BMP15基因对绒山羊mGSCs增殖分化的影响

发布时间：2018-10-17 09:53

【摘要】：骨形态发生蛋白15(Bone morphogenetic protein 15,BMP15)是转化生长因子β(transforming growth factorβ,TGFβ)超家族成员,早期研究认为BMP15只特异性的在卵母细胞中表达,对雌性动物的繁殖力有重要的调控作用。但随着研究的深入,发现在一些物种的垂体、下丘脑、睾丸及其他组织中发现有BMP15表达,提示BMP15可能调节其他组织的发育。但目前对BMP15在卵巢上的功能作用研究较多,而在垂体、下丘脑、睾丸及其他组织中的功能作用研究较少。本研究通过BMP15基因及其编码蛋白的结构和功能预测,过表达BMP15对mGSCs增殖和分化的影响,为进一步研究BMP15蛋白的功能及其在睾丸发育中的功能作用提供参考。本研究主要获得以下结果:1.采用混合酶消化、差速贴壁的方法分离纯化了陕北白绒山羊mGSCs。差速贴壁培养7 d后经AKP染色,发现阳性克隆约占60%,经CD49f和Oct4免疫组织化学染色后,发现细胞CD49f和Oct4大量表达,表明得到mGSCs纯度较高。悬滴培养未分化mGSCs,发现拟胚体(EBs)出现,将EBs贴壁培养7d后,发现有脂肪、成骨和软骨细胞出现,确定体外培养的陕北白绒山羊mGSCs有较好的分化潜能。2.运用生物信息学分析软件分析BMP15基因序列及其编码蛋白的结构功能表明,山羊BMP15基因编码区长1185 bp,编码394个氨基酸,其基因序列及蛋白序列与绵羊、牛的同源性分别为99%和98%,亲缘关系近。根据信号肽分析,预测出在25和26位氨基酸之间可能存在最佳剪切位点,D值为0.815,提示BMP15蛋白可能是生长因子,属于分泌性蛋白,具有信号转导功能。利用Smart在线网站程序对BMP15蛋白功能结构域进行预测分析,表明BMP15有TGFβ功能结构域,位于293-394氨基酸区域。利用ProtFun在线网站程序对BMP15蛋白功能预测,得到山羊BMP15具有细胞外被膜功能的可能性最大,达到12.822,推测其合成后可能被转运到细胞外膜上进行功能反应。基因本体范畴分析发现,BMP15作为激素在生理活动中传递信息的可能性最大。3.通过RT-PCR检测BMP15在成年绒山羊不同组织中的表达量,发现BMP15在公羊睾丸、十二指肠、瘤胃等组织上均有一定量的表达,因此推测BMP15在睾丸等组织生理过程中有调节作用。在mGSCs中过表达BMP15后发现c-Kit表达量有所增加,PLZF表达量有所降低,但均与对照组差异不显著(P0.05),推测BMP15对mGSCs增殖分化有一定的影响。本研究结果表明,体外培养的绒山羊mGSCs具有较好的多向分化的潜能;BMP15有TGFβ功能结构域,具有信号转导功能,对mGSCs增殖分化有一定的影响。
[Abstract]:Bone morphogenetic protein 15 (Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor 尾 (TGF 尾) superfamily. Early studies showed that BMP15 was expressed only specifically in oocytes and played an important role in regulating the reproductive ability of female animals. However, BMP15 expression was found in pituitary, hypothalamus, testis and other tissues of some species, suggesting that BMP15 may regulate the development of other tissues. However, there are many studies on the function of BMP15 in ovary, but less on pituitary, hypothalamus, testis and other tissues. This study predicted the structure and function of BMP15 gene and its encoded protein and the effect of overexpression of BMP15 on the proliferation and differentiation of mGSCs. This study provides a reference for further study on the function of BMP15 protein and its function in testicular development. The main results of this study are as follows: 1. MGSCs. of Northern Shaanxi White Cashmere Goat was isolated and purified by mixed enzyme digestion and differential adherence. After 7 days of differential adherent culture, the positive clones were found to be about 60% by AKP staining. After CD49f and Oct4 immunohistochemical staining, the high expression of CD49f and Oct4 was found, which indicated that the purity of mGSCs was high. (EBs) was found in undifferentiated mGSCs, and fat, osteogenesis and chondrocytes were found in EBs adherent culture for 7 days. It was confirmed that mGSCs cultured in vitro had good differentiation potential. 2. The structure and function of BMP15 gene and its encoded protein were analyzed by bioinformatics analysis software. The results showed that goat BMP15 gene coding region 1185 bp, encodes 394 amino acids, and its gene sequence and protein sequence are similar to that of sheep. The homology of cattle was 99% and 98% respectively. According to the signal peptide analysis, it was predicted that there might be the best shear site between 25 and 26 amino acids, and the D value was 0.815, which suggested that the BMP15 protein might be a growth factor and a secretory protein with signal transduction function. The functional domain of BMP15 protein was predicted and analyzed by Smart online website program. The results showed that BMP15 had TGF 尾 functional domain, which was located in 293-394 amino acid region. Using ProtFun online website program to predict the function of BMP15 protein, it was found that goat BMP15 had the most possibility of extracellular envelope function, reaching 12.822. It was speculated that goat BMP15 might be transported to the extracellular membrane for functional response after synthesis. Noumenon category analysis showed that BMP15, as a hormone, was most likely to transmit information in physiological activities. RT-PCR was used to detect the expression of BMP15 in different tissues of adult cashmere goats. It was found that BMP15 was expressed in testis, duodenum, rumen and other tissues of male sheep. Therefore, it was speculated that BMP15 could regulate the physiological process of testis. After overexpression of BMP15 in mGSCs, it was found that the expression of c-Kit increased and the expression of PLZF decreased, but there was no significant difference between the two groups (P0.05). It was speculated that BMP15 had a certain effect on the proliferation and differentiation of mGSCs. The results showed that the cultured Cashmere Goat mGSCs had the potential of multidirectional differentiation and BMP15 had TGF 尾 functional domain and signal transduction function, which had a certain effect on the proliferation and differentiation of mGSCs.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

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