本文關鍵詞：應用siRNA沉默共培養(yǎng)生精細胞uPAR基因表達的研究　出處：《華中科技大學》2007年博士論文　論文類型：學位論文

  更多相關文章： 大鼠 精子發(fā)生 uPAR 實時熒光定量聚合酶鏈反應 大鼠 睪丸支持細胞 培養(yǎng) 原位雜交 鑒定 大鼠 生精細胞 支持細胞 共培養(yǎng) 大鼠 生精細胞 共培養(yǎng) siRNA RNAi

【摘要】： 精子發(fā)生是個復雜、多步驟的過程,可分為三個過程:精原細胞的增殖更新、精母細胞的成熟分裂和精子細胞變態(tài)為精子三個階段。目前發(fā)現(xiàn)在曲細精管中uPAR主要表達在精子細胞,可能與精子的排放有關。Tong Zhang等應用原位雜交技術檢測發(fā)現(xiàn)獼猴睪丸支持細胞在精子發(fā)生的特定階段(VII～VIII期)表達uPA mRNA;uPAR mRNA由睪丸生精細胞產(chǎn)生,但是不同發(fā)育階段及不同生殖細胞表達的差異表明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認為支持細胞這種時期特異性表達的uPA與生殖細胞uPAR結(jié)合后可能是通過激活uPAR介導的信號傳導通路作為生長因子而起作用。但是uPAR是通過激活何種信號傳導通路來調(diào)節(jié)精子的發(fā)生過程目前尚不清楚。uPAR是否還通過信號傳導來刺激生精細胞的發(fā)生及分化功能呢?因此,uPAR在精子發(fā)生中的作用機制尚待進一步研究。 然而由于睪丸組織結(jié)構和功能的復雜性,一般情況下很難在整體情況下對生殖細胞的生理生化功能和與其他細胞的相互作用進行深入研究。生精細胞培養(yǎng)技術的建立為深入地了解生精細胞發(fā)育過程,研究生精細胞自身及與其它生精細胞的關系,提供了一種新的、強有力的工具。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)新型機制,可導致靶基因mRNA的降解及特異性基因沉默信號的擴增。目前siRNA已經(jīng)成為選擇性沉默體外培養(yǎng)的哺乳細胞基因表達的一個有力工具。 本實驗首先應用實時RT-PCR和Western Blot檢測出生后不同發(fā)育階段大鼠睪丸中uPAR基因及蛋白表達的變化,然后建立的生精細胞體外培養(yǎng)體系,最后應用siRNA沉默技術阻斷體外培養(yǎng)生精細胞uPAR的基因表達,為研究uPAR在精子發(fā)生中作用機制及信號傳導通路的研究提供新的重要研究手段和技術平臺。 一、uPAR在大鼠睪丸第一精子發(fā)生波中的表達變化 目的:為探討uPAR在大鼠精子發(fā)生中的作用,研究在第一個精子發(fā)生波中大鼠睪丸組織uPAR的mRNA表達及蛋白表達變化。方法:將SD大鼠按生后年齡分組,以出生當天為d0,取出生后d0、d5、d10、d15、d21、d28、d35、d42、d49、d56大鼠睪丸組織,用實時熒光定量分析方法檢測各年齡組大鼠uPAR mRNA表達,并用Western印跡法檢測各年齡組大鼠uPAR蛋白表達。結(jié)果:大鼠睪丸組織uPAR mRNA表達與蛋白表達表現(xiàn)出相似的趨勢:剛出生時表達水平較高,后逐漸下降,約在d15天達最低,到d28時開始增加,到d35時到達高峰,隨后d42下降,后保持一定的水平。雙變量回歸相關分析顯示uPAR mRNA表達與蛋白表達為中度正相關。結(jié)論:大鼠睪丸uPAR表達在第一精子發(fā)生波中出現(xiàn)兩個高峰,第一高峰在剛出生時,這個時期生殖母細胞向基底部遷移的時期,這在精子以后的發(fā)生中起著至關重要的作用,說明uPAR與精子發(fā)生的啟動有著密切聯(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期,這說明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、大鼠睪丸生殖細胞培養(yǎng)體系的建立 大鼠睪丸生殖細胞培養(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。本實驗通過建立支持細胞及生精細胞兩種培養(yǎng)體系,為進一步研究uPAR在精子發(fā)生中的作用奠定基礎。 (一)大鼠睪丸支持細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胞,并應用檢測ABP mRNA原位雜交方法鑒定分離培養(yǎng)的支持細胞。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胞,放于32℃5%CO2的培養(yǎng)箱培養(yǎng),48小時后用20mmol Tris—HCl低滲處理培養(yǎng)細胞。