【摘要】：骨形態(tài)發(fā)生蛋白15(Bone morphogenetic protein 15,BMP15)是轉(zhuǎn)化生長(zhǎng)因子β(transforming growth factorβ,TGFβ)超家族成員,早期研究認(rèn)為BMP15只特異性的在卵母細(xì)胞中表達(dá),對(duì)雌性動(dòng)物的繁殖力有重要的調(diào)控作用。但隨著研究的深入,發(fā)現(xiàn)在一些物種的垂體、下丘腦、睪丸及其他組織中發(fā)現(xiàn)有BMP15表達(dá),提示BMP15可能調(diào)節(jié)其他組織的發(fā)育。但目前對(duì)BMP15在卵巢上的功能作用研究較多,而在垂體、下丘腦、睪丸及其他組織中的功能作用研究較少。本研究通過(guò)BMP15基因及其編碼蛋白的結(jié)構(gòu)和功能預(yù)測(cè),過(guò)表達(dá)BMP15對(duì)mGSCs增殖和分化的影響,為進(jìn)一步研究BMP15蛋白的功能及其在睪丸發(fā)育中的功能作用提供參考。本研究主要獲得以下結(jié)果:1.采用混合酶消化、差速貼壁的方法分離純化了陜北白絨山羊mGSCs。差速貼壁培養(yǎng)7 d后經(jīng)AKP染色,發(fā)現(xiàn)陽(yáng)性克隆約占60%,經(jīng)CD49f和Oct4免疫組織化學(xué)染色后,發(fā)現(xiàn)細(xì)胞CD49f和Oct4大量表達(dá),表明得到mGSCs純度較高。懸滴培養(yǎng)未分化mGSCs,發(fā)現(xiàn)擬胚體(EBs)出現(xiàn),將EBs貼壁培養(yǎng)7d后,發(fā)現(xiàn)有脂肪、成骨和軟骨細(xì)胞出現(xiàn),確定體外培養(yǎng)的陜北白絨山羊mGSCs有較好的分化潛能。2.運(yùn)用生物信息學(xué)分析軟件分析BMP15基因序列及其編碼蛋白的結(jié)構(gòu)功能表明,山羊BMP15基因編碼區(qū)長(zhǎng)1185 bp,編碼394個(gè)氨基酸,其基因序列及蛋白序列與綿羊、牛的同源性分別為99%和98%,親緣關(guān)系近。根據(jù)信號(hào)肽分析,預(yù)測(cè)出在25和26位氨基酸之間可能存在最佳剪切位點(diǎn),D值為0.815,提示BMP15蛋白可能是生長(zhǎng)因子,屬于分泌性蛋白,具有信號(hào)轉(zhuǎn)導(dǎo)功能。利用Smart在線網(wǎng)站程序?qū)MP15蛋白功能結(jié)構(gòu)域進(jìn)行預(yù)測(cè)分析,表明BMP15有TGFβ功能結(jié)構(gòu)域,位于293-394氨基酸區(qū)域。利用ProtFun在線網(wǎng)站程序?qū)MP15蛋白功能預(yù)測(cè),得到山羊BMP15具有細(xì)胞外被膜功能的可能性最大,達(dá)到12.822,推測(cè)其合成后可能被轉(zhuǎn)運(yùn)到細(xì)胞外膜上進(jìn)行功能反應(yīng)�；虮倔w范疇分析發(fā)現(xiàn),BMP15作為激素在生理活動(dòng)中傳遞信息的可能性最大。3.通過(guò)RT-PCR檢測(cè)BMP15在成年絨山羊不同組織中的表達(dá)量,發(fā)現(xiàn)BMP15在公羊睪丸、十二指腸、瘤胃等組織上均有一定量的表達(dá),因此推測(cè)BMP15在睪丸等組織生理過(guò)程中有調(diào)節(jié)作用。在mGSCs中過(guò)表達(dá)BMP15后發(fā)現(xiàn)c-Kit表達(dá)量有所增加,PLZF表達(dá)量有所降低,但均與對(duì)照組差異不顯著(P0.05),推測(cè)BMP15對(duì)mGSCs增殖分化有一定的影響。本研究結(jié)果表明,體外培養(yǎng)的絨山羊mGSCs具有較好的多向分化的潛能;BMP15有TGFβ功能結(jié)構(gòu)域,具有信號(hào)轉(zhuǎn)導(dǎo)功能,對(duì)mGSCs增殖分化有一定的影響。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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本文編號(hào)：2276290