本文選題：A型精原細胞 + 細胞分化��； 參考：《華中科技大學》2008年博士論文

【摘要】： 哺乳動物的生精過程精細而復雜,多種調(diào)節(jié)因素參與生精細胞的產(chǎn)生、增殖和分化。精原干細胞是具有干細胞活性的生精細胞,是生精過程的起始。了解精原干細胞的分化機制將有益于揭示生精過程的起源,有益于人類利用精原干細胞、改造精原干細胞,從而促進醫(yī)學、生物學以及畜牧業(yè)的發(fā)展。 已證實:在整個生精過程中,生精細胞的發(fā)育過程經(jīng)歷了A0型精原細胞、A1-A4型精原細胞、In型(中間型)精原細胞、B型精原細胞、初級精母細胞、次級精母細胞、精子細胞、精子。精原細胞的各個亞型細胞中,僅A0型精原細胞具有干細胞特性,即精原干細胞,是生精過程的起始,是一種未分化的精原細胞,由它而分化出A1-A4精原細胞及后續(xù)的各種生精細胞。然而從未分化的精原細胞分化為A1型精原細胞是一個非常很關(guān)鍵的時期。隱睪、VitA缺乏、Sertoli細胞中毒、放射損傷都能導致這個分化步驟的阻斷。然而對如何調(diào)控未分化精原細胞的分化、在以上過程中這個步驟是如何停滯的還不得而知。 c-kit是由原癌基因W locus編碼的跨膜酪氨酸激酶受體,其配體是干細胞因子(SCF,由sl基因編碼),c-kit受體和它的配體SCF在生精細胞的發(fā)育和分化過程中起著重要作用。在缺乏c-kit受體存在的情況下,精原干細胞無法分化。有研究表明:未分化的精原細胞不表達c-kit,而分化中的精原細胞有c-kit表達。精原細胞從未分化走向分化,c-kit從不表達走向表達,這其中c-kit與精原細胞分化過程的聯(lián)系成為研究熱點。研究并利用c-kit在精原細胞發(fā)育過程中的表達特點,對闡明精原細胞的分化過程將起重要作用。 精原干細胞具有不對稱分裂的特性,即可向下分化與自我更新,在如何維持精原干細胞自我更新功能的研究中,plzf的作用引人注目。已證實plzf缺陷的雄性小鼠出生后會出現(xiàn)漸進性的不育,表現(xiàn)為生精細胞耗竭,即精原干細胞自我更新能力缺失。作為一種抑制性的轉(zhuǎn)錄因子,研究plzf在睪丸發(fā)育過程中表達的變化、與代表分化事件的c-kit表達水平的對比,將從新的角度探索精原干細胞的發(fā)育過程。 本課題通過研究不同發(fā)育階段的大鼠睪丸內(nèi)c-kit在核酸水平、蛋白水平上表達,探討c-kit與精原細胞分化的相關(guān)性;在此基礎(chǔ)上利用c-kit分選出A1-A4型精原細胞,探討精原細胞分化比例;研究了與精原細胞自我更新有重要關(guān)系的plzf表達,且與c-kit的表達特點相對比,揭示未分化精原細胞走向分化與走向更新之間命運選擇特點,并初步探討其機制。 一、c-kit在發(fā)育中的睪丸內(nèi)的表達 實驗選擇出生后1日齡組、9日齡組、30日齡組雄性SD大鼠,分別使用免疫組化和western-blot檢測其睪丸內(nèi)c-kit蛋白表達、RT-PCR半定量檢測c-kit mRNA表達,并通過流式細胞技術(shù)檢測了9日齡大鼠睪丸細胞內(nèi)c-kit陽性細胞的比例。結(jié)果如下: 1、c-kit蛋白和mRNA在大鼠睪丸發(fā)育過程中持續(xù)表達;表達位置和水平存在變化,出生后9日左右存在一個較高的水平表達,mRNA與蛋白表達變化一致; 2、根據(jù)文獻報道精原細胞發(fā)育的時相特點,結(jié)合本實驗觀察結(jié)果,9日齡睪丸出現(xiàn)的c-kit表達增高與精原細胞在此階段出現(xiàn)大量分化相關(guān); 3、在9日齡大鼠睪丸中,光鏡下可見的僅有支持細胞和精原細胞,通過流式細胞儀檢測此特定日齡的大鼠睪丸細胞,結(jié)合此階段的細胞類型,可認為:此時的精原細胞已出現(xiàn)分化,有(3.16±0.84)%的睪丸細胞表達c-kit,即分化的A1-A4型精原細胞,為進一步分離此亞型細胞提供了依據(jù)。 