本文選題：ANKRD49 + 精子發(fā)生��； 參考：《山西醫(yī)科大學》2017年碩士論文

【摘要】：目的:運用分子克隆技術構建ankrd49真核表達重組質粒并建立ankrd49穩(wěn)定過表達的小鼠精原細胞系(GC-1)細胞模型;分析ankrd49過表達對UV誘導GC-1細胞凋亡的影響,探討NF-κB信號通路是否參與ANKRD49對UV誘導GC-1細胞凋亡的調控,并進一步探討ANKRD49蛋白激活NF-κB信號通路的具體機制,為深入研究該基因的作用及其在精子發(fā)生中的調控機制提供思路。方法:1.利用分子克隆技術構建ankrd49真核表達重組質粒pMSCVpuro-ankrd49-flag;2.建立ankrd49穩(wěn)定過表達的GC-1細胞模型,并采用RT-PCR和Western blotting檢測ankrd49 mRNA和蛋白質的表達水平;3.通過Hoechst33258染色對紫外線(UV)誘導的GC-1細胞凋亡狀況進行分析;4.運用流式細胞術檢測過表達ankrd49基因對UV誘導的GC-1細胞凋亡率和線粒體膜電位下降率的影響;5.運用Caspase-3酶活性檢測試劑盒檢測過表達ankrd49基因對GC-1細胞Caspase-3酶活性的影響;6.利用Western blotting檢測過表達ankrd49基因后GC-1細胞內凋亡相關蛋白多聚ADP核糖聚合酶—PARP(poly ADP-ribose polymerase)、Cleaved-Caspase-3、Bcl-xL和Bax的表達變化;7.利用流式細胞術、caspase-3酶活性檢測試劑盒和免疫印跡技術分別檢測過表達ankrd49的gc-1細胞中加入nf-κb信號通路抑制劑(pdtc)后,gc-1細胞的凋亡率、caspase-3酶活性和凋亡相關蛋白parp、cleaved-caspase-3、bcl-xl和bax的表達變化情況;8.分別提取過表達組和空載體組細胞胞漿總蛋白和核蛋白,并采用westernblotting檢測p65蛋白的亞細胞定位。結果:1.pcr擴增、雙酶切分析及dna序列測定結果均表明真核表達重組質粒pmscvpuro-ankrd49-flag構建成功;2.rt-pcr和westernblotting檢測結果顯示ankrd49基因在gc-1細胞中實現(xiàn)了穩(wěn)定過表達;3.hoechst33258染色結果顯示,ankrd49穩(wěn)定過表達組細胞的凋亡百分比為(0.03±0.01)×100%,明顯低于空載體組(0.23±0.05)×100%和裸細胞組(0.25±0.04)×100%,(p0.01);4.流式細胞術結果顯示過表達ankrd49組細胞的早期凋亡率為2.71%±0.50%,明顯低于空載體組11.66%±1.01%和裸細胞組11.31%±1.74%,過表達ankrd49組細胞的線粒體膜電位下降率為11.51%±1.53%,明顯低于空載體組和裸細胞組(30.54%±2.42%和28.99%±1.61%),(p0.01);5.caspase-3酶活性實驗結果顯示過表達ankrd49組細胞caspase-3酶活性為1.09±0.15,明顯低于空載體組(3.01±0.32)和裸細胞組(3.12±0.23),(p0.01);6.westernblotting檢測結果顯示ankrd49穩(wěn)定過表達組cleaved-parp、cleaved-caspase-3蛋白的表達水平明顯低于對照組,而bcl-xl蛋白表達水平顯著高于對照組;7.過表達ankrd49的gc-1細胞中加入nf-κb信號通路抑制劑(pdtc)后,經(jīng)過流式細胞術檢測表明pdtc處理組細胞早期凋亡率明顯高于pdtc未處理組(p0.01),caspase-3酶活性實驗結果顯示pdtc處理組caspase-3酶活性明顯升高(p0.01),westernblotting檢測結果顯示pdtc處理組細胞內cleaved-parp、Cleaved-Caspase-3蛋白的表達水平明顯增高,而Bcl-x L蛋白表達水平明顯降低(P0.05);8.提取空載體組和過表達組細胞胞漿和核蛋白進行Western blotting檢測,結果表明ANKRD49可以促進p65蛋白入核(P0.01)。結論:1.成功構建了ankrd49的真核表達重組質粒,并建立ankrd49穩(wěn)定過表達GC-1細胞模型。2.ANKRD49蛋白對UV誘導GC-1細胞凋亡具有抑制作用。3.ANKRD49蛋白通過激活NF-κB信號通路抑制GC-1細胞凋亡。
[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.

【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R698.2

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