BMP4在人睾丸Sertoli细胞的表达、功能、机制及与无精子症和精原细胞瘤的相关性研究

发布时间：2018-07-31 19:42

【摘要】：目的:无精子症和精原细胞瘤均可以伴随精子发生障碍,是男性不育的重要原因。导致精子发生障碍的病因中,除生殖细胞本身的问题外,睾丸精子发生微环境的异常也很常见。Sertoli细胞是精子发生微环境的关键组成成分。近年发现,Sertoli细胞在精子发生异常患者中表现出细胞数量、形态、成熟度和生物学功能的改变。因此,研究影响Sertoli细胞生长及其功能的因子已成为当前的一个重点。BMP4在Sertoli细胞有大量的表达,对精原细胞增殖和分化起重要作用,但其对Sertoli细胞的功能及其作用机制目前尚无报道。本学位论文首次研究了BMP4在人睾丸Sertoli细胞的表达、功能、作用机制及其与非梗阻性无精子症和精原细胞瘤的相关性。方法:我们首先利用逆转录PCR(RT-PCR)、免疫印迹(Western blots)、免疫细胞化学(Immunocytochemistry)、免疫组织化学(Immunohistochemistry)等方法分别从基因和蛋白质水平在人睾丸组织和分选的Sertoli细胞中检测BMP4及其受体的表达。然后外源添加BMP4或其拮抗剂noggin,或通过转染BMP4的小分子干扰RNA(si RNA)后,结合细胞增殖(CCK-8)和Brd U渗入等实验方法,检测其对人睾丸Sertoli细胞的数量和生物学功能的作用及其机制。最后,我们用组织芯片检测不同类型无精子症和精原细胞瘤患者的睾丸生精小管BMP4的表达水平。本研究采用χ2检验方法统计所获得的数据,采用t检验进行单因素的比较,设定P0.05具有统计学差异。实验结果采用Prism GraphPad5作图。结果:研究发现,BMP4和其三个常见受体,即ALK3、ALK6和BMPRII,在人睾丸Sertoli细胞中均有表达,提示BMP4可能对人Sertoli细胞起作用。CCK-8细胞增殖检测、BrdU渗入实验、PCNA免疫细胞化学染色等实验的结果表明,BMP4促进人Sertoli细胞的DNA合成和细胞增殖。相反,在体外培养的人Sertoli细胞中外源性加入BMP4的拮抗剂noggin,或者对Sertoli细胞内源性BMP4合成进行siRNA干扰,发现人Sertoli细胞的分裂和增殖减慢。并且,siRNA干扰降低BMP4表达后在转录水平发现一些因子,如FGF2、SCF、claudin 11和ZO-1的表达均有下调。而且,我们检测了BMP4作用的信号通路,包括Smad1/5通路、PI3K/AKT和ERK通路在人睾丸Sertoli细胞的变化。我们发现,BMP4通过激活Smad1/5通路,上调ID2和ID3表达,并降低p27Kip1表达对人Sertoli细胞发挥作用;BMP4并不激活人Sertoli细胞的PI3K/AKT和ERK通路。值得注意的是,睾丸组织芯片的免疫组化染色发现,与梗阻性无精子症(有正常精子发生)相比,BMP4在包括唯支持细胞综合征、精子成熟停滞于精原细胞或精母细胞阶段在内的非梗阻性无精子症睾丸组织中表达增高,而在人精原细胞瘤的睾丸组织不表达。结论:该研究首次发现BMP4通过激活Smad1/5通路,上调ID2和ID3,降低p27Kip1调节人睾丸Sertoli细胞的生长和生物学功能,并且,BMP4在非梗阻性无精子症患者和精原细胞瘤的睾丸中异常表达。由此推测,异常表达的BMP4可能通过破坏男性睾丸微环境平衡而引起男性精子发生障碍和精原细胞瘤,从而为男性不育病因学提供理论依据。
[Abstract]:Objective: azoospermia and spermatogonial tumor can be accompanied by spermatogenesis, which is an important cause of male infertility. In the cause of spermatogenesis disorder, except for the problems of the germ cell itself, the abnormal microenvironment of spermatogenesis is also a key component of the sperm microenvironment. In recent years, the.Sertoli cell is found to be a key component of the spermatogenic microenvironment. Serto Li cells show the changes in cell number, morphology, maturity and biological function in patients with abnormal spermatogenesis. Therefore, the study of factors affecting the growth and function of Sertoli cells has become a key.BMP4 in Sertoli cells and plays an important role in the cell proliferation and differentiation of spermatogonial cells, but it is fine to Sertoli. The function and mechanism of the cell are not yet reported. This dissertation first studies the expression, function, mechanism of BMP4 in human testicular Sertoli cells and its correlation with non obstructive azoospermia and spermatogonial tumor. Methods: we first use reverse transcription PCR (RT-PCR), Western blots (Western blots), immunocytochemistry (Immun) Ocytochemistry), immuno histochemistry (Immunohistochemistry) and other methods, respectively, detect the expression of BMP4 and its receptor in the human testicular tissue and the selected Sertoli cells from the gene and protein level. Then add BMP4 or its antagonist noggin, or interfere with RNA (Si