培養(yǎng)一周后應用伊紅染色、吖啶橙熒光染色、Feulgen染色對所培養(yǎng)支持細胞進行鑒定,同時應用原位雜交檢測ABP mRNA方法鑒定分離培養(yǎng)的支持細胞。結(jié)果培養(yǎng)一周后所獲培養(yǎng)的支持細胞純度達95%以上。分離培養(yǎng)的支持細胞ABP mRNA表達陽性,其形態(tài)結(jié)構特征與用其它方法鑒定為支持細胞的形態(tài)結(jié)構特征一致。結(jié)論采用三酶連續(xù)消化及低滲處理法分離培養(yǎng)的支持細胞純度高,而且應用原位雜交方法檢測ABP mRNA是一種新的、特異、有效的鑒定支持細胞及其功能的方法。 (二)大鼠睪丸支持細胞/生精細胞共培養(yǎng)系統(tǒng)的建立 目的建立生精細胞體外分化培養(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,再重復用1 mg/ml膠原酶消化5～10min。細胞用內(nèi)含10%胎牛血清及各種營養(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)。相差顯微鏡下動態(tài)觀察細胞生長、形態(tài)等變化,及定期應用伊紅染色、Brdu染色法對所培養(yǎng)生精細胞/支持細胞進行染色鑒定。結(jié)果在生精細胞/支持細胞共培養(yǎng)體系中,雙室培養(yǎng)2周后,部分生精細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)量的生精細胞附著在支持細胞上,部分生精細胞上可見鞭毛。單室培養(yǎng)的生精細胞在培養(yǎng)第4-5天開始出現(xiàn)明顯脫落,培養(yǎng)1周后大多數(shù)生精細胞發(fā)生脫落死亡,生精細胞上未見鞭毛出現(xiàn),但在培養(yǎng)體系中可見次級精母細胞及精子細胞。結(jié)論:應用雙室培養(yǎng),生精細胞可存活達一月之久,而且部分生精細胞尾部出現(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、應用siRNA沉默共培養(yǎng)生精細胞uPAR的基因表達 目的利用小分子干擾RNA(siRNA)技術沉默共培養(yǎng)生精細胞uPAR的基因表達,為uPAR在精子發(fā)生中的作用研究提供一個很好的研究工具。方法siRNA混合雞尾酒是根據(jù)ShortCut RNAi Kit手冊來制備。簡單的說,首先通過RT-PCR應用加有T7啟動子的特異性引物來合成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酶消化來制備siRNAs混合物。在培養(yǎng)48小時用Transfect轉(zhuǎn)染試劑分別將15nM、30nM siRNA混合物轉(zhuǎn)染到共培養(yǎng)的生精細胞,48小時應用實時熒光定量RT-PCR方法檢測共培養(yǎng)生精細胞uPAR的基因表達。該試驗設立兩個對照組:空白對照(未加siRNA及轉(zhuǎn)染試劑)及陰性對照組(只加轉(zhuǎn)染試劑,無siRNA)。結(jié)果15nM及30nM siRNA轉(zhuǎn)染組uPAR mRNA表達量均明顯低于陰性對照組及空白對照組(p0.05),其中以30nMsiRNA轉(zhuǎn)染組更為明顯;相對于陰性對照組,15nM及30nM siRNA轉(zhuǎn)染組uPAR的基因表達抑制率分別為63.5%及76.7%。結(jié)論應用ShortCut RNase III法制備的siRNAs雞尾酒能有效地抑制共培養(yǎng)生精細胞uPAR基因的表達。 五、結(jié)論 在本研究中我們發(fā)現(xiàn)uPAR在第一精子發(fā)生波中表達最高峰為出生后35天,此時為圓形精子轉(zhuǎn)變?yōu)樯扉L精子,并開始向管腔排放的時期。在我們的共培養(yǎng)體系中,來自20～22天大小的大鼠睪丸組織的生精細胞在培養(yǎng)二周后能分化成精子細胞及變形成為伸長型精子。而這些變化相應于體內(nèi)uPAR的表達出現(xiàn)高峰期所發(fā)生的變化。因此在本培養(yǎng)體系中,應用siRNA沉默uPAR的表達,可以為研究uPAR在精子發(fā)生特定時期中的作用及機制提供一個很好的研究工具,如其信號傳導通路等。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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R321

