發(fā)布時間：2018-05-14 17:28

  本文選題：spata3 + 精子發(fā)生��； 參考：《山西醫(yī)科大學》2017年碩士論文

【摘要】：目的:明確spata3基因m RNA及其編碼蛋白產(chǎn)物在小鼠睪丸組織中的特異表達特性;運用分子克隆技術構建spata3的真核表達質粒并建立spata3過表達HEK293T細胞模型;分析spata3過表達HEK293T細胞中細胞凋亡和細胞自噬相關蛋白的表達;進一步構建spata3過表達慢病毒載體并建立穩(wěn)定表達GC2-spd細胞模型;分析spata3穩(wěn)定表達GC2-spd細胞中細胞自噬相關蛋白的表達;為進一步探究在精子發(fā)生過程中spata3所發(fā)揮的的作用及意義奠定基礎。方法:1.采用RT-PCR分析spata3基因m RNA在小鼠各組織中的表達;2.利用Western blotting分析SPATA3蛋白在小鼠各組織中的表達;3.應用免疫組織化學染色檢測SPATA3蛋白在小鼠睪丸組織中的細胞定位;4.通過間接免疫熒光染色觀察SPATA3蛋白在小鼠生精細胞中的定位分布;5.利用分子克隆技術構建spata3真核表達重組質粒p LV-EGFP(2A)Purospata3;6.采用RT-PCR和Western blotting分析重組質粒p LV-EGFP(2A)Puro-spata3轉染HEK293T細胞后spata3 m RNA和蛋白的表達水平;7.采用間接免疫熒光染色檢測SPATA3蛋白在過表達HEK293T細胞中的定位分布;8.采用Western blotting檢測細胞凋亡相關蛋白Caspase3、PARP、Bax和Bcl-2以及細胞自噬相關蛋白Lc3A/B的表達水平;9.過表達慢病毒載體瞬時感染HEK293T細胞;采用RT-PCR和Western blotting檢測spata3在HEK293T細胞中的瞬時表達;10.建立穩(wěn)定表達spata3的GC2-spd細胞模型;采用RT-PCR和Western blotting檢測spata3在GC2-spd細胞中的穩(wěn)定表達;11.采用Western blotting檢測自噬相關蛋白Lc3A/B在spata3穩(wěn)定表達GC2-spd細胞中的表達水平。12.采用間接免疫熒光染色檢測自噬相關蛋白Lc3A/B在spata3穩(wěn)定表達GC2-spd細胞中的分布。結果:1.RT-PCR結果顯示spata3基因m RNA在小鼠睪丸組織特異表達;2.Western blotting結果顯示SPATA3蛋白在小鼠睪丸組織特異表達;3.免疫組織化學染色結果表明SPATA3蛋白主要定位在曲精小管內(nèi)圓形精子細胞、粗線期精母細胞和長形精子細胞中,間質細胞、支持細胞、精原細胞、前細線期精母細胞中未見SPATA3明顯陽性信號;4.免疫熒光分析顯示粗線期精母細胞和圓形精子細胞的胞漿與胞核均有顯著SPATA3蛋白的陽性著色,長形精子細胞的胞漿也有大量分布;5.DNA測序結果表明重組質粒p LV-EGFP(2A)Puro-spata3構建成功;6.重組質粒轉染HEK293T細胞后,經(jīng)RT-PCR和Western blotting檢測表明spata3基因在HEK293T內(nèi)過表達;7.細胞免疫熒光染色分析顯示在HEK293T細胞內(nèi)過表達的SPATA3蛋白分布廣泛,但核周熒光信號明顯強于胞核;8.Western blotting結果表明過表達spata3的293T細胞中細胞凋亡相關蛋白及自噬相關蛋白Caspase3、PARP無明顯變化,Bax、Lc3A/B蛋白水平高于對照組(P0.01),而Bcl-2蛋白含量低于對照組(P0.01),差異具有統(tǒng)計學意義;9.RT-PCR和Western blotting結果表明spata3慢病毒載體在HEK293T細胞中過表達,表明慢病毒載體構建成功;10.RT-PCR和Western blotting結果表明spata3慢病毒載體在GC2-spd細胞中過表達,spata3過表達GC2-spd穩(wěn)定細胞模型構建成功;11.Western blotting結果表明過表達spata3的GC2-spd穩(wěn)定細胞中自噬相關蛋白Lc3A/B蛋白含量高于對照組(P0.01),差異具有統(tǒng)計學意義。12.細胞免疫熒光染色分析顯示過表達spata3的GC2-spd穩(wěn)定細胞中自噬相關蛋白Lc3A/B熒光信號在核周呈點狀廣泛分布;結論:1.spata3基因在小鼠睪丸組織大量表達,并具有生精細胞定位特異性。2.成功建立了spata3穩(wěn)定過表達GC2-spd細胞模型。3.spata3基因過表達具有促進HEK293T細胞自噬的作用,而與細胞凋亡沒有直接關系。spata3基因過表達能夠促進GC2-spd細胞自噬,可能與哺乳類動物精子發(fā)生期間的生精細胞自噬密切相關。
[Abstract]:Objective: to clarify the specific expression characteristics of spata3 gene m RNA and its encoded protein products in mouse testicular tissue; construct eukaryotic expression plasmid of spata3 by molecular cloning technique and establish spata3 overexpressed HEK293T cell model; analyze the expression of cell apoptosis and autophagy related protein expression in spata3 overexpressed HEK293T cells; further construct the expression of cell autophagy related proteins in HEK293T cells of spata3 overexpression; Spata3 overexpressed lentivirus vector and established a stable expression of GC2-spd cell model, and analyzed the expression of autophagy related protein in GC2-spd cells by spata3, and laid the foundation for further exploring the role and significance of spata3 in the process of spermatogenesis. Method: 1. RT-PCR analysis of spata3 gene m RNA in mice in each group Expression in the fabric; 2. Western blotting was used to analyze the expression of SPATA3 protein in various tissues of mice; 3. the localization of SPATA3 protein in mouse testis tissue was detected by immunohistochemical staining; 4. by indirect immunofluorescence staining, the location and distribution of SPATA3 protein in the murine fine cells; and 5. by molecular cloning technology. The recombinant plasmid P LV-EGFP (2A) Purospata3 was expressed by spata3; 6. the expression level of the recombinant plasmid P LV-EGFP (2A) was analyzed by RT-PCR and Western blotting, and the expression level of the protein in the cells was detected by indirect immunofluorescence staining; and 8. was used to detect the distribution of the protein in the overexpressed cells. N blotting detected the expression of apoptosis related proteins Caspase3, PARP, Bax and Bcl-2, and the expression level of