RNA) through the small molecules transfected with BMP4, and combine cell proliferation (CCK-8). The effect and mechanism of Brd U infiltration on the number and biological function of human testicular Sertoli cells were detected. Finally, we detected the expression level of BMP4 in the testicular seminiferous tubules of the patients with different types of azoospermia and spermatogonial tumor by tissue chip. The data obtained by the chi 2 test were used in this study, and t was used. The results of a single factor comparison were statistically different. The results of P0.05 were statistically different. The results of the experiment were Prism GraphPad5. Results: the results showed that BMP4 and its three common receptors, namely, ALK3, ALK6 and BMPRII, were expressed in the human testicular Sertoli cells, suggesting that BMP4 may play a role in the detection of.CCK-8 cell proliferation in the human Sertoli cells, and BrdU infiltrate into reality. The results of PCNA immunocytochemical staining showed that BMP4 promoted the DNA synthesis and cell proliferation of human Sertoli cells. On the contrary, the BMP4 antagonist noggin was added to the human Sertoli cells in vitro, or the endogenous BMP4 synthesis in Sertoli cells was interfered with siRNA, and the division and proliferation of human Sertoli cells were found to be slowed down. Furthermore, the expression of FGF2, SCF, claudin 11 and ZO-1 were downregulated at the transcriptional level after the siRNA interference reduced BMP4 expression. Furthermore, we detected the signaling pathway of the BMP4 action, including the Smad1/5 pathway, the PI3K/AKT and ERK pathways in the human testicular Sertoli cells. And ID3 expression, and reduce p27Kip1 expression to the human Sertoli cells; BMP4 does not activate the PI3K/AKT and ERK pathways of human Sertoli cells. It is worth noting that the immunohistochemical staining of the testicular tissue microarray shows that BMP4 is included in the only support cell syndrome, and the sperm maturation is stagnant compared with the obstructive azoospermia (normal spermatogenesis). The expression in the testicular tissue of non obstructive azoospermia in spermatogonial cell or spermatogonial stage is higher, but it is not expressed in the testicular tissue of human spermatogonial tumor. Conclusion: This study was the first to find that BMP4 regulates the growth and biological function of Sertoli cells in human testis pill by activating the Smad1/5 pathway and reducing the growth and biological function of p27Kip1 in the Sertoli cells of human testis pill, and BMP 4 in the testicles of non obstructive azoospermia patients and spermatogonial tumors, the abnormal expression of BMP4 may result in male spermatogenesis and spermatogonial tumor by destroying the balance of male testicular microenvironment, which provides a theoretical basis for male infertility etiology.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R698.2

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