蛋白質(zhì)代謝通路對雞雄性生殖細胞分化的調(diào)控
發(fā)布時間：2018-07-21 12:45
【摘要】：【目的】探究蛋白質(zhì)代謝在雞雄性生殖細胞分化過程中的作用機制,為完善雞胚胎干細胞(embryonic stem cell,ESC)體外向雄性生殖細胞誘導分化體系研究提供依據(jù)�！痉椒ā坎捎昧魇椒诌x的方法獲取高純度的ESC、原始生殖細胞(primordial germ cells,PGCs)和精原干細胞(spermatogonia stem cell,SSCs),分別提取細胞的總RNA,采用RNA-Seq方法對3種細胞的轉(zhuǎn)錄本進行深度測序,然后進行WEGO(web gene ontology)和KEGG(kyoto encyclopedia of genes and genomes)通路富集分析,篩選出雞雄性生殖細胞分化過程中參與蛋白質(zhì)代謝的關鍵通路及其關鍵基因,RT-q PCR(Real time Quantitative PCR)檢測部分關鍵差異基因的表達變化,并與RNA-Seq(RNA sequencing)結(jié)果進行比較分析,同時分別從體內(nèi)和體外水平對關鍵基因NOS2進行抑制,觀察各分組不同天數(shù)的細胞形態(tài)變化及檢測NOS2和C-kit、Cvh、Stra8、Dazl、integrinα6和integrinβ1等生殖標記基因表達變化情況�！窘Y(jié)果】在雄性生殖細胞分化的整個階段,有697個差異基因參與生物代謝,顯著富集于精氨酸-脯氨酸代謝通路、酪氨酸代謝通路以及色氨酸代謝通路,在這3條通路上篩選出NOS2、FAH和IDO等關鍵性基因,這些基因的在ESCs向SSCs分化過程中表達變化趨勢與其在RNA-Seq中的結(jié)果一致。體內(nèi)抑制NOS2基因后,NOS2及C-kit、Cvh、Stra8、Dazl、integrinα6和integrinβ1等生殖標記基因在空白組和對照組之間無顯著性的差異,而在抑制劑組中,NOS2及C-kit、Cvh、Stra8、Dazl、integrinα6和integrinβ1等生殖標記基因的m RNA表達量均出現(xiàn)了降低;而體外抑制NOS2基因后,對照組中的ESCs在2、4、6、8和10d內(nèi)細胞不斷增殖,但是未出現(xiàn)類胚體;RA誘導組中,2d出現(xiàn)小的類胚體,4d類胚體增大,且數(shù)量增多,6d類胚體邊緣開始出現(xiàn)破裂,8d類胚體解體,10d出現(xiàn)類精原樣細胞;抑制劑組中,ESCs在2、4、6、8和10d內(nèi)無類胚體出現(xiàn),且相較于對照組細胞增殖緩慢;RA+抑制劑組中,2和4d內(nèi)無類胚體出現(xiàn),細胞增殖緩慢,6d出現(xiàn)小的類胚體,8d類胚體數(shù)量少量增多,且體積稍顯增大,10d類胚體開始裂解。并且經(jīng)過抑制劑的抑制后,NOS2及C-kit、Cvh、Stra8、Dazl、integrinα6和integrinβ1等生殖標記基因的表達量在RA誘導組、抑制劑組和RA+抑制劑組中相對于對照組均呈顯著性或極顯著性的下調(diào)趨勢�！窘Y(jié)論】基于RNA-Seq技術及生物信息學方法篩選出ESCs向雄性生殖細胞分化過程中精氨酸-脯氨酸代謝通路及關鍵基因NOS2的基礎上,通過對NOS2基因在雞體內(nèi)和體外的抑制,發(fā)現(xiàn)NOS2在被抑制后,ESCs向雄性生殖細胞分化的過程受到抑制。說明了精氨酸-脯氨酸代謝通路及關鍵基因NOS2對ESCs向雄性生殖細胞分化過程中起到重要的調(diào)節(jié)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.
【作者單位】： 揚州大學動物科學與技術學院/江蘇省動物繁育與分子設計重點實驗室;
【基金】：國家自然科學基金(31272429) 高等學校博士學科點專項科研基金資助課題(20103250110006) 江蘇省“六大人才高峰” 江蘇省優(yōu)勢學科
【分類號】：S831
本文編號：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.
【作者單位】： 揚州大學動物科學與技術學院/江蘇省動物繁育與分子設計重點實驗室;
【基金】：國家自然科學基金(31272429) 高等學校博士學科點專項科研基金資助課題(20103250110006) 江蘇省“六大人才高峰” 江蘇省優(yōu)勢學科
【分類號】：S831
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