小鼠精原干細胞體內(nèi)增殖和分化階段睪丸組織基因的差異表達
發(fā)布時間：2018-11-22 15:19
【摘要】： 研究背景：精原干細胞是一群具有高度自我更新能力和分化潛能的成體干細胞,是精子發(fā)生的起始。揭示精原干細胞增殖和分化的調(diào)控機制是促進精原干細胞體外培養(yǎng)和移植以及精子發(fā)生機制研究的重要基礎(chǔ)。由于精原干細胞在生精細胞中的含量極低且缺乏特異性的表面標(biāo)志,以及精子發(fā)生存在不同步性(在小鼠中精子發(fā)生的12個階段同時存在于曲細精管生精上皮),這兩方面因素使得直接研究精原干細胞在體內(nèi)增殖和分化的分子調(diào)控機制非常困難。研究顯示烷化劑白消安對生精細胞的殺傷作用具有劑量和物種相關(guān)性。若能利用白消安殺滅大部分精原干細胞,那么,為了恢復(fù)干細胞池的穩(wěn)定,精子再生初期精原干細胞的更新會得到加強而其分化會被延緩,精原干細胞體內(nèi)增殖與分化的相對同步就可能再這一過程中得到實現(xiàn)。 目的：探討建立昆明小鼠精子再生模型的白消安適宜劑量,通過該劑量建立的較理想的精子再生模型實現(xiàn)精子再生初期精原干細胞增殖與分化的相對同步。在此基礎(chǔ)上,采用基因芯片檢測精原干細胞增殖和分化階段小鼠睪丸組織基因表達譜的差異,初步探討精原干細胞增殖和分化的分子調(diào)控機制。 方法：8-10周齡雄性昆明白小鼠按區(qū)組隨機分為實驗組(168只)和對照組(8只)。實驗組小鼠接受白消安腹腔注射,根據(jù)不同給藥劑量進一步分為4組：A、B兩組各36只,分別接受10mg/kg和20mg/kg白消安單次注射；C、D兩組各48只,分別接受10mg/kg和15mg/kg白消安間隔24d二次注射。對照組小鼠接受單次等量溶劑腹腔注射。于給藥結(jié)束后1、2、3、4、6和8周,分別將各實驗組小鼠按期頸椎脫臼處死(組內(nèi)等量),對照組小鼠在注射后1周一次性處死。取出小鼠雙側(cè)睪丸。一側(cè)睪丸采用HE染色觀察曲細精管的組織形態(tài)學(xué)變化和免疫組化法檢測C、D組和對照組生精細胞Ki-67表達;對側(cè)睪丸組織采用電鏡觀察超微結(jié)構(gòu)。通過前述研究判斷建立精子再生模型的白消安適宜劑量和模型分期。以此適宜劑量白消安再次建立精子再生模型,分別選取精原干細胞處于增殖和分化階段的睪丸組織標(biāo)本,Trizol一步法提取組織總RNA并進行純化和熒光標(biāo)記。將經(jīng)前述反應(yīng)得到的標(biāo)記DNA于42℃在芯片雜交儀上與36kMouse Genome Array雜交過夜。用雙通道激光掃描儀掃描雜交芯片,對各芯片進行片間線性校正和片內(nèi)歸一化處理。對篩選得到的差異表達基因進行GO和KEGG信號通路分析。參照www. sabiosciences. com/gene_array_product/HTML/OMM-405. html.篩選差異表達的干細胞相關(guān)基因。實時定量PCR驗證Kit、bFGF和Oct4表達。免疫組化檢測Oct4和Thy-1在生精上皮的表達。 結(jié)果：第一周C、D兩組生精上皮的損傷程度介于A、B兩組之間。第二周,C、D兩組生精上皮僅基底膜部殘留以單個型精原細胞(As型)為主精原細胞和Sertoli細胞,D組生精上皮部分Sertoli細胞空泡變性;第三周,C、D兩組As型精原細胞均增多;第四周,C、D兩組均出現(xiàn)分化精原細胞和精母細胞；6-8周,兩組均出現(xiàn)精子發(fā)生,C組生精上皮結(jié)構(gòu)逐步恢復(fù)正常,而D組曲細精管直徑和生精上皮厚度仍顯著低于對照組。C、D兩組精原細胞Ki-67陽性率在第一、二周顯著低于對照組,在第三周極度增高,第四周開始下降,6-8周與對照組無顯著差異,第二、三周D組精原細胞Ki-67陽性率均低于C組�；蛐酒囼灲Y(jié)果顯示911個基因在精原干細胞增殖和分化階段的睪丸組織中表達存在差異,其中,上調(diào)608個(增殖期／分化期),下調(diào)303個。這些差異表達基因分別涉及生物學(xué)過程、分子功能和分子組成。84個信號通路功能改變具有統(tǒng)計學(xué)意義(P0.05),包括Notch和Wnt信號通路。與干細胞相關(guān)的差異基因有56個,上調(diào)40個,下調(diào)16個。部分干細胞的陽性標(biāo)記物(如Cd9, Stra8, Itgb1,Oct4和Thyl)和部分生長因子(如Fgf2, Csfl和Pdgfa)上調(diào)。免疫組化染色結(jié)果顯示Oct4抗原表達于曲細精管基底膜的精原細胞且在精原干細胞增殖期表達顯著高于分化期。 結(jié)論間隔24天10mg/kg白消安二次腹腔注射是建立小鼠精子再生模型的理想劑量。該模型可實現(xiàn)精子發(fā)生的相對同步：二次給藥后3周主要為精原干細胞增殖期,4周為分化期,6-8周為精子發(fā)生恢復(fù)期。小鼠精原干細胞增殖和分化過程的調(diào)控涉及許多基因(分屬不同信號通路)的差異表達,進一步研究這些基因(和通路)功能有助于揭示精原干細胞增殖和分化的調(diào)控機制。
[Abstract]:Background: Spermatogonial stem cells are a group of adult stem cells with high self-renewal and differentiation potential, which is the beginning of spermatogenesis. The mechanism of regulating the proliferation and differentiation of spermatogonial stem cells is an important basis for promoting the in vitro culture and transplantation of spermatogonial stem cells and the mechanism of spermatogenesis. Since the content of the spermatogonial stem cells in the spermatogenic cells is extremely low and the specific surface marker is lacking, and the occurrence of spermatogenesis is not synchronized (there are 12 stages of the spermatogenesis in the mouse at the same time in the spermatogenic epithelium of the fine sperm tube), These two factors make it very difficult to directly study the molecular regulation and regulation mechanism of spermatogonial stem cells in vivo proliferation and differentiation. The study shows that the anti-killing effect of the alkylating agent, the white spirit of the alkylating agent, on the spermatogenic cells has a dose and a species-related relationship. if it is possible to kill most of the spermatogonial stem cells by using the white anhydride, in order to restore the stability of the stem cell pool, the regeneration of the spermatogonial stem cells at the early stage of the sperm regeneration will be enhanced and the differentiation thereof will be delayed, The relative synchronization of the proliferation and differentiation of the spermatogonial stem cells may be achieved in this process. Objective: To study the appropriate dosage of the white Astian in the sperm regeneration model of Kunming mice, and to realize the proliferation and differentiation of the spermatogonial stem cells in the early stage of the sperm regeneration