本文關(guān)鍵詞：應(yīng)用siRNA沉默共培養(yǎng)生精細(xì)胞uPAR基因表達(dá)的研究　出處：《華中科技大學(xué)》2007年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 大鼠 精子發(fā)生 uPAR 實(shí)時(shí)熒光定量聚合酶鏈反應(yīng) 大鼠 睪丸支持細(xì)胞 培養(yǎng) 原位雜交 鑒定 大鼠 生精細(xì)胞 支持細(xì)胞 共培養(yǎng) 大鼠 生精細(xì)胞 共培養(yǎng) siRNA RNAi

【摘要】： 精子發(fā)生是個(gè)復(fù)雜、多步驟的過程,可分為三個(gè)過程:精原細(xì)胞的增殖更新、精母細(xì)胞的成熟分裂和精子細(xì)胞變態(tài)為精子三個(gè)階段。目前發(fā)現(xiàn)在曲細(xì)精管中uPAR主要表達(dá)在精子細(xì)胞,可能與精子的排放有關(guān)。Tong Zhang等應(yīng)用原位雜交技術(shù)檢測(cè)發(fā)現(xiàn)獼猴睪丸支持細(xì)胞在精子發(fā)生的特定階段(VII～VIII期)表達(dá)uPA mRNA;uPAR mRNA由睪丸生精細(xì)胞產(chǎn)生,但是不同發(fā)育階段及不同生殖細(xì)胞表達(dá)的差異表明uPA/uPAR系統(tǒng)可能在精子發(fā)生過程中的特定階段起作用。Vassalli認(rèn)為支持細(xì)胞這種時(shí)期特異性表達(dá)的uPA與生殖細(xì)胞uPAR結(jié)合后可能是通過激活uPAR介導(dǎo)的信號(hào)傳導(dǎo)通路作為生長(zhǎng)因子而起作用。但是uPAR是通過激活何種信號(hào)傳導(dǎo)通路來(lái)調(diào)節(jié)精子的發(fā)生過程目前尚不清楚。uPAR是否還通過信號(hào)傳導(dǎo)來(lái)刺激生精細(xì)胞的發(fā)生及分化功能呢?因此,uPAR在精子發(fā)生中的作用機(jī)制尚待進(jìn)一步研究。 然而由于睪丸組織結(jié)構(gòu)和功能的復(fù)雜性,一般情況下很難在整體情況下對(duì)生殖細(xì)胞的生理生化功能和與其他細(xì)胞的相互作用進(jìn)行深入研究。生精細(xì)胞培養(yǎng)技術(shù)的建立為深入地了解生精細(xì)胞發(fā)育過程,研究生精細(xì)胞自身及與其它生精細(xì)胞的關(guān)系,提供了一種新的、強(qiáng)有力的工具。RNA干擾(RNAi)是一種由雙鏈小干擾RNA(siRNA)引發(fā)的轉(zhuǎn)錄后基因沉默(PTGS)新型機(jī)制,可導(dǎo)致靶基因mRNA的降解及特異性基因沉默信號(hào)的擴(kuò)增。目前siRNA已經(jīng)成為選擇性沉默體外培養(yǎng)的哺乳細(xì)胞基因表達(dá)的一個(gè)有力工具。 本實(shí)驗(yàn)首先應(yīng)用實(shí)時(shí)RT-PCR和Western Blot檢測(cè)出生后不同發(fā)育階段大鼠睪丸中uPAR基因及蛋白表達(dá)的變化,然后建立的生精細(xì)胞體外培養(yǎng)體系,最后應(yīng)用siRNA沉默技術(shù)阻斷體外培養(yǎng)生精細(xì)胞uPAR的基因表達(dá),為研究uPAR在精子發(fā)生中作用機(jī)制及信號(hào)傳導(dǎo)通路的研究提供新的重要研究手段和技術(shù)平臺(tái)。 一、uPAR在大鼠睪丸第一精子發(fā)生波中的表達(dá)變化 目的:為探討uPAR在大鼠精子發(fā)生中的作用,研究在第一個(gè)精子發(fā)生波中大鼠睪丸組織uPAR的mRNA表達(dá)及蛋白表達(dá)變化。方法:將SD大鼠按生后年齡分組,以出生當(dāng)天為d0,取出生后d0、d5、d10、d15、d21、d28、d35、d42、d49、d56大鼠睪丸組織,用實(shí)時(shí)熒光定量分析方法檢測(cè)各年齡組大鼠uPAR mRNA表達(dá),并用Western印跡法檢測(cè)各年齡組大鼠uPAR蛋白表達(dá)。結(jié)果:大鼠睪丸組織uPAR mRNA表達(dá)與蛋白表達(dá)表現(xiàn)出相似的趨勢(shì):剛出生時(shí)表達(dá)水平較高,后逐漸下降,約在d15天達(dá)最低,到d28時(shí)開始增加,到d35時(shí)到達(dá)高峰,隨后d42下降,后保持一定的水平。雙變量回歸相關(guān)分析顯示uPAR mRNA表達(dá)與蛋白表達(dá)為中度正相關(guān)。結(jié)論:大鼠睪丸uPAR表達(dá)在第一精子發(fā)生波中出現(xiàn)兩個(gè)高峰,第一高峰在剛出生時(shí),這個(gè)時(shí)期生殖母細(xì)胞向基底部遷移的時(shí)期,這在精子以后的發(fā)生中起著至關(guān)重要的作用,說(shuō)明uPAR與精子發(fā)生的啟動(dòng)有著密切聯(lián)系。