以上結(jié)果表明: c-kit表達與精原細胞分化關(guān)系密切,精原細胞分化活動增多時,c-kit的表達升高,c-kit是精原細胞分化的標志物,其表達水平可代表精原細胞分化活動的強弱。 二、A1-A4亞型精原細胞的分選 實驗選擇9日齡雄性SD大鼠5只,無菌取其睪丸兩步法消化成細胞懸液,percoll不連續(xù)密度梯度分離初步純化出精原細胞,流式細胞分選技術(shù)分離出c-kit陽性細胞。結(jié)果如下: 1、9日齡大鼠睪丸內(nèi)出現(xiàn)了分化中的精原細胞,即A1-A4亞型精原細胞;其含量為(3.16±0.84)%; 2、通過percoll不連續(xù)密度梯度分離,A1-A4亞型細胞含量可增加至(18.65±1.69)%,起到了明顯的純化作用; 3、以c-kit為標志通過流式細胞分選,可成功分離出A1-A4亞型細胞,分離后的細胞活性良好,形態(tài)正常。 以上結(jié)果表明: 選擇合適日齡動物,以percoll不連續(xù)密度梯度純化精原細胞,再以c-kit作為標志物,通過流式細胞分選技術(shù)可成功分離A1-A4亞型精原細胞,為亞型精原細胞體外純化策略探索了條件、提供了實驗依據(jù)。 三、精原干細胞增殖與分化的調(diào)節(jié)因素 選擇出生后1日齡、10日齡、30日齡雄性SD大鼠,RT-PCR、western-blot檢測其睪丸內(nèi)plzf表達水平,并與同日齡組c-kit表達特點相對比,結(jié)果如下: 1、plzf在發(fā)育中的大鼠睪丸內(nèi)有表達,1日齡睪丸中出現(xiàn)較高水平表達,隨日齡增加,plzf表達水平降低; 2、對比c-kit的表達情況,可知,在9日齡之前,c-kit表達隨日齡增加而增加,plzf隨日齡增加而下降;9日齡之后,c-kit與plzf表達水平均有下降; 3、從1日齡到9日齡,plzf表達明顯下降,同時c-kit表達明顯升高,與此相對的發(fā)育過程是精原細胞增殖減少,而分化增多,反應出plzf參與維持干細胞自我復制,抑制分化的功能特點。 以上結(jié)果表明: Plzf與精原細胞保持干細胞活性,即維持自我更新密切相關(guān);c-kit與精原細胞分化密切相關(guān),兩者在精原細胞發(fā)育方式的選擇中起著重要作用;plzf與c-kit表達的水平代表了精原細胞發(fā)育的方向,為今后體外調(diào)節(jié)精原細胞發(fā)育、人工干預生精過程提供了恰當?shù)陌悬c和實驗依據(jù)。 結(jié)論 1、c-kit在精原細胞分化過程中起重要作用,是精原細胞分化的標志,其表達水平可代表精原細胞分化活動的強弱;以c-kit為標志可成功分離A1-A4亞型精原細胞; 2、plzf在精原細胞自我更新中起重要作用,一定時期內(nèi)與c-kit表達水平相反,其表達水平可代表精原細胞自我更新活動的強弱。 3、本文為進一步研究精原細胞發(fā)育方式的體外調(diào)控提供了實驗依據(jù)。
[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 鹵 0.84 ) % .



2 . The content of A1 - A4 subtype cells can be increased to ( 18.65 鹵 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，

本文編號：2010794