c-kit与精原干细胞分化机制的研究

发布时间：2018-06-12 19:21

  本文选题：A型精原细胞 + 细胞分化　； 参考：《华中科技大学》2008年博士论文

【摘要】： 哺乳动物的生精过程精细而复杂,多种调节因素参与生精细胞的产生、增殖和分化。精原干细胞是具有干细胞活性的生精细胞,是生精过程的起始。了解精原干细胞的分化机制将有益于揭示生精过程的起源,有益于人类利用精原干细胞、改造精原干细胞,从而促进医学、生物学以及畜牧业的发展。 已证实:在整个生精过程中,生精细胞的发育过程经历了A0型精原细胞、A1-A4型精原细胞、In型(中间型)精原细胞、B型精原细胞、初级精母细胞、次级精母细胞、精子细胞、精子。精原细胞的各个亚型细胞中,仅A0型精原细胞具有干细胞特性,即精原干细胞,是生精过程的起始,是一种未分化的精原细胞,由它而分化出A1-A4精原细胞及后续的各种生精细胞。然而从未分化的精原细胞分化为A1型精原细胞是一个非常很关键的时期。隐睾、VitA缺乏、Sertoli细胞中毒、放射损伤都能导致这个分化步骤的阻断。然而对如何调控未分化精原细胞的分化、在以上过程中这个步骤是如何停滞的还不得而知。 c-kit是由原癌基因W locus编码的跨膜酪氨酸激酶受体,其配体是干细胞因子(SCF,由sl基因编码),c-kit受体和它的配体SCF在生精细胞的发育和分化过程中起着重要作用。在缺乏c-kit受体存在的情况下,精原干细胞无法分化。有研究表明:未分化的精原细胞不表达c-kit,而分化中的精原细胞有c-kit表达。精原细胞从未分化走向分化,c-kit从不表达走向表达,这其中c-kit与精原细胞分化过程的联系成为研究热点。研究并利用c-kit在精原细胞发育过程中的表达特点,对阐明精原细胞的分化过程将起重要作用。 精原干细胞具有不对称分裂的特性,即可向下分化与自我更新,在如何维持精原干细胞自我更新功能的研究中,plzf的作用引人注目。已证实plzf缺陷的雄性小鼠出生后会出现渐进性的不育,表现为生精细胞耗竭,即精原干细胞自我更新能力缺失。作为一种抑制性的转录因子,研究plzf在睾丸发育过程中表达的变化、与代表分化事件的c-kit表达水平的对比,将从新的角度探索精原干细胞的发育过程。 本课题通过研究不同发育阶段的大鼠睾丸内c-kit在核酸水平、蛋白水平上表达,探讨c-kit与精原细胞分化的相关性;在此基础上利用c-kit分选出A1-A4型精原细胞,探讨精原细胞分化比例;研究了与精原细胞自我更新有重要关系的plzf表达,且与c-kit的表达特点相对比,揭示未分化精原细胞走向分化与走向更新之间命运选择特点,并初步探讨其机制。 一、c-kit在发育中的睾丸内的表达 实验选择出生后1日龄组、9日龄组、30日龄组雄性SD大鼠,分别使用免疫组化和western-blot检测其睾丸内c-kit蛋白表达、RT-PCR半定量检测c-kit mRNA表达,并通过流式细胞技术检测了9日龄大鼠睾丸细胞内c-kit阳性细胞的比例。结果如下: 1、c-kit蛋白和mRNA在大鼠睾丸发育过程中持续表达;表达位置和水平存在变化,出生后9日左右存在一个较高的水平表达,mRNA与蛋白表达变化一致; 2、根据文献报道精原细胞发育的时相特点,结合本实验观察结果,9日龄睾丸出现的c-kit表达增高与精原细胞在此阶段出现大量分化相关; 3、在9日龄大鼠睾丸中,光镜下可见的仅有支持细胞和精原细胞,通过流式细胞仪检测此特定日龄的大鼠睾丸细胞,结合此阶段的细胞类型,可认为:此时的精原细胞已出现分化,有(3.16±0.84)%的睾丸细胞表达c-kit,即分化的A1-A4型精原细胞,为进一步分离此亚型细胞提供了依据。 以上结果表明: c-kit表达与精原细胞分化关系密切,精原细胞分化活动增多时,c-kit的表达升高,c-kit是精原细胞分化的标志物,其表达水平可代表精原细胞分化活动的强弱。 二、A1-A4亚型精原细胞的分选 实验选择9日龄雄性SD大鼠5只,无菌取其睾丸两步法消化成细胞悬液,percoll不连续密度梯度分离初步纯化出精原细胞,流式细胞分选技术分离出c-kit阳性细胞。结果如下: 1、9日龄大鼠睾丸内出现了分化中的精原细胞,即A1-A4亚型精原细胞;其含量为(3.16±0.84)%; 2、通过percoll不连续密度梯度分离,A1-A4亚型细胞含量可增加至(18.65±1.69)%,起到了明显的纯化作用; 3、以c-kit为标志通过流式细胞分选,可成功分离出A1-A4亚型细胞,分离后的细胞活性良好,形态正常。 以上结果表明: 选择合适日龄动物,以percoll不连续密度梯度纯化精原细胞,再以c-kit作为标志物,通过流式细胞分选技术可成功分离A1-A4亚型精原细胞,为亚型精原细胞体外纯化策略探索了条件、提供了实验依据。 三、精原干细胞增殖与分化的调节因素 选择出生后1日龄、10日龄、30日龄雄性SD大鼠,RT-PCR、western-blot检测其睾丸内plzf表达水平,并与同日龄组c-kit表达特点相对比,结果如下: 1、plzf在发育中的大鼠睾丸内有表达,1日龄睾丸中出现较高水平表达,随日龄增加,plzf表达水平降低; 2、对比c-kit的表达情况,可知,在9日龄之前,c-kit表达随日龄增加而增加,plzf随日龄增加而下降;9日龄之后,c-kit与plzf表达水平均有下降; 3、从1日龄到9日龄,plzf表达明显下降,同时c-kit表达明显升高,与此相对的发育过程是精原细胞增殖减少,而分化增多,反应出plzf参与维持干细胞自我复制,抑制分化的功能特点。 以上结果表明: Plzf与精原细胞保持干细胞活性,即维持自我更新密切相关;c-kit与精原细胞分化密切相关,两者在精原细胞发育方式的选择中起着重要作用;plzf与c-kit表达的水平代表了精原细胞发育的方向,为今后体外调节精原细胞发育、人工干预生精过程提供了恰当的靶点和实验依据。 结论 1、c-kit在精原细胞分化过程中起重要作用,是精原细胞分化的标志,其表达水平可代表精原细胞分化活动的强弱;以c-kit为标志可成功分离A1-A4亚型精原细胞; 2、plzf在精原细胞自我更新中起重要作用,一定时期内与c-kit表达水平相反,其表达水平可代表精原细胞自我更新活动的强弱。 3、本文为进一步研究精原细胞发育方式的体外调控提供了实验依据。
[Abstract]:The spermatogonial stem cell is a spermatogenic cell with stem cell activity , which is the starting point of the spermatogenic process . It is beneficial to reveal the origin of spermatogenic process , which is beneficial to the human use of spermatogonial stem cells and the transformation of spermatogonial stem cells , thus promoting the development of medicine , biology and animal husbandry .