第二高峰是在第5周升高明顯,第5周為精子變形并開始向管腔排放的時(shí)期,這說(shuō)明uPAR可能參與精子排放過程中的組織重塑及精子變態(tài)過程。 二、大鼠睪丸生殖細(xì)胞培養(yǎng)體系的建立 大鼠睪丸生殖細(xì)胞培養(yǎng)體系的建立是體外研究睪丸精子發(fā)生過程中各種調(diào)控因素的的重要工具。本實(shí)驗(yàn)通過建立支持細(xì)胞及生精細(xì)胞兩種培養(yǎng)體系,為進(jìn)一步研究uPAR在精子發(fā)生中的作用奠定基礎(chǔ)。 (一)大鼠睪丸支持細(xì)胞的分離純化和鑒定 目的培養(yǎng)高純度的大鼠睪丸支持細(xì)胞,并應(yīng)用檢測(cè)ABP mRNA原位雜交方法鑒定分離培養(yǎng)的支持細(xì)胞。方法選用18～22天齡SD雄性大鼠睪丸,采用0.25%胰蛋白酶、0.1%透明質(zhì)酸酶、0.1%膠原酶三酶依次消化法分離支持細(xì)胞,放于32℃5%CO2的培養(yǎng)箱培養(yǎng),48小時(shí)后用20mmol Tris—HCl低滲處理培養(yǎng)細(xì)胞。培養(yǎng)一周后應(yīng)用伊紅染色、吖啶橙熒光染色、Feulgen染色對(duì)所培養(yǎng)支持細(xì)胞進(jìn)行鑒定,同時(shí)應(yīng)用原位雜交檢測(cè)ABP mRNA方法鑒定分離培養(yǎng)的支持細(xì)胞。結(jié)果培養(yǎng)一周后所獲培養(yǎng)的支持細(xì)胞純度達(dá)95%以上。分離培養(yǎng)的支持細(xì)胞ABP mRNA表達(dá)陽(yáng)性,其形態(tài)結(jié)構(gòu)特征與用其它方法鑒定為支持細(xì)胞的形態(tài)結(jié)構(gòu)特征一致。結(jié)論采用三酶連續(xù)消化及低滲處理法分離培養(yǎng)的支持細(xì)胞純度高,而且應(yīng)用原位雜交方法檢測(cè)ABP mRNA是一種新的、特異、有效的鑒定支持細(xì)胞及其功能的方法。 (二)大鼠睪丸支持細(xì)胞/生精細(xì)胞共培養(yǎng)系統(tǒng)的建立 目的建立生精細(xì)胞體外分化培養(yǎng)體系,為uPAR在精子發(fā)生中作用的體外研究提供一個(gè)很好的研究模型。方法取20～22天齡SD雄性大鼠睪丸,去被膜及血管,放在預(yù)冷的Hank’s液中洗兩次,然后用加1 mg/ml膠原酶的F12/DMEM液32℃消化15～20min。用眼科剪將睪丸剪碎成非常小的片斷,再重復(fù)用1 mg/ml膠原酶消化5～10min。細(xì)胞用內(nèi)含10%胎牛血清及各種營(yíng)養(yǎng)因子的HamF12/ DMEM培養(yǎng)液懸浮,約按1*106CELL/cm2的密度接種于雙室培養(yǎng)槽或放有蓋玻片的六孔板中,在32℃、5%CO2條件下培養(yǎng)。相差顯微鏡下動(dòng)態(tài)觀察細(xì)胞生長(zhǎng)、形態(tài)等變化,及定期應(yīng)用伊紅染色、Brdu染色法對(duì)所培養(yǎng)生精細(xì)胞/支持細(xì)胞進(jìn)行染色鑒定。結(jié)果在生精細(xì)胞/支持細(xì)胞共培養(yǎng)體系中,雙室培養(yǎng)2周后,部分生精細(xì)胞胞體一端有鞭毛出現(xiàn),培養(yǎng)4周后,培養(yǎng)體系中仍有一定數(shù)量的生精細(xì)胞附著在支持細(xì)胞上,部分生精細(xì)胞上可見鞭毛。單室培養(yǎng)的生精細(xì)胞在培養(yǎng)第4-5天開始出現(xiàn)明顯脫落,培養(yǎng)1周后大多數(shù)生精細(xì)胞發(fā)生脫落死亡,生精細(xì)胞上未見鞭毛出現(xiàn),但在培養(yǎng)體系中可見次級(jí)精母細(xì)胞及精子細(xì)胞。結(jié)論:應(yīng)用雙室培養(yǎng),生精細(xì)胞可存活達(dá)一月之久,而且部分生精細(xì)胞尾部出現(xiàn)鞭毛,從形態(tài)上發(fā)生了減數(shù)分裂及精子變態(tài)過程。 三、應(yīng)用siRNA沉默共培養(yǎng)生精細(xì)胞uPAR的基因表達(dá) 目的利用小分子干擾RNA(siRNA)技術(shù)沉默共培養(yǎng)生精細(xì)胞uPAR的基因表達(dá),為uPAR在精子發(fā)生中的作用研究提供一個(gè)很好的研究工具。方法siRNA混合雞尾酒是根據(jù)ShortCut RNAi Kit手冊(cè)來(lái)制備。簡(jiǎn)單的說(shuō),首先通過RT-PCR應(yīng)用加有T7啟動(dòng)子的特異性引物來(lái)合成DNA模板,然后DNA模板通過體外轉(zhuǎn)錄過程產(chǎn)生雙鏈RNA,最后通過ShortCut RNaseIII酶消化來(lái)制備siRNAs混合物。