【摘要】：目的:無(wú)精子癥和精原細(xì)胞瘤均可以伴隨精子發(fā)生障礙,是男性不育的重要原因。導(dǎo)致精子發(fā)生障礙的病因中,除生殖細(xì)胞本身的問(wèn)題外,睪丸精子發(fā)生微環(huán)境的異常也很常見(jiàn)。Sertoli細(xì)胞是精子發(fā)生微環(huán)境的關(guān)鍵組成成分。近年發(fā)現(xiàn),Sertoli細(xì)胞在精子發(fā)生異常患者中表現(xiàn)出細(xì)胞數(shù)量、形態(tài)、成熟度和生物學(xué)功能的改變。因此,研究影響Sertoli細(xì)胞生長(zhǎng)及其功能的因子已成為當(dāng)前的一個(gè)重點(diǎn)。BMP4在Sertoli細(xì)胞有大量的表達(dá),對(duì)精原細(xì)胞增殖和分化起重要作用,但其對(duì)Sertoli細(xì)胞的功能及其作用機(jī)制目前尚無(wú)報(bào)道。本學(xué)位論文首次研究了BMP4在人睪丸Sertoli細(xì)胞的表達(dá)、功能、作用機(jī)制及其與非梗阻性無(wú)精子癥和精原細(xì)胞瘤的相關(guān)性。方法:我們首先利用逆轉(zhuǎn)錄PCR(RT-PCR)、免疫印跡(Western blots)、免疫細(xì)胞化學(xué)(Immunocytochemistry)、免疫組織化學(xué)(Immunohistochemistry)等方法分別從基因和蛋白質(zhì)水平在人睪丸組織和分選的Sertoli細(xì)胞中檢測(cè)BMP4及其受體的表達(dá)。然后外源添加BMP4或其拮抗劑noggin,或通過(guò)轉(zhuǎn)染BMP4的小分子干擾RNA(si RNA)后,結(jié)合細(xì)胞增殖(CCK-8)和Brd U滲入等實(shí)驗(yàn)方法,檢測(cè)其對(duì)人睪丸Sertoli細(xì)胞的數(shù)量和生物學(xué)功能的作用及其機(jī)制。最后,我們用組織芯片檢測(cè)不同類(lèi)型無(wú)精子癥和精原細(xì)胞瘤患者的睪丸生精小管BMP4的表達(dá)水平。本研究采用χ2檢驗(yàn)方法統(tǒng)計(jì)所獲得的數(shù)據(jù),采用t檢驗(yàn)進(jìn)行單因素的比較,設(shè)定P0.05具有統(tǒng)計(jì)學(xué)差異。實(shí)驗(yàn)結(jié)果采用Prism GraphPad5作圖。結(jié)果:研究發(fā)現(xiàn),BMP4和其三個(gè)常見(jiàn)受體,即ALK3、ALK6和BMPRII,在人睪丸Sertoli細(xì)胞中均有表達(dá),提示BMP4可能對(duì)人Sertoli細(xì)胞起作用。CCK-8細(xì)胞增殖檢測(cè)、BrdU滲入實(shí)驗(yàn)、PCNA免疫細(xì)胞化學(xué)染色等實(shí)驗(yàn)的結(jié)果表明,BMP4促進(jìn)人Sertoli細(xì)胞的DNA合成和細(xì)胞增殖。相反,在體外培養(yǎng)的人Sertoli細(xì)胞中外源性加入BMP4的拮抗劑noggin,或者對(duì)Sertoli細(xì)胞內(nèi)源性BMP4合成進(jìn)行siRNA干擾,發(fā)現(xiàn)人Sertoli細(xì)胞的分裂和增殖減慢。并且,siRNA干擾降低BMP4表達(dá)后在轉(zhuǎn)錄水平發(fā)現(xiàn)一些因子,如FGF2、SCF、claudin 11和ZO-1的表達(dá)均有下調(diào)。而且,我們檢測(cè)了BMP4作用的信號(hào)通路,包括Smad1/5通路、PI3K/AKT和ERK通路在人睪丸Sertoli細(xì)胞的變化。我們發(fā)現(xiàn),BMP4通過(guò)激活Smad1/5通路,上調(diào)ID2和ID3表達(dá),并降低p27Kip1表達(dá)對(duì)人Sertoli細(xì)胞發(fā)揮作用;BMP4并不激活人Sertoli細(xì)胞的PI3K/AKT和ERK通路。值得注意的是,睪丸組織芯片的免疫組化染色發(fā)現(xiàn),與梗阻性無(wú)精子癥(有正常精子發(fā)生)相比,BMP4在包括唯支持細(xì)胞綜合征、精子成熟停滯于精原細(xì)胞或精母細(xì)胞階段在內(nèi)的非梗阻性無(wú)精子癥睪丸組織中表達(dá)增高,而在人精原細(xì)胞瘤的睪丸組織不表達(dá)。結(jié)論:該研究首次發(fā)現(xiàn)BMP4通過(guò)激活Smad1/5通路,上調(diào)ID2和ID3,降低p27Kip1調(diào)節(jié)人睪丸Sertoli細(xì)胞的生長(zhǎng)和生物學(xué)功能,并且,BMP4在非梗阻性無(wú)精子癥患者和精原細(xì)胞瘤的睪丸中異常表達(dá)。由此推測(cè),異常表達(dá)的BMP4可能通過(guò)破壞男性睪丸微環(huán)境平衡而引起男性精子發(fā)生障礙和精原細(xì)胞瘤,從而為男性不育病因?qū)W提供理論依據(jù)。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R698.2

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