It has been confirmed that the growth process of spermatogenic cells in the whole process of spermatogenic cells has undergone the development of A0 - type spermatogonia , type A1 - A4 spermatogonia , In - type ( intermediate ) spermatogonia , B - type spermatogonia , primary spermatocytes , secondary spermatocytes , spermatids , sperm , and spermatogenic cells .



c - kit is a transmembrane tyrosine kinase receptor encoded by proto - oncogene w locus . Its ligand is stem cell factor ( SCF , encoded by sl gene ) , c - kit receptor and its ligand SCF play an important role in the development and differentiation of spermatogenic cells .



In the study of how to maintain the self - renewal function of spermatogonial stem cells , the effect of plzf is remarkable . In the study of how to maintain the self - renewal function of spermatogonial stem cells , it has been proved that the male mice with plzf deficiency have the loss of self - renewal ability . As an inhibitory transcription factor , the study of plzf ' s expression in the course of testis development , and the expression level of c - kit representing differentiation events , will explore the developmental process of spermatogonial stem cells from a new angle .



In this study , we studied the relationship between c - kit and spermatogenic cell differentiation by studying the expression of c - kit in rat testis at different developmental stages .



Expression of one , c - kit in the development of testis



The c - kit protein expression was detected by immunohistochemistry and western - blot . c - kit mRNA expression was detected by RT - PCR semi - quantitative polymerase chain reaction ( RT - PCR ) , and the ratio of c - kit positive cells in the testis cells of 9 - day - old rats was detected by flow cytometry .



1 , c - kit protein and mRNA were expressed continuously during the rat testis development . There was a high level of expression of c - kit protein and mRNA in the testis during postnatal 9 days .



2 . According to the characteristics of the time phase of the development of spermatogonia , the expression of c - kit in the testis of 9 - day - old was associated with the proliferation of spermatogonia in this stage .



3 . In the testis of 9 - day - old rats , only the supporting cells and the spermatogonia were observed under the light microscope , and the rat testis cells were detected by flow cytometry .



The above results show that :



c - kit expression is closely related to the differentiation of spermatogonia , and the expression of c - kit is increased , c - kit is a marker for the differentiation of spermatogonia , and the expression level of c - kit can represent the strength and weakness of spermatogenic cell differentiation activity .



II . sorting of the A1 - A4 subtype spermatogonia



5 rats of 9 - day - old male SD rats were randomly divided into cell suspension by two - step method .



1 . In 9 - day - old rat testis , spermatogonia , i.e . , A1 - A4 spermatogonia , were found in the testis , and the content was ( 3.16 卤 0.84 ) % .



2 . The content of A1 - A4 subtype cells can be increased to ( 18.65 卤 1 . 69 ) % by percollindiscontinuous density gradient separation , which plays a significant role in purification .



3 . The cells of A1 - A4 were isolated by flow cytometry with c - kit as the marker , and the isolated cells had good activity and normal morphology .



The above results show that :



A suitable day - old animal was selected to purify the spermatogonia with percollindiscontinuous density gradient , and c - kit was used as a marker to successfully isolate the A1 - A4 spermatogonia by flow cytometry , and the conditions were explored for the in vitro purification strategy of the subtype spermatogonia , and the experimental basis was provided .



Regulation factors of proliferation and differentiation of spermatogonial stem cells



閫夋嫨鍑虹敓鍚，

本文编号：2010794