spata3基因参与小鼠生殖细胞发育及其初步功能研究

发布时间：2018-05-14 17:28

  本文选题：spata3 + 精子发生　； 参考：《山西医科大学》2017年硕士论文

【摘要】：目的:明确spata3基因m RNA及其编码蛋白产物在小鼠睾丸组织中的特异表达特性;运用分子克隆技术构建spata3的真核表达质粒并建立spata3过表达HEK293T细胞模型;分析spata3过表达HEK293T细胞中细胞凋亡和细胞自噬相关蛋白的表达;进一步构建spata3过表达慢病毒载体并建立稳定表达GC2-spd细胞模型;分析spata3稳定表达GC2-spd细胞中细胞自噬相关蛋白的表达;为进一步探究在精子发生过程中spata3所发挥的的作用及意义奠定基础。方法:1.采用RT-PCR分析spata3基因m RNA在小鼠各组织中的表达;2.利用Western blotting分析SPATA3蛋白在小鼠各组织中的表达;3.应用免疫组织化学染色检测SPATA3蛋白在小鼠睾丸组织中的细胞定位;4.通过间接免疫荧光染色观察SPATA3蛋白在小鼠生精细胞中的定位分布;5.利用分子克隆技术构建spata3真核表达重组质粒p LV-EGFP(2A)Purospata3;6.采用RT-PCR和Western blotting分析重组质粒p LV-EGFP(2A)Puro-spata3转染HEK293T细胞后spata3 m RNA和蛋白的表达水平;7.采用间接免疫荧光染色检测SPATA3蛋白在过表达HEK293T细胞中的定位分布;8.采用Western blotting检测细胞凋亡相关蛋白Caspase3、PARP、Bax和Bcl-2以及细胞自噬相关蛋白Lc3A/B的表达水平;9.过表达慢病毒载体瞬时感染HEK293T细胞;采用RT-PCR和Western blotting检测spata3在HEK293T细胞中的瞬时表达;10.建立稳定表达spata3的GC2-spd细胞模型;采用RT-PCR和Western blotting检测spata3在GC2-spd细胞中的稳定表达;11.采用Western blotting检测自噬相关蛋白Lc3A/B在spata3稳定表达GC2-spd细胞中的表达水平。12.采用间接免疫荧光染色检测自噬相关蛋白Lc3A/B在spata3稳定表达GC2-spd细胞中的分布。结果:1.RT-PCR结果显示spata3基因m RNA在小鼠睾丸组织特异表达;2.Western blotting结果显示SPATA3蛋白在小鼠睾丸组织特异表达;3.免疫组织化学染色结果表明SPATA3蛋白主要定位在曲精小管内圆形精子细胞、粗线期精母细胞和长形精子细胞中,间质细胞、支持细胞、精原细胞、前细线期精母细胞中未见SPATA3明显阳性信号;4.免疫荧光分析显示粗线期精母细胞和圆形精子细胞的胞浆与胞核均有显著SPATA3蛋白的阳性着色,长形精子细胞的胞浆也有大量分布;5.DNA测序结果表明重组质粒p LV-EGFP(2A)Puro-spata3构建成功;6.重组质粒转染HEK293T细胞后,经RT-PCR和Western blotting检测表明spata3基因在HEK293T内过表达;7.细胞免疫荧光染色分析显示在HEK293T细胞内过表达的SPATA3蛋白分布广泛,但核周荧光信号明显强于胞核;8.Western blotting结果表明过表达spata3的293T细胞中细胞凋亡相关蛋白及自噬相关蛋白Caspase3、PARP无明显变化,Bax、Lc3A/B蛋白水平高于对照组(P0.01),而Bcl-2蛋白含量低于对照组(P0.01),差异具有统计学意义;9.RT-PCR和Western blotting结果表明spata3慢病毒载体在HEK293T细胞中过表达,表明慢病毒载体构建成功;10.RT-PCR和Western blotting结果表明spata3慢病毒载体在GC2-spd细胞中过表达,spata3过表达GC2-spd稳定细胞模型构建成功;11.Western blotting结果表明过表达spata3的GC2-spd稳定细胞中自噬相关蛋白Lc3A/B蛋白含量高于对照组(P0.01),差异具有统计学意义。12.细胞免疫荧光染色分析显示过表达spata3的GC2-spd稳定细胞中自噬相关蛋白Lc3A/B荧光信号在核周呈点状广泛分布;结论:1.spata3基因在小鼠睾丸组织大量表达,并具有生精细胞定位特异性。2.成功建立了spata3稳定过表达GC2-spd细胞模型。3.spata3基因过表达具有促进HEK293T细胞自噬的作用,而与细胞凋亡没有直接关系。spata3基因过表达能够促进GC2-spd细胞自噬,可能与哺乳类动物精子发生期间的生精细胞自噬密切相关。
[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.

【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

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