【相似文獻】

相關期刊論文 前10條

1 張杰;李克勇;朱紅雁;梁繼仁;李翠珍;周志俊;吳慶;;六氯聯(lián)苯誘導新生雄性大鼠睪丸細胞凋亡的體內(nèi)體外研究[J];環(huán)境與職業(yè)醫(yī)學;2011年06期

2 顏秋霞;唐愛發(fā);葛頌;余州;李文杰;陳靜;蔡志明;桂耀庭;;精子頂體小泡蛋白-1(ACRV1)在小鼠睪丸組織中的表達與定位[J];生殖與避孕;2011年09期

3 袁東智;于海禮;丁小玲;張金虎;何亞平;岳利民;;周期素cyclin G1在小鼠睪丸生精上皮中的表達特點[J];四川大學學報(醫(yī)學版);2011年04期

4 張懿;劉剛;;大鼠生精相關基因TSARG1原核表達載體的構建和表達[J];實用預防醫(yī)學;2011年08期

5 林莉;;附睪避孕靶蛋白研究進展[J];咸寧學院學報(醫(yī)學版);2011年04期

6 林釵英;陶曉倩;柳海燕;時姍姍;路榕;姚兵;;Annexin5對雄性SD大鼠生殖相關指標和睪酮分泌的影響[J];中國男科學雜志;2010年11期

7 王達利;;重視陰莖陰囊皮膚撕脫傷修復方式的選擇[J];中華損傷與修復雜志(電子版);2011年03期

8 胡艷秋;;人RGS22蛋白表達及多克隆抗體制備[J];細胞與分子免疫學雜志;2011年09期

9 洪文;陳澤林;;抑制素B在男性不育癥中的應用進展[J];中國社區(qū)醫(yī)師(醫(yī)學專業(yè));2011年25期

10 于春梅;張平;王靜;劉明兮;王暉;周作民;沙家豪;;Annexin A7在精原干細胞中的表達研究[J];中華男科學雜志;2011年06期

相關會議論文 前10條

1 洪水根;倪子綿;薛如;孫濤;劉桂杰;韓俊雁;;中國鱟精子發(fā)生的研究[A];中國細胞生物學學會第五次會議論文摘要匯編[C];1992年

2 何瑩;侯林;楊萬喜;;Myosin Va在中華絨螯蟹精子發(fā)生過程中的空間分布與表達[A];“基因、進化與生理功能多樣性”海內(nèi)外學術研討會暨中國生理學會第七屆比較生理學學術會議論文摘要[C];2009年

3 王彥平;王亮;李俊杰;周光斌;朱士恩;;冷凍對精子超活化水平的影響[A];中國畜牧獸醫(yī)學會動物繁殖學分會第十五屆學術研討會論文集（下冊）[C];2010年

4 袁秀堂;周一兵;;墨西哥灣扇貝精子發(fā)生的超微結(jié)構和細胞化學[A];貝類學會第七次會員代表大會暨第十一次學術討論會摘要[C];2003年

5 馬全紅;郭睿;王海坤;王會珍;葛曄華;馬靜;陳詠梅;韓代書;;一個在小鼠精子發(fā)生后期特異表達基因(SRG-L)的克隆及分析[A];中國細胞生物學學會2005年學術大會、青年學術研討會論文摘要集[C];2005年

6 彭彬;張仁東;鄧顯忠;萬勇;楊正偉;;彌猴輸精管結(jié)扎對精子發(fā)生的影響——體視學定量研究[A];解剖學雜志——中國解剖學會2002年年會文摘匯編[C];2002年

7 張明鳳;趙云龍;;隆線n灳臃⑸俺墑煬擁某⒔峁筟A];中國動物學會甲殼動物學分會、中國海洋與湖沼學會甲殼動物學分會2004年甲殼動物學分會會員代表大會暨學術年會論文摘要集[C];2004年

8 孫廣峰;王達利;魏在榮;羅志軍;聶開瑜;金文虎;;游離皮片移植修復陰囊撕脫傷對精子發(fā)生影響的實驗研究及臨床觀察[A];中國藥理學會第七屆全國生殖藥理學術交流會論文摘要匯編[C];2011年

9 桂耀庭;唐愛發(fā);余振東;張立兵;張鍵榮;李賢新;蔡志明;;精子發(fā)生相關基因的篩選和功能研究[A];第一屆中華醫(yī)學會生殖醫(yī)學分會、中國動物學會生殖生物學分會聯(lián)合年會論文匯編[C];2007年

