应用siRNA沉默共培养生精细胞uPAR基因表达的研究

发布时间：2018-01-01 06:04

本文关键词：应用siRNA沉默共培养生精细胞uPAR基因表达的研究　出处：《华中科技大学》2007年博士论文　论文类型：学位论文

【摘要】： 精子发生是个复杂、多步骤的过程,可分为三个过程:精原细胞的增殖更新、精母细胞的成熟分裂和精子细胞变态为精子三个阶段。目前发现在曲细精管中uPAR主要表达在精子细胞,可能与精子的排放有关。Tong Zhang等应用原位杂交技术检测发现猕猴睾丸支持细胞在精子发生的特定阶段(VII～VIII期)表达uPA mRNA;uPAR mRNA由睾丸生精细胞产生,但是不同发育阶段及不同生殖细胞表达的差异表明uPA/uPAR系统可能在精子发生过程中的特定阶段起作用。Vassalli认为支持细胞这种时期特异性表达的uPA与生殖细胞uPAR结合后可能是通过激活uPAR介导的信号传导通路作为生长因子而起作用。但是uPAR是通过激活何种信号传导通路来调节精子的发生过程目前尚不清楚。uPAR是否还通过信号传导来刺激生精细胞的发生及分化功能呢?因此,uPAR在精子发生中的作用机制尚待进一步研究。然而由于睾丸组织结构和功能的复杂性,一般情况下很难在整体情况下对生殖细胞的生理生化功能和与其他细胞的相互作用进行深入研究。生精细胞培养技术的建立为深入地了解生精细胞发育过程,研究生精细胞自身及与其它生精细胞的关系,提供了一种新的、强有力的工具。RNA干扰(RNAi)是一种由双链小干扰RNA(siRNA)引发的转录后基因沉默(PTGS)新型机制,可导致靶基因mRNA的降解及特异性基因沉默信号的扩增。目前siRNA已经成为选择性沉默体外培养的哺乳细胞基因表达的一个有力工具。本实验首先应用实时RT-PCR和Western Blot检测出生后不同发育阶段大鼠睾丸中uPAR基因及蛋白表达的变化,然后建立的生精细胞体外培养体系,最后应用siRNA沉默技术阻断体外培养生精细胞uPAR的基因表达,为研究uPAR在精子发生中作用机制及信号传导通路的研究提供新的重要研究手段和技术平台。一、uPAR在大鼠睾丸第一精子发生波中的表达变化目的:为探讨uPAR在大鼠精子发生中的作用,研究在第一个精子发生波中大鼠睾丸组织uPAR的mRNA表达及蛋白表达变化。方法:将SD大鼠按生后年龄分组,以出生当天为d0,取出生后d0、d5、d10、d15、d21、d28、d35、d42、d49、d56大鼠睾丸组织,用实时荧光定量分析方法检测各年龄组大鼠uPAR mRNA表达,并用Western印迹法检测各年龄组大鼠uPAR蛋白表达。结果:大鼠睾丸组织uPAR mRNA表达与蛋白表达表现出相似的趋势:刚出生时表达水平较高,后逐渐下降,约在d15天达最低,到d28时开始增加,到d35时到达高峰,随后d42下降,后保持一定的水平。双变量回归相关分析显示uPAR mRNA表达与蛋白表达为中度正相关。结论:大鼠睾丸uPAR表达在第一精子发生波中出现两个高峰,第一高峰在刚出生时,这个时期生殖母细胞向基底部迁移的时期,这在精子以后的发生中起着至关重要的作用,说明uPAR与精子发生的启动有着密切联系。第二高峰是在第5周升高明显,第5周为精子变形并开始向管腔排放的时期,这说明uPAR可能参与精子排放过程中的组织重塑及精子变态过程。二、大鼠睾丸生殖细胞培养体系的建立大鼠睾丸生殖细胞培养体系的建立是体外研究睾丸精子发生过程中各种调控因素的的重要工具。本实验通过建立支持细胞及生精细胞两种培养体系,为进一步研究uPAR在精子发生中的作用奠定基础。 (一)大鼠睾丸支持细胞的分离纯化和鉴定目的培养高纯度的大鼠睾丸支持细胞,并应用检测ABP mRNA原位杂交方法鉴定分离培养的支持细胞。方法选用18～22天龄SD雄性大鼠睾丸,采用0.25%胰蛋白酶、0.1%透明质酸酶、0.1%胶原酶三酶依次消化法分离支持细胞,放于32℃5%CO2的培养箱培养,48小时后用20mmol Tris—HCl低渗处理培养细胞。