【摘要】：lpa-miR-nov-66是羊駝皮膚中發(fā)現(xiàn)的新miRNA,在黑色素生成過(guò)程中起重要的調(diào)控作用。a-MSH是一種重要的神經(jīng)內(nèi)分泌激素,通過(guò)調(diào)控MC1R影響皮膚黑色素的生成和分布。本實(shí)驗(yàn)通過(guò)在黑色素細(xì)胞內(nèi)轉(zhuǎn)染lpa-miR-nov-66和外源性添加一定濃度的a-MSH,分析二者在羊駝黑色素細(xì)胞內(nèi)對(duì)黑色素生成的影響,以探討二者的相互作用。主要實(shí)驗(yàn)結(jié)果如下：1用實(shí)時(shí)細(xì)胞檢測(cè)儀監(jiān)測(cè)細(xì)胞增殖,實(shí)驗(yàn)35小時(shí)后,與對(duì)照組相比,各實(shí)驗(yàn)組表現(xiàn)出不同的增殖情況,但沒(méi)有顯著差異。2用qRT-PCR技術(shù)和Western blotting檢測(cè)基因情況和和蛋白表達(dá)量,結(jié)果如下：添加a-MSH后.lpa-miR-nov-66表達(dá)量是正常羊駝黑色素細(xì)胞的0.24±0.012倍,并呈差異極顯著(P0.01)；與對(duì)照組相比,轉(zhuǎn)染組、添加組、轉(zhuǎn)染添加組中MITF的基因和蛋白表達(dá)量分別下降0.16±0.014倍/0.90±0.07倍、2.03±0.051倍/1.24±0.09倍、0.30±0.013倍/1.12±0.06倍,并呈差異極顯著(P0.01); TYR的表達(dá)量分別下降了0.73±0.033倍/0.85±0.05倍、上調(diào)了4.67±0.14倍/1.69±0.14倍、2.54±0.21倍/1.27±0.11倍,并呈差異極顯著(P0.01)。3用ELISA方法檢測(cè)細(xì)胞內(nèi)cGMP和cAMP含量的變化情況,結(jié)果發(fā)現(xiàn)：與對(duì)照組相比,轉(zhuǎn)染組、添加組、轉(zhuǎn)染添加組中cGMP和cAMP的含量分別是對(duì)照組的0.149±0.012倍、2.68±0.17倍和2.267±0.12倍：0.57±0.042倍、2.07±0.19倍和1.36±0.124倍,且均呈差異極顯著(P0.01)。4用酶標(biāo)法檢測(cè)各實(shí)驗(yàn)組中黑色素產(chǎn)量變化,結(jié)果發(fā)現(xiàn)：與對(duì)照組相比,黑色素的產(chǎn)量分別是對(duì)照組的0.58±0.043倍、3.27±0.27倍和1.81±0.16倍,且呈差異極顯著(P0.01)。由此得出結(jié)論：lpa-miR-nov-66和a-MSH協(xié)同作用,通過(guò)調(diào)控羊駝黑色素細(xì)胞中毛色形成的cAMP路徑,從而影響黑色素的產(chǎn)生。
[Abstract]:Lpa-miR-nov-66 is a new miRNA, found in alpaca skin, which plays an important role in melanogenesis. A-MSH is an important neuroendocrine hormone, which affects the production and distribution of melanin in alpaca skin by regulating MC1R. In this study, the effects of lpa-miR-nov-66 and exogenous a / MSH on melanin production in alpaca melanocytes were analyzed in order to explore the interaction between them. The main results were as follows: 1 the proliferation of cells was monitored by real-time cell detector. 35 hours later, compared with the control group, each experimental group showed different proliferation, but there was no significant difference. 2 the gene and protein expression were detected by qRT-PCR and Western blotting, the results were as follows: after adding a-MSH, the expression of LPA-miR-nov-66 was 0.24 鹵0.012 times higher than that of normal alpaca melanocytes. The difference was very significant (P 0.01). Compared with the control group, the expression of MITF gene and protein decreased by 0.16 鹵0.014 times / 0.90 鹵0.07 times, 2.03 鹵0.051 times / 1.24 鹵0.09 times, 0.30 鹵0.013 times / 1.12 鹵0.06 times, respectively, and the difference was very significant (P 0.01). The expression of TYR decreased by 0.73 鹵0.033 times / 0. 85 times, increased by 4. 67 鹵0. 14 times / 1. 69 鹵0. 14 times, 2. 54 鹵0. 21 times / 1. 27 鹵0. 11 times, and the difference was very significant (P01). 3 the changes of cGMP and cAMP contents in the cells were detected by ELISA method, and the results showed that compared with the control group, the transfection group, the addition group, The contents of cGMP and cAMP in the control group were 0.149 鹵0.012 times, 2.68 鹵0.17 times and 2.267 鹵0.12 times as much as those in the control group: 0.57 鹵0.042 times, 2.07 鹵0.19 times and 1.36 鹵0.124 times, respectively, and there were significant differences (P 0.01). 4 the melanin production in each experimental group was detected by enzyme labeling method, and the results showed that the melanin yield was 0.58 鹵0.043 times higher than that in the control group, respectively. 3.27 鹵0.27 times and 1.81 鹵0.16 times, and the difference was very significant (P 0.01). It is concluded that the synergistic effect of lpa-miR-nov-66 and a-MSH affects the production of melanin by regulating the cAMP pathway of hair color formation in alpaca melanocytes.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S829.9

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