by the ideal sperm regeneration model established by this dose. On the basis of this, the difference of the expression profiles of the testis tissue genes in the spermatogonial stem cells in the stage of proliferation and differentiation of the spermatogonial stem cells was detected by using the gene chip, and the molecules of the spermatogonial stem cell proliferation and differentiation were primarily discussed. Methods: 8-10-week-old male Kunming white mice were randomly divided into experimental group (168 and the control group (8 rats). The experimental group mice were divided into 4 groups according to the different administration dose, and the control group was further divided into 4 groups according to the different administration dose: 36 of the groups A and B received 10 mg/ kg and 20 mg/ kg of Bai Xiao an injection, and 48 of the C and D groups received 10mg/ kg and 15mg/ kg of Bai Xiao an, respectively. Interval 24d Secondary injection. Control group mouse receiving single The mice were sacrificed at 1, 2, 3, 4, 6 and 8 weeks after the end of the administration. One-time sacrifice at one week after injection The expression of Ki-67 in C, D and control group was detected by HE staining and the expression of Ki-67 in C, D and control group was detected by HE staining. The ultrastructure was observed by electron microscope. It was determined by the above-mentioned study to establish a model for the regeneration of the sperm. The appropriate dose and model stage were established. The sperm regeneration model was set up again with the appropriate dose of Bai Xiao-an, and the testis tissue specimens of the spermatogonial stem cells in the stage of proliferation and differentiation were respectively selected, and the total RNA of the tissue was extracted by Trizol one-step one-step method. Purification and fluorescence labeling were performed. The labeled DNA obtained by the foregoing reaction was compared to 36kMuse Genome on a chip hybridization apparatus at 42.degree. C. Array hybridization overnight. The hybridization chip was scanned with a two-channel laser scanner, and each wafer was subjected to inter-chip linear correction and normalizing treatment in the positive and the negative plates, and GO and K are carried out on the differentially expressed genes obtained by screening. EGG signal path analysis. See www.sabi osciences. com/gene_array_product/HTM L/ OMM-405. html. Filter the difference table up-to-date stem cell-related genes. Real-time quantitative PCR validation kit, bF GF and Oct4 expression. Immunohistochemistry was used to detect Oct4 and Thy-1. The expression of C and D in the first week. The degree of injury was between the two groups of A and B. In the second week, there were only a single type of spermatogonial cells (As-type) and Sertoli cells, and vacuolation of Sertoli cells in the D group, and the third week, the C, and the D two groups of As. Spermatogonial cells and spermatocytes were all in the peripheral, C, and D groups. Spermatogenesis occurred in both groups at 6-8 weeks, and the structure of the C-group spermatogenic epithelium gradually returned to normal, and the diameter of the D-group curved fine-fine tube was the same as that of the D-group. The positive rate of Ki-67 in the C and D group was significantly lower than that in the control group. The positive rate of Ki-67 in the group C and D was significantly lower than that in the control group. The positive rate of Ki-67 was lower than that of group C. The results of gene chip test showed that the expression of the 911 genes in the testis of spermatogonial stem cell proliferation and differentiation stage was different, of which 608 (increased) were up-regulated. The expression of these differentially expressed genes involved in the biological process, the molecular function and the molecular composition, respectively. The change of the function of 84 signal pathways was of statistical significance (P0.05). BNotch and Wnt signaling pathways. The differential genes associated