10 王玉鳳;朱朝兵;;羅氏沼蝦精子發(fā)生超微結(jié)構的研究[A];新世紀 新機遇 新挑戰(zhàn)——知識創(chuàng)新和高新技術產(chǎn)業(yè)發(fā)展（上冊）[C];2001年

相關重要報紙文章 前10條

1 上海交通大學醫(yī)學院附屬仁濟醫(yī)院上海市人類精子庫 杜宏偉　上海市男科學研究所 李錚;人體最激情的舞者——精子[N];東方早報;2011年

2 劉長亮;“附睪炎致不孕”內(nèi)幕揭秘[N];農(nóng)村醫(yī)藥報（漢）;2008年

3 易善永;治療不孕為何還要先避孕[N];健康報;2006年

4 段恩奎;人造精子用于臨床不能過于樂觀[N];科技日報;2006年

5 楊孝文;揭開海馬的繁衍生息之謎[N];中國漁業(yè)報;2007年

6 省畜牧局高級畜牧師 王學君;采精公豬常見問題的解決辦法（下）[N];河南科技報;2006年

7 ;前列腺炎導致不育怎么辦？[N];樂山日報;2006年

8 潘文;對人工精子的臨床前景不能過于樂觀[N];中國醫(yī)藥報;2006年

9 馮桃莉;避孕套能治不孕癥？[N];大眾衛(wèi)生報;2004年

10 華中農(nóng)業(yè)大學 彭健 博士;憑借什么讓豬繁殖高效？[N];中國畜牧獸醫(yī)報;2006年

相關博士學位論文 前10條

1 黃東暉;應用siRNA沉默共培養(yǎng)生精細胞uPAR基因表達的研究[D];華中科技大學;2007年

2 李慕軍;不同類型精子發(fā)生障礙全基因組甲基化差異分析與重要功能基因的表達驗證[D];廣西醫(yī)科大學;2012年

3 邢俊;VAMP4在精子運動中的功能研究[D];南京醫(yī)科大學;2009年

4 葛少欽;十足目甲殼動物精子發(fā)生及頂體反應過程中堿性蛋白和乙�；疕4蛋白的變化[D];河北大學;2008年

5 劉邊疆;人類精子中電壓依賴性陰離子通道蛋白的基礎和臨床研究[D];南京醫(yī)科大學;2011年

6 沙家豪　;人類睪丸cDNA array制備及睪丸發(fā)育/精子發(fā)生相關基因表達譜系構建[D];南京師范大學;2003年

7 周作民;人睪丸發(fā)育/精子發(fā)生相關基因的研究[D];南京師范大學;2003年

8 邱為民;小鼠znf230和人TCP11基因的克隆和功能研究[D];四川大學;2004年

9 劉順利;鼠精子纖維鞘蛋白FSCB的磷酸化在獲能和超活化中的作用研究[D];第三軍醫(yī)大學;2012年

10 馬靜;Smads在腎陽虛大鼠精子發(fā)生中的作用及溫陽生精湯對Smads表達的影響[D];第四軍醫(yī)大學;2005年

相關碩士學位論文 前10條

1 晏正碧;稀有泩鯽（Gobiocypris rarus）精子發(fā)生的初步研究[D];西南大學;2011年

2 魯可可;人類hUFP基因初步研究[D];南京醫(yī)科大學;2004年

3 袁媧;NORPEG基因不同轉(zhuǎn)錄本在人睪丸中的表達研究[D];南京醫(yī)科大學;2005年

4 曹志國;睪丸特異蛋白GGN1與GGNBP2的相互作用研究[D];中國人民解放軍軍事醫(yī)學科學院;2005年

5 張立飛;4種十足目甲殼動物精子發(fā)生的比較研究[D];浙江大學;2003年

6 任艷玲;孕酮、γ-氨基丁酸誘發(fā)馬鹿精子頂體反應及對離子轉(zhuǎn)運調(diào)節(jié)的研究[D];東北林業(yè)大學;2006年

7 余周;細胞凋亡抑制因子survivin在精子發(fā)生過程中的作用研究[D];重慶醫(yī)科大學;2007年

8 楊立;“生精寶”對小鼠少精子癥模型的作用及機理探討[D];昆明醫(yī)學院;2008年

9 柳強;奶牛X-，Y-精子抑制消減文庫的構建[D];內(nèi)蒙古農(nóng)業(yè)大學;2009年

10 周其趙;基因芯片篩選成年男性精子活力不足分子標記物[D];南方醫(yī)科大學;2010年

，

本文編號：1363223