在培養(yǎng)48小時(shí)用Transfect轉(zhuǎn)染試劑分別將15nM、30nM siRNA混合物轉(zhuǎn)染到共培養(yǎng)的生精細(xì)胞,48小時(shí)應(yīng)用實(shí)時(shí)熒光定量RT-PCR方法檢測(cè)共培養(yǎng)生精細(xì)胞uPAR的基因表達(dá)。該試驗(yàn)設(shè)立兩個(gè)對(duì)照組:空白對(duì)照(未加siRNA及轉(zhuǎn)染試劑)及陰性對(duì)照組(只加轉(zhuǎn)染試劑,無(wú)siRNA)。結(jié)果15nM及30nM siRNA轉(zhuǎn)染組uPAR mRNA表達(dá)量均明顯低于陰性對(duì)照組及空白對(duì)照組(p0.05),其中以30nMsiRNA轉(zhuǎn)染組更為明顯;相對(duì)于陰性對(duì)照組,15nM及30nM siRNA轉(zhuǎn)染組uPAR的基因表達(dá)抑制率分別為63.5%及76.7%。結(jié)論應(yīng)用ShortCut RNase III法制備的siRNAs雞尾酒能有效地抑制共培養(yǎng)生精細(xì)胞uPAR基因的表達(dá)。 五、結(jié)論 在本研究中我們發(fā)現(xiàn)uPAR在第一精子發(fā)生波中表達(dá)最高峰為出生后35天,此時(shí)為圓形精子轉(zhuǎn)變?yōu)樯扉L(zhǎng)精子,并開始向管腔排放的時(shí)期。在我們的共培養(yǎng)體系中,來(lái)自20～22天大小的大鼠睪丸組織的生精細(xì)胞在培養(yǎng)二周后能分化成精子細(xì)胞及變形成為伸長(zhǎng)型精子。而這些變化相應(yīng)于體內(nèi)uPAR的表達(dá)出現(xiàn)高峰期所發(fā)生的變化。因此在本培養(yǎng)體系中,應(yīng)用siRNA沉默uPAR的表達(dá),可以為研究uPAR在精子發(fā)生特定時(shí)期中的作用及機(jī)制提供一個(gè)很好的研究工具,如其信號(hào)傳導(dǎo)通路等。此研究模型的建立也有助于減數(shù)分裂及精子變態(tài)過程的其它調(diào)控因素的研究。
[Abstract]:Spermatogenesis is a complex multistep process, can be divided into three processes: the proliferation of spermatogonia renewal, meiosis of spermatocytes and spermatid sperm three stages. Currently found in the seminiferous tubules of uPAR mainly expressed in sperm cells, sperm may detection and related to the emission of.Tong the application of Zhang in situ hybridization found in rhesus monkey Sertoli cells at specific stages of spermatogenesis (VII ~ VIII) expression of uPA mRNA; uPAR mRNA produced by spermatogenic cells, but in different developmental stages and different expression of germ cells showed that uPA/uPAR system may be in spermatogenesis specific stages in the course of action that.Vassalli combined with the support of specific expression of uPA in the period of the cells and germ cells after uPAR may be through the activation of uPAR mediated signal transduction pathways as growth factors play a role. But uPAR is. It is unclear whether any signal transduction pathway regulates spermatogenesis. It is not clear whether.UPAR can stimulate the function of spermatogenesis and differentiation through signal transduction. Therefore, the mechanism of uPAR in spermatogenesis is still to be further studied.