培养一周后应用伊红染色、吖啶橙荧光染色、Feulgen染色对所培养支持细胞进行鉴定,同时应用原位杂交检测ABP mRNA方法鉴定分离培养的支持细胞。结果培养一周后所获培养的支持细胞纯度达95%以上。分离培养的支持细胞ABP mRNA表达阳性,其形态结构特征与用其它方法鉴定为支持细胞的形态结构特征一致。结论采用三酶连续消化及低渗处理法分离培养的支持细胞纯度高,而且应用原位杂交方法检测ABP mRNA是一种新的、特异、有效的鉴定支持细胞及其功能的方法。 (二)大鼠睾丸支持细胞/生精细胞共培养系统的建立目的建立生精细胞体外分化培养体系,为uPAR在精子发生中作用的体外研究提供一个很好的研究模型。方法取20～22天龄SD雄性大鼠睾丸,去被膜及血管,放在预冷的Hank’s液中洗两次,然后用加1 mg/ml胶原酶的F12/DMEM液32℃消化15～20min。用眼科剪将睾丸剪碎成非常小的片断,再重复用1 mg/ml胶原酶消化5～10min。细胞用内含10%胎牛血清及各种营养因子的HamF12/ DMEM培养液悬浮,约按1*106CELL/cm2的密度接种于双室培养槽或放有盖玻片的六孔板中,在32℃、5%CO2条件下培养。相差显微镜下动态观察细胞生长、形态等变化,及定期应用伊红染色、Brdu染色法对所培养生精细胞/支持细胞进行染色鉴定。结果在生精细胞/支持细胞共培养体系中,双室培养2周后,部分生精细胞胞体一端有鞭毛出现,培养4周后,培养体系中仍有一定数量的生精细胞附着在支持细胞上,部分生精细胞上可见鞭毛。单室培养的生精细胞在培养第4-5天开始出现明显脱落,培养1周后大多数生精细胞发生脱落死亡,生精细胞上未见鞭毛出现,但在培养体系中可见次级精母细胞及精子细胞。结论:应用双室培养,生精细胞可存活达一月之久,而且部分生精细胞尾部出现鞭毛,从形态上发生了减数分裂及精子变态过程。三、应用siRNA沉默共培养生精细胞uPAR的基因表达目的利用小分子干扰RNA(siRNA)技术沉默共培养生精细胞uPAR的基因表达,为uPAR在精子发生中的作用研究提供一个很好的研究工具。方法siRNA混合鸡尾酒是根据ShortCut RNAi Kit手册来制备。简单的说,首先通过RT-PCR应用加有T7启动子的特异性引物来合成DNA模板,然后DNA模板通过体外转录过程产生双链RNA,最后通过ShortCut RNaseIII酶消化来制备siRNAs混合物。在培养48小时用Transfect转染试剂分别将15nM、30nM siRNA混合物转染到共培养的生精细胞,48小时应用实时荧光定量RT-PCR方法检测共培养生精细胞uPAR的基因表达。该试验设立两个对照组:空白对照(未加siRNA及转染试剂)及阴性对照组(只加转染试剂,无siRNA)。结果15nM及30nM siRNA转染组uPAR mRNA表达量均明显低于阴性对照组及空白对照组(p0.05),其中以30nMsiRNA转染组更为明显;相对于阴性对照组,15nM及30nM siRNA转染组uPAR的基因表达抑制率分别为63.5%及76.7%。结论应用ShortCut RNase III法制备的siRNAs鸡尾酒能有效地抑制共培养生精细胞uPAR基因的表达。五、结论在本研究中我们发现uPAR在第一精子发生波中表达最高峰为出生后35天,此时为圆形精子转变为伸长精子,并开始向管腔排放的时期。在我们的共培养体系中,来自20～22天大小的大鼠睾丸组织的生精细胞在培养二周后能分化成精子细胞及变形成为伸长型精子。而这些变化相应于体内uPAR的表达出现高峰期所发生的变化。因此在本培养体系中,应用siRNA沉默uPAR的表达,可以为研究uPAR在精子发生特定时期中的作用及机制提供一个很好的研究工具,如其信号传导通路等。此研究模型的建立也有助于减数分裂及精子变态过程的其它调控因素的研究。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R321

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