with stem cells are 5 6, up to 40, down 16. Positive markers of some stem cells (e.g., Cd9, Stra8, Igb1, Oct4, and Thyl) and some growth factors (e.g., Fgf2 The results of the immunohistochemical staining showed that the Oct4 antigen was expressed in the spermatogonial cells of the basement membrane of the fine-fine tube and dried in the fine form. The expression of the cell proliferative phase was significantly higher than that of the differentiation period. the injection of the cavity is an ideal dose for establishing a mouse sperm regeneration model, and the model can realize the relative synchronization of the spermatogenesis: the 3-week after the secondary administration is mainly the spermatogonial stem cell proliferation period, The regulation of the proliferation and differentiation of the spermatogonial stem cells involved many genes, which belong to different signal pathways, and further study the function of these genes (and pathways).
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329
本文編號：2349741
[Abstract]:Background: Spermatogonial stem cells are a group of adult stem cells with high self-renewal and differentiation potential, which is the beginning of spermatogenesis. The mechanism of regulating the proliferation and differentiation of spermatogonial stem cells is an important basis for promoting the in vitro culture and transplantation of spermatogonial stem cells and the mechanism of spermatogenesis. Since the content of the spermatogonial stem cells in the spermatogenic cells is extremely low and the specific surface marker is lacking, and the occurrence of spermatogenesis is not synchronized (there are 12 stages of the spermatogenesis in the mouse at the same time in the spermatogenic epithelium of the fine sperm tube), These two factors make it very difficult to directly study the molecular regulation and regulation mechanism of spermatogonial stem cells in vivo proliferation and differentiation. The study shows that the anti-killing effect of the alkylating agent, the white spirit of the alkylating agent, on the spermatogenic cells has a dose and a species-related relationship. if it is possible to kill most of the spermatogonial stem cells by using the white anhydride, in order to restore the stability of the stem cell pool, the regeneration of the spermatogonial stem cells at the early stage of the sperm regeneration will be enhanced and the differentiation thereof will be delayed, The relative synchronization of the proliferation and differentiation of the spermatogonial stem cells may be achieved in this process. Objective: To study the appropriate dosage of the white Astian in the sperm regeneration model of Kunming mice, and to realize the proliferation and differentiation of the spermatogonial stem cells in the early stage of the sperm regeneration by the ideal sperm regeneration model established by this dose. On the basis of this, the difference of the expression profiles of the testis tissue genes in the spermatogonial stem cells in the stage of proliferation and differentiation of the spermatogonial stem cells was detected by using the gene chip, and the molecules of the spermatogonial stem cell proliferation and differentiation were primarily discussed. Methods: 8-10-week-old male Kunming white mice were randomly divided into experimental group (168 and the control group (8 rats). The experimental group mice were divided into 4 groups according to the different administration dose, and the control group was further divided into 4 groups according to the different administration dose: 36 of the groups A and B received 10 mg/ kg and 20 mg/ kg of Bai Xiao an injection, and 48 of the C and D groups received 10mg/ kg and 15mg/ kg of Bai Xiao an, respectively. Interval 24d Secondary injection. Control group mouse receiving single The mice were sacrificed at 1, 2, 3, 4, 6 and 8 weeks after the end of the administration. One-time sacrifice at one week after injection The expression of Ki-67 in C, D and control group was detected by HE staining