However, due to the complexity of the structure and function of testis tissue, it is difficult to study the interaction in the overall condition of physiological and biochemical functions of germ cells and other cells in the general case. Establish ofspermatogenic cell culture system for in-depth understanding of the process of spermatogenesis, spermatogenic cells and its relationship with other spermatogenic cells, provides a new, powerful tool of.RNA interference (RNAi) is a double stranded small interfering RNA (siRNA) caused by post transcriptional gene silencing (PTGS) mechanism, can lead to degradation of amplification of target gene mRNA and specific gene silencing signal. Now siRNA has become a powerful tool for the expression of genes in cultured mammalian cells in vitro selective silence.
The first change by real-time RT-PCR and Western Blot expression of uPAR in the testis of rats in different developmental stages of gene and protein detection after birth, then set up the training system of spermatogenic cells in vitro, finally the application of siRNA silencing in vitro blockade of spermatogenic cells uPAR gene expression of uPAR in spermatogenesis and mechanism research signal transduction pathways provide a new study method and technology platform.
Expression of uPAR in the first spermatogenesis wave of rat testis
Objective: To investigate the effect of uPAR in rat spermatogenesis in the role of the expression of mRNA and protein expression in the first uPAR of spermatogenesis in testis of rats in the wave. Methods: SD rats at postnatal age group, to the day of birth was d0, was d0, D5, D10, D15. D21, D28, D35, d42, d49, D56 in the testicular tissue of rats in each age group, expression of rat uPAR mRNA detection method using real-time fluorescence quantitative Western, and Western blot was used to detect the uPAR protein expression in rats with different age groups. Results: the expression of mRNA in rat testis tissue uPAR and protein expression showed a similar trend: high expression level at birth, then decreased gradually, about D15 in Tianda is the lowest, D28 began to increase at D35 reached the peak, then decreased after d42, to maintain a certain level. Regression and correlation analysis showed that uPAR mRNA expression was positively correlated with the expression of uP in rat testis. Conclusion: The expression of AR in the first wave of spermatogenesis in two peaks, the first peak at birth, the period of germ cell migration to the basal part of the period, after the occurrence of the sperm plays a vital role in uPAR and spermatogenesis started are closely linked. The second peak was significantly increased in fifth week, Fifth weeks for spermiogenesis and began to spermiation period, indicating that uPAR may be involved in sperm emission in the process of tissue remodeling and spermiogenesis.
Two, the establishment of the germ cell culture system of rat testis
Rat testicular germ cell culture system establishment is an important tool in the process of regulatory factors in vitro testicular sperm. The Sertoli cells and spermatogenic cells coculture system two, which lays a foundation for further study of role of uPAR in spermatogenesis.
