小鼠精原干细胞体内增殖和分化阶段睾丸组织基因的差异表达
发布时间：2018-11-22 15:19
【摘要】： 研究背景：精原干细胞是一群具有高度自我更新能力和分化潜能的成体干细胞,是精子发生的起始。揭示精原干细胞增殖和分化的调控机制是促进精原干细胞体外培养和移植以及精子发生机制研究的重要基础。由于精原干细胞在生精细胞中的含量极低且缺乏特异性的表面标志,以及精子发生存在不同步性(在小鼠中精子发生的12个阶段同时存在于曲细精管生精上皮),这两方面因素使得直接研究精原干细胞在体内增殖和分化的分子调控机制非常困难。研究显示烷化剂白消安对生精细胞的杀伤作用具有剂量和物种相关性。若能利用白消安杀灭大部分精原干细胞,那么,为了恢复干细胞池的稳定,精子再生初期精原干细胞的更新会得到加强而其分化会被延缓,精原干细胞体内增殖与分化的相对同步就可能再这一过程中得到实现。 目的：探讨建立昆明小鼠精子再生模型的白消安适宜剂量,通过该剂量建立的较理想的精子再生模型实现精子再生初期精原干细胞增殖与分化的相对同步。在此基础上,采用基因芯片检测精原干细胞增殖和分化阶段小鼠睾丸组织基因表达谱的差异,初步探讨精原干细胞增殖和分化的分子调控机制。 方法：8-10周龄雄性昆明白小鼠按区组随机分为实验组(168只)和对照组(8只)。实验组小鼠接受白消安腹腔注射,根据不同给药剂量进一步分为4组：A、B两组各36只,分别接受10mg/kg和20mg/kg白消安单次注射；C、D两组各48只,分别接受10mg/kg和15mg/kg白消安间隔24d二次注射。对照组小鼠接受单次等量溶剂腹腔注射。于给药结束后1、2、3、4、6和8周,分别将各实验组小鼠按期颈椎脱臼处死(组内等量),对照组小鼠在注射后1周一次性处死。取出小鼠双侧睾丸。一侧睾丸采用HE染色观察曲细精管的组织形态学变化和免疫组化法检测C、D组和对照组生精细胞Ki-67表达;对侧睾丸组织采用电镜观察超微结构。通过前述研究判断建立精子再生模型的白消安适宜剂量和模型分期。以此适宜剂量白消安再次建立精子再生模型,分别选取精原干细胞处于增殖和分化阶段的睾丸组织标本,Trizol一步法提取组织总RNA并进行纯化和荧光标记。将经前述反应得到的标记DNA于42℃在芯片杂交仪上与36kMouse Genome Array杂交过夜。用双通道激光扫描仪扫描杂交芯片,对各芯片进行片间线性校正和片内归一化处理。对筛选得到的差异表达基因进行GO和KEGG信号通路分析。参照www. sabiosciences. com/gene_array_product/HTML/OMM-405. html.筛选差异表达的干细胞相关基因。实时定量PCR验证Kit、bFGF和Oct4表达。免疫组化检测Oct4和Thy-1在生精上皮的表达。 结果：第一周C、D两组生精上皮的损伤程度介于A、B两组之间。第二周,C、D两组生精上皮仅基底膜部残留以单个型精原细胞(As型)为主精原细胞和Sertoli细胞,D组生精上皮部分Sertoli细胞空泡变性;第三周,C、D两组As型精原细胞均增多;第四周,C、D两组均出现分化精原细胞和精母细胞；6-8周,两组均出现精子发生,C组生精上皮结构逐步恢复正常,而D组曲细精管直径和生精上皮厚度仍显著低于对照组。C、D两组精原细胞Ki-67阳性率在第一、二周显著低于对照组,在第三周极度增高,第四周开始下降,6-8周与对照组无显著差异,第二、三周D组精原细胞Ki-67阳性率均低于C组。基因芯片试验结果显示911个基因在精原干细胞增殖和分化阶段的睾丸组织中表达存在差异,其中,上调608个(增殖期／分化期),下调303个。这些差异表达基因分别涉及生物学过程、分子功能和分子组成。84个信号通路功能改变具有统计学意义(P0.05),包括Notch和Wnt信号通路。与干细胞相关的差异基因有56个,上调40个,下调16个。部分干细胞的阳性标记物(如Cd9, Stra8, Itgb1,Oct4和Thyl)和部分生长因子(如Fgf2, Csfl和Pdgfa)上调。免疫组化染色结果显示Oct4抗原表达于曲细精管基底膜的精原细胞且在精原干细胞增殖期表达显著高于分化期。 结论间隔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剂量。该模型可实现精子发生的相对同步：二次给药后3周主要为精原干细胞增殖期,4周为分化期,6-8周为精子发生恢复期。小鼠精原干细胞增殖和分化过程的调控涉及许多基因(分属不同信号通路)的差异表达,进一步研究这些基因(和通路)功能有助于揭示精原干细胞增殖和分化的调控机制。
[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).
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
本文编号：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).
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前1条
1 申复进;张茨;杨嗣星;熊云鹤;廖文彪;杜贤进;王玲珑;;BALB/c小鼠精原干细胞体外长期培养和鉴定[J];中华男科学杂志;2008年11期
，本文编号：2349741
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