autophagy related protein Lc3A/B; 9. over expressed lentivirus vectors were transient infection of HEK293T cells; RT-PCR and Western blotting were used to detect the instantaneous expression of spata3 in the cells; and 10. to establish a stable cell model for stable expression. RT-PCR and Western blotting were used to detect the stable expression of spata3 in GC2-spd cells; 11. the expression level of autophagy related protein Lc3A/B in spata3 stable expression GC2-spd cells was detected by Western blotting, and indirect immunofluorescence staining was used to detect the distribution of autophagy associated protein Lc3A/B in the stable expression of cells. Fruit: 1.RT-PCR results showed that the spata3 gene m RNA was specifically expressed in mouse testicular tissue; 2.Western blotting results showed that the SPATA3 protein was expressed specifically in the mouse testis tissue; 3. the results of immunohistochemical staining showed that SPATA3 protein was mainly located in the round spermatocyte in the tubuloseminiferous, the roughage spermatocyte and the long spermatocyte. The SPATA3 positive signals were not found in the cytoplasm, the supporting cells, the spermatogonial cells and the pre fine line spermatocytes; 4. the immunofluorescence analysis showed that the cytoplasm and the nucleus of the rounded spermatocytes and round spermatocytes were positive for SPATA3 protein, and the cytoplasm of the elongated spermatocytes also had a large number of distribution; the 5.DNA sequencing results showed that the recombinant plasmids were recombinant. P LV-EGFP (2A) Puro-spata3 was constructed successfully; 6. after transfection of recombinant plasmid to HEK293T cells, RT-PCR and Western blotting detection showed that the spata3 gene was overexpressed in HEK293T; the 7. cell immunofluorescence staining analysis showed that the SPATA3 protein overexpressed in the HEK293T cells was widely distributed, but the fluorescence signal of the nuclear peri was stronger than the nucleus. The results of tting showed that the apoptosis related protein and autophagy related protein Caspase3, PARP in the 293T cells expressing spata3 had no significant change, Bax, Lc3A/B protein level was higher than that of the control group (P0.01), and the content of Bcl-2 protein was lower than that of the control group (P0.01), and the difference was statistically significant. 9.RT-PCR and Western results showed that the lentivirus carrier was the carrier. The overexpression in HEK293T cells showed that the lentivirus vector was constructed successfully, and the results of 10.RT-PCR and Western blotting showed that the spata3 lentivirus vector was overexpressed in GC2-spd cells, and the spata3 overexpressed GC2-spd stable cell model construction successfully; 11.Western blotting results showed that the autophagy related protein in the spata3 GC2-spd stable cells expressed the spata3. The content of /B protein was higher than that of the control group (P0.01), and the difference was statistically significant. The.12. cell immunofluorescence staining analysis showed that the autophagy related protein Lc3A/B fluorescent signal in the GC2-spd stable cells expressed spata3 was widely distributed in the nuclear week, and the conclusion: the 1.spata3 gene was expressed in a large number of mouse testis and was specific for the localization of spermatogenic cells. Sex.2. has successfully established the spata3 stable overexpression GC2-spd cell model.3.spata3 gene overexpression to promote the autophagy of HEK293T cells, but not directly related to cell apoptosis, the overexpression of.Spata3 gene can promote autophagy of GC2-spd cells, which may be closely related to the autophagy of spermatogenic cells during the spermatogenesis of mammalian animals.

【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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