(1) isolation, purification and identification of rat testis support cells
Objective to develop a high purity of rat Sertoli cells, and the application of identification method for the detection of ABP mRNA in situ hybridization of cultured cells. Methods 18 ~ 22 day old male SD rat testis, using 0.25% trypsin, 0.1% hyaluronidase and 0.1% collagenase three enzyme digestion to separate Sertoli cells in culture, box at the temperature of 32 5%CO2 after 48 hours of culture, 20mmol Tris - HCl hypotonic treatment of cultured cells. Cultivating eosin staining one week after acridine orange fluorescent staining, Feulgen staining of the cultured Sertoli cells were identified, at the same time the application of in situ hybridization detection of ABP mRNA method for identification of cultured cells. Results the cultured Sertoli cells the purity of the training after a week of more than 95%. Cultured cells ABP mRNA expression, its morphological characteristics and identification using other methods for morphological characteristics of Sertoli cells induced by a node. On the basis of three enzyme continuous digestion and low osmotic treatment, the purity of Sertoli cells was high, and the detection of ABP mRNA by in situ hybridization is a new, specific and effective method to identify supporting cells and their functions.
(two) the establishment of co culture system of rat testis support cells / spermatogenic cells
Objective to establish a culture system of differentiation of germ cells in vitro, provide a good research model for the in vitro study on the role of uPAR in spermatogenesis. Methods 20~22 day old male SD rat testis, to be membrane and blood vessels, washing two times in the pre cooling Hank 's solution, and then use the F12/DMEM solution add 1 mg/ ml collagenase 32 c digestion 15 ~ 20min. with ophthalmic scissor testis was cut into very small pieces, and then repeat with 1 mg/ml collagenase with 5 ~ containing 10% fetal bovine serum and various nutritional factors of HamF12/ cultured in DMEM 10min. cell suspension, at about 1*106CELL/cm2 were inoculated in the dual chamber culture tank or put the coverslips in 6-well plates, at 32 degrees, under the condition of 5%CO2 culture. The dynamic growth of the cells was observed under phase contrast microscope, the morphological changes, and the regular application of eosin staining, Brdu staining of the cultured cells were stained sperm cells support identification results / students. In spermatogenic cells / Sertoli cell co culture system, 2 weeks after the end of the dual chamber culture, part of the spermatogenic cells with flagella, after 4 weeks of training, training system still has a certain number of spermatogenic cells attached to the supporting cells, part of the spermatogenic cells visible on the flagella. Dan Shipei raised students sperm cells in culture 4-5 days began to show off, after 1 weeks of culture, the majority of spermatogenic cell death occurred off, no flagella of spermatogenic cells, but in the culture system of visible secondary spermatocytes and sperm cells. Conclusion: the application of dual chamber culture, germ cell can survive for a month, and the tail part of the spermatogenic cells appeared morphologically flagella, occurred in meiosis and spermiogenesis.
Three, gene expression of uPAR with siRNA silencing co cultured cultured spermatogenic cells
The purpose of using small interfering RNA (siRNA) expression silencing in co cultured spermatogenic cells uPAR gene, provides a good tool for studying the role of uPAR in spermatogenesis. Methods siRNA cocktails were RNAi according to the ShortCut Kit manual prepared. Simply put, first through the RT-PCR application with specific primers of T7 promoter to synthesis of DNA template, and DNA template by in vitro transcription process of double stranded RNA, finally by ShortCut RNaseIII enzyme digestion to prepare siRNAs mixture. After 48 hours with Transfect transfection reagent respectively 15nM, 30nM and siRNA mixture was transfected into cultured spermatogenic cells, the expression of real time 48 hours fluorescence quantitative RT-PCR method for detection of co cultured spermatogenic cells uPAR gene. The study consisted of two control groups: blank control (without siRNA and transfection reagent) and negative control group (only with transfection reagent, No siRNA). The expression of 15nM and 30nM siRNA uPAR mRNA transfection group were significantly lower than the negative control group and blank control group (P0.05), which was transfected with 30nMsiRNA group was more obvious; compared with the negative control group, 15nM and 30nM siRNA transfected uPAR gene expression inhibition rate was 63.5% and 76.7%. conclusion ShortCut RNase III prepared siRNAs cocktail can effectively inhibit the expression of co cultured spermatogenic cells uPAR gene.
Five. Conclusion
In this study we found that uPAR in the first wave of spermatogenesis in the expression peak for 35 days after birth, when round sperm into elongated sperm, and began to spermiation period. In the co culture system, from 20 to 22 day old rat testis spermatogenic cell differentiation a sperm cell and transformed to elongate sperm after two weeks in culture. The expression of these changes corresponding to the uPAR in appearance change peak. In this culture system, siRNA silence the expression of uPAR, for the study of uPAR provides a good research tool in sperm function and mechanism of students in a specific period, as the signal transduction pathway. The establishment of this model also has other factors contribute to the regulation of meiosis and spermiogenesis.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R321

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