and the expression of Ki-67 in C, D and control group was detected by HE staining. The ultrastructure was observed by electron microscope. It was determined by the above-mentioned study to establish a model for the regeneration of the sperm. The appropriate dose and model stage were established. The sperm regeneration model was set up again with the appropriate dose of Bai Xiao-an, and the testis tissue specimens of the spermatogonial stem cells in the stage of proliferation and differentiation were respectively selected, and the total RNA of the tissue was extracted by Trizol one-step one-step method. Purification and fluorescence labeling were performed. The labeled DNA obtained by the foregoing reaction was compared to 36kMuse Genome on a chip hybridization apparatus at 42.degree. C. Array hybridization overnight. The hybridization chip was scanned with a two-channel laser scanner, and each wafer was subjected to inter-chip linear correction and normalizing treatment in the positive and the negative plates, and GO and K are carried out on the differentially expressed genes obtained by screening. EGG signal path analysis. See www.sabi osciences. com/gene_array_product/HTM L/ OMM-405. html. Filter the difference table up-to-date stem cell-related genes. Real-time quantitative PCR validation kit, bF GF and Oct4 expression. Immunohistochemistry was used to detect Oct4 and Thy-1. The expression of C and D in the first week. The degree of injury was between the two groups of A and B. In the second week, there were only a single type of spermatogonial cells (As-type) and Sertoli cells, and vacuolation of Sertoli cells in the D group, and the third week, the C, and the D two groups of As. Spermatogonial cells and spermatocytes were all in the peripheral, C, and D groups. Spermatogenesis occurred in both groups at 6-8 weeks, and the structure of the C-group spermatogenic epithelium gradually returned to normal, and the diameter of the D-group curved fine-fine tube was the same as that of the D-group. The positive rate of Ki-67 in the C and D group was significantly lower than that in the control group. The positive rate of Ki-67 in the group C and D was significantly lower than that in the control group. The positive rate of Ki-67 was lower than that of group C. The results of gene chip test showed that the expression of the 911 genes in the testis of spermatogonial stem cell proliferation and differentiation stage was different, of which 608 (increased) were up-regulated. The expression of these differentially expressed genes involved in the biological process, the molecular function and the molecular composition, respectively. The change of the function of 84 signal pathways was of statistical significance (P0.05). BNotch and Wnt signaling pathways. The differential genes associated with stem cells are 5 6, up to 40, down 16. Positive markers of some stem cells (e.g., Cd9, Stra8, Igb1, Oct4, and Thyl) and some growth factors (e.g., Fgf2 The results of the immunohistochemical staining showed that the Oct4 antigen was expressed in the spermatogonial cells of the basement membrane of the fine-fine tube and dried in the fine form. The expression of the cell proliferative phase was significantly higher than that of the differentiation period. the injection of the cavity is an ideal dose for establishing a mouse sperm regeneration model, and the model can realize the relative synchronization of the spermatogenesis: the 3-week after the secondary administration is mainly the spermatogonial stem cell proliferation period, The regulation of the proliferation and differentiation of the spermatogonial stem cells involved many genes, which belong to different signal pathways, and further study the function of these genes (and pathways).
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329
【參考文獻】
相關(guān)期刊論文 前1條
1 申復(fù)進;張茨;楊嗣星;熊云鶴;廖文彪;杜賢進;王玲瓏;;BALB/c小鼠精原干細胞體外長期培養(yǎng)和鑒定[J];中華男科學(xué)雜志;2008年11期
，本文編號：2349741
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2349741.html
最近更新
教材專著
