蛋白质代谢通路对鸡雄性生殖细胞分化的调控
发布时间：2018-07-21 12:45
【摘要】：【目的】探究蛋白质代谢在鸡雄性生殖细胞分化过程中的作用机制,为完善鸡胚胎干细胞(embryonic stem cell,ESC)体外向雄性生殖细胞诱导分化体系研究提供依据。【方法】采用流式分选的方法获取高纯度的ESC、原始生殖细胞(primordial germ cells,PGCs)和精原干细胞(spermatogonia stem cell,SSCs),分别提取细胞的总RNA,采用RNA-Seq方法对3种细胞的转录本进行深度测序,然后进行WEGO(web gene ontology)和KEGG(kyoto encyclopedia of genes and genomes)通路富集分析,筛选出鸡雄性生殖细胞分化过程中参与蛋白质代谢的关键通路及其关键基因,RT-q PCR(Real time Quantitative PCR)检测部分关键差异基因的表达变化,并与RNA-Seq(RNA sequencing)结果进行比较分析,同时分别从体内和体外水平对关键基因NOS2进行抑制,观察各分组不同天数的细胞形态变化及检测NOS2和C-kit、Cvh、Stra8、Dazl、integrinα6和integrinβ1等生殖标记基因表达变化情况。【结果】在雄性生殖细胞分化的整个阶段,有697个差异基因参与生物代谢,显著富集于精氨酸-脯氨酸代谢通路、酪氨酸代谢通路以及色氨酸代谢通路,在这3条通路上筛选出NOS2、FAH和IDO等关键性基因,这些基因的在ESCs向SSCs分化过程中表达变化趋势与其在RNA-Seq中的结果一致。体内抑制NOS2基因后,NOS2及C-kit、Cvh、Stra8、Dazl、integrinα6和integrinβ1等生殖标记基因在空白组和对照组之间无显著性的差异,而在抑制剂组中,NOS2及C-kit、Cvh、Stra8、Dazl、integrinα6和integrinβ1等生殖标记基因的m RNA表达量均出现了降低;而体外抑制NOS2基因后,对照组中的ESCs在2、4、6、8和10d内细胞不断增殖,但是未出现类胚体;RA诱导组中,2d出现小的类胚体,4d类胚体增大,且数量增多,6d类胚体边缘开始出现破裂,8d类胚体解体,10d出现类精原样细胞;抑制剂组中,ESCs在2、4、6、8和10d内无类胚体出现,且相较于对照组细胞增殖缓慢;RA+抑制剂组中,2和4d内无类胚体出现,细胞增殖缓慢,6d出现小的类胚体,8d类胚体数量少量增多,且体积稍显增大,10d类胚体开始裂解。并且经过抑制剂的抑制后,NOS2及C-kit、Cvh、Stra8、Dazl、integrinα6和integrinβ1等生殖标记基因的表达量在RA诱导组、抑制剂组和RA+抑制剂组中相对于对照组均呈显著性或极显著性的下调趋势。【结论】基于RNA-Seq技术及生物信息学方法筛选出ESCs向雄性生殖细胞分化过程中精氨酸-脯氨酸代谢通路及关键基因NOS2的基础上,通过对NOS2基因在鸡体内和体外的抑制,发现NOS2在被抑制后,ESCs向雄性生殖细胞分化的过程受到抑制。说明了精氨酸-脯氨酸代谢通路及关键基因NOS2对ESCs向雄性生殖细胞分化过程中起到重要的调节作用。
[Abstract]:[Objective] to explore the mechanism of protein metabolism in the differentiation of male reproductive cells of chickens, and to provide a basis for improving the differentiation system of embryonic stem cell (ESC) to the male reproductive cells in vitro. [Methods] high purity ESC was obtained by flow separation method, and the original germ cells (primordial germ) were obtained. Cells, PGCs) and spermatogonial stem cells (spermatogonia stem cell, SSCs), respectively extract the total RNA of the cells. The RNA-Seq method was used to sequenced the transcriptional transcripts of the 3 cells, and then the WEGO (WEB gene ontology) and the enrichment analysis were carried out to screen the differentiation process of the male reproductive cells. The key pathway and key genes involved in protein metabolism, RT-q PCR (Real time Quantitative PCR), were used to detect the changes in the expression of some key differentially differentially genes, and compared with the results of RNA-Seq (RNA sequencing). At the same time, the key basis was suppressed by NOS2 in vivo and in vitro, and the cell shape of different days in each group was observed. Changes in the expression of NOS2 and C-kit, Cvh, Stra8, Dazl, integrin alpha 6 and integrin beta 1. [results] in the whole stage of the differentiation of male reproductive cells, there were 697 different genes involved in biological metabolism, significantly enriched in the arginine proline metabolite pathway, tyrosine metabolic pathway and tryptophan Xie Tong The key genes such as NOS2, FAH and IDO were screened on these 3 pathways. The expression of these genes in the process of ESCs to SSCs was consistent with the results in RNA-Seq. After the inhibition of the NOS2 gene in vivo, the reproductive marker genes such as NOS2 and C-kit, Cvh, Stra8, Dazl, alpha 6 and beta 1 were not found between the blank group and the control group. In the inhibitor group, in the inhibitor group, the m RNA expression of NOS2 and C-kit, Cvh, Stra8, Dazl, integrin alpha 6 and integrin beta 1 all decreased, and the ESCs in the control group was proliferating in 2,4,6,8 and inner cells after inhibiting the NOS2 gene in the control group, but there was a small embryoid body in the induction group, 4 The D embryoid body increased, and the number increased, the edge of 6D type embryo began to rupture, 8D like body was disintegrated, and 10d appeared sperm like primitive cells. In the inhibitor group, ESCs had no embryoid body in 2,4,6,8 and 10d, and the cell proliferation was slow compared to the control group. In RA+ inhibitor group, no embryoid body appeared in 2 and 4D, the cell proliferation was slow, 6D appeared small embryoid. The number of 8D embryoid bodies increased slightly and the volume slightly increased, and the 10d embryoid body began to cleavage. And after inhibition of the inhibitors, the expression of NOS2 and C-kit, Cvh, Stra8, Dazl, integrin a 6 and integrin beta 1 were expressed in the RA induction group, and the inhibitor group and the RA+ inhibitor group were both significant or significant relative to the control group. [Conclusion] Based on RNA-Seq and bioinformatics methods, based on the screening of the arginine proline metabolic pathway and the key gene NOS2 during the differentiation of male reproductive cells from ESCs to the male reproductive cells, the inhibition of the NOS2 gene in the chicken and in vitro was found, and the differentiation process of ESCs into the male reproductive cells was found after the inhibition of NOS2. It is inhibited that arginine proline metabolic pathway and key gene NOS2 play an important regulatory role in the differentiation of ESCs into male germ cells.
