significantly lower than that in the group (0.23 + 0.05) x 100% and the naked cell group (0.25 + 0.04) x 100% (P0.01), and 4. flow cytometry showed that the early apoptosis rate of the overexpressed ankrd49 group was 2.71% + 0.50%, It was significantly lower than that in the group of 11.66% + 1.01% and 11.31% + 1.74% in the naked cell group. The decrease rate of mitochondrial membrane potential in the overexpressed ankrd49 group was 11.51% + 1.53%, which was significantly lower than that of the unloaded group and the naked cell group (30.54% + 2.42% and 28.99% + 1.61%), (P0.01), and the results of the 5.caspase-3 enzyme activity showed that the caspase-3 enzyme activity in the ankrd49 group was expressed. 1.09 + 0.15, significantly lower than the no-load group (3.01 + 0.32) and naked cell group (3.12 + 0.23), (P0.01), 6.westernblotting detection results showed that ankrd49 stable overexpression group cleaved-parp, cleaved-caspase-3 protein expression level was significantly lower than the control group, and Bcl-xL egg white expression level was significantly higher than the control group; 7. over the ankrd49 GC-1 cells. After adding nf- kappa B signaling pathway inhibitor (PDTC), flow cytometry showed that the early apoptosis rate of PDTC treated group was significantly higher than that of PDTC untreated group (P0.01). The results of Caspase-3 enzyme activity test showed that the caspase-3 enzyme activity in PDTC treatment group increased significantly (P0.01), westernblotting detection results showed that the PDTC treated group was within the cells. PARP, the expression level of Cleaved-Caspase-3 protein was significantly increased, while the expression level of Bcl-x L protein decreased significantly (P0.05). 8. the cytoplasm and nucleoprotein of the overexpressed group and the overexpressed group were detected by Western blotting detection. The results showed that ANKRD49 could promote the nucleation of p65 protein (P0.01). Conclusion: 1. successfully constructed the ankrd49 eukaryotic expression and recombination. Plasmids and the establishment of ankrd49 stable overexpressed GC-1 cell model.2.ANKRD49 protein inhibiting the apoptosis of GC-1 cells induced by UV,.3.ANKRD49 protein inhibits GC-1 cell apoptosis by activating the NF- kappa B signaling pathway.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R698.2

【参考文献】

相关期刊论文 前5条

1 Chuck C K Chao;;Inhibition of apoptosis by oncogenic hepatitis B virus X protein: Implications for the treatment of hepatocellular carcinoma[J];World Journal of Hepatology;2016年25期

2 Na ZHANG;Xiao-jie XIE;Jian-an WANG;;心锚重复蛋白的研究进展（英文）[J];Journal of Zhejiang University-Science B(Biomedicine & Biotechnology);2016年05期

3 庞敏;郭民;袁丽蓉;郭睿;王海龙;;ANKRD49在非小细胞肺癌的表达[J];基础医学与临床;2016年01期

4 张明谦;刘文斌;陈洪强;刘晋yN;;ANKRD18B基因在大肠癌中的甲基化与表达[J];昆明医科大学学报;2014年11期

5 Qian Li;Pan Zhang;Chao Zhang;Ying Wang;Ru Wan;Ye Yang;Xuejiang Guo;Ran Huo;Min Lin;Zuomin Zhou;Jiahao Sha;;DDX3X regulates cell survival and cell cycle during mouse early embryonic development[J];The Journal of Biomedical Research;2014年04期

，

本文编号：1887837