【作者单位】： 扬州大学动物科学与技术学院/江苏省动物繁育与分子设计重点实验室;
【基金】：国家自然科学基金(31272429) 高等学校博士学科点专项科研基金资助课题(20103250110006) 江苏省“六大人才高峰” 江苏省优势学科
【分类号】：S831
本文编号：2135573
[Abstract]:[Objective] to explore the mechanism of protein metabolism in the differentiation of male reproductive cells of chickens, and to provide a basis for improving the differentiation system of embryonic stem cell (ESC) to the male reproductive cells in vitro. [Methods] high purity ESC was obtained by flow separation method, and the original germ cells (primordial germ) were obtained. Cells, PGCs) and spermatogonial stem cells (spermatogonia stem cell, SSCs), respectively extract the total RNA of the cells. The RNA-Seq method was used to sequenced the transcriptional transcripts of the 3 cells, and then the WEGO (WEB gene ontology) and the enrichment analysis were carried out to screen the differentiation process of the male reproductive cells. The key pathway and key genes involved in protein metabolism, RT-q PCR (Real time Quantitative PCR), were used to detect the changes in the expression of some key differentially differentially genes, and compared with the results of RNA-Seq (RNA sequencing). At the same time, the key basis was suppressed by NOS2 in vivo and in vitro, and the cell shape of different days in each group was observed. Changes in the expression of NOS2 and C-kit, Cvh, Stra8, Dazl, integrin alpha 6 and integrin beta 1. [results] in the whole stage of the differentiation of male reproductive cells, there were 697 different genes involved in biological metabolism, significantly enriched in the arginine proline metabolite pathway, tyrosine metabolic pathway and tryptophan Xie Tong The key genes such as NOS2, FAH and IDO were screened on these 3 pathways. The expression of these genes in the process of ESCs to SSCs was consistent with the results in RNA-Seq. After the inhibition of the NOS2 gene in vivo, the reproductive marker genes such as NOS2 and C-kit, Cvh, Stra8, Dazl, alpha 6 and beta 1 were not found between the blank group and the control group. In the inhibitor group, in the inhibitor group, the m RNA expression of NOS2 and C-kit, Cvh, Stra8, Dazl, integrin alpha 6 and integrin beta 1 all decreased, and the ESCs in the control group was proliferating in 2,4,6,8 and inner cells after inhibiting the NOS2 gene in the control group, but there was a small embryoid body in the induction group, 4 The D embryoid body increased, and the number increased, the edge of 6D type embryo began to rupture, 8D like body was disintegrated, and 10d appeared sperm like primitive cells. In the inhibitor group, ESCs had no embryoid body in 2,4,6,8 and 10d, and the cell proliferation was slow compared to the control group. In RA+ inhibitor group, no embryoid body appeared in 2 and 4D, the cell proliferation was slow, 6D appeared small embryoid. The number of 8D embryoid bodies increased slightly and the volume slightly increased, and the 10d embryoid body began to cleavage. And after inhibition of the inhibitors, the expression of NOS2 and C-kit, Cvh, Stra8, Dazl, integrin a 6 and integrin beta 1 were expressed in the RA induction group, and the inhibitor group and the RA+ inhibitor group were both significant or significant relative to the control group. [Conclusion] Based on RNA-Seq and bioinformatics methods, based on the screening of the arginine proline metabolic pathway and the key gene NOS2 during the differentiation of male reproductive cells from ESCs to the male reproductive cells, the inhibition of the NOS2 gene in the chicken and in vitro was found, and the differentiation process of ESCs into the male reproductive cells was found after the inhibition of NOS2. It is inhibited that arginine proline metabolic pathway and key gene NOS2 play an important regulatory role in the differentiation of ESCs into male germ cells.
【作者单位】： 扬州大学动物科学与技术学院/江苏省动物繁育与分子设计重点实验室;
【基金】：国家自然科学基金(31272429) 高等学校博士学科点专项科研基金资助课题(20103250110006) 江苏省“六大人才高峰” 江苏省优势学科
【分类号】：S831
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