【摘要】：本試驗(yàn)以大通牦牛和天祝白牦牛兩種毛色牦牛為研究對(duì)象,采用了熒光定量PCR、石蠟切片、建立B16細(xì)胞模型等試驗(yàn)方法。利用熒光定量PCR技術(shù)測定Agouti、MC1R、MITF和TY尺基因在不同毛色牦牛皮膚組織中相對(duì)表達(dá)量;通過甲苯胺藍(lán)和HE染色方法觀察黑色素顆粒在牦牛不同毛色皮膚組織中分布;用B16細(xì)胞作為生物模型,特異性shRNA重組質(zhì)粒載體導(dǎo)入B16細(xì)胞抑制MC1R基因mRNA表達(dá)量,然后對(duì)轉(zhuǎn)染B16細(xì)胞Agouti、MC1R、MITF和TYR基因表達(dá)量進(jìn)行測定和測定B16細(xì)胞中黑色素顆粒含量。研究結(jié)果如下:(1)實(shí)時(shí)熒光定量PCR分析得到,Agouti基因表達(dá)量大通牦牛與天祝白牦牛無顯著差異;MC1R基因表達(dá)量大通牦牛是天祝白牦牛的2.4倍,差異極顯著(P0.01);MITF基因表達(dá)量在天祝白牦牛比大通牦牛高,差異顯著;TYR基因表達(dá)量大通牦牛比天祝白牦牛高,差異顯著。TYR對(duì)黑色被毛有一定影響,MITF對(duì)白色被毛有一定影響;推測黑色被毛與MC1R的高表達(dá)量有重要關(guān)系。(2)甲苯胺藍(lán)染色比HE染色較容易觀察到黑色素顆粒的分布和皮膚分層結(jié)構(gòu),但對(duì)黑色素細(xì)胞進(jìn)行染色不理想;HE染色對(duì)黑色素細(xì)胞染色充分,細(xì)胞核和細(xì)胞質(zhì)區(qū)分明顯,能夠看到黑色素細(xì)胞的細(xì)胞核和細(xì)胞質(zhì),黑色素顆粒在細(xì)胞中分布。黑色素細(xì)胞出現(xiàn)在表皮與真皮連接處,另外毛囊周圍也大量分布。HE染色顯示大通牦牛毛囊中與毛發(fā)相連毛乳頭附近的黑色素含量比毛囊周圍其他部位含量高。表皮層黑色素細(xì)胞有形成空泡趨勢,呈現(xiàn)不規(guī)則形態(tài),其一般細(xì)胞核比較大。天祝白牦牛表皮的基底層發(fā)現(xiàn)少量黑色素顆粒分布,但在毛囊毛球部及周圍沒有黑色素顆粒分布。在大通牦牛中大量黑色素顆粒分布在表皮基底層和毛囊周圍。白毛色是天祝白牦牛毛囊毛球部的黑色素細(xì)胞無法生成黑色素顆粒,致使毛纖維中不存在黑色素顆粒呈現(xiàn)白色。(3)本研究用B16細(xì)胞作為生物模型,用shRNA對(duì)細(xì)胞中的目的基因MC1R表達(dá)抑制。測定細(xì)胞中黑色素顆粒含量,轉(zhuǎn)染shRNA重組質(zhì)粒后的細(xì)胞形態(tài)沒有發(fā)生變化,活性沒有受到影響。試驗(yàn)分為三組:對(duì)目的基因特異性抑制的shRNA重組質(zhì)粒試驗(yàn)組、含有無效序列shRNA重組質(zhì)粒陰性對(duì)照組和空白對(duì)照組。轉(zhuǎn)染特異性shRNA重組質(zhì)粒后試驗(yàn)組B16細(xì)胞合成的黑色素顆粒含量降低,與空白對(duì)照組和陰性對(duì)照組差異極顯著。從蛋白水平,空白對(duì)照和陰性對(duì)照分別是試驗(yàn)組的7.5倍和6.83倍,抑制率達(dá)到87%。MC1R基因表達(dá)量空白對(duì)照和陰性對(duì)照分別是試驗(yàn)組的11.21倍和10.19倍,試驗(yàn)組表達(dá)量低于其它兩組差異極顯著(P0.01),抑制率91%以上。天祝白牦牛毛囊分布著黑色素細(xì)胞及表皮少量黑色素顆粒;MC1R基因表達(dá)量對(duì)黑色素顆粒的形成起著重要的調(diào)控作用。
[Abstract]:In this experiment, Datong yak and Tianzhu white yak were used as research objects. Fluorescence quantitative PCR, paraffin sections were used to establish B16 cell model. Fluorescence quantitative PCR technique was used to detect the relative expression of Agouti,MC1R,MITF and TY ulnar genes in different hairy yak skin tissues, and to observe the distribution of melanin granules in yak skin tissues with different coat color by toluidine blue and HE staining. B16 cells were used as biological model. The specific shRNA recombinant plasmid vector was introduced into B16 cells to inhibit the expression of MC1R gene mRNA. Then the expression of Agouti,MC1R,MITF and TYR genes in B16 cells was measured and the content of melanin granules in B16 cells was measured. The results were as follows: (1) the expression of Agouti gene in Datong yak and Tianzhu white yak was not significantly different from that in Tianzhu white yak by real-time fluorescence quantitative PCR analysis, and the expression of MC1R gene in Datong yak was 2.4 times higher than that in Tianzhu white yak (P0.01). The expression of MITF gene in Tianzhu white yak was higher than that in Datong yak, and the expression of TYR gene in Datong yak was higher than that in Tianzhu white yak, the difference was significant. TYR had a certain effect on black coat, MITF on white coat. (2) toluidine blue staining was easier than HE staining to observe the distribution of melanin particles and skin layering structure, but the staining of melanocytes was not ideal; HE staining showed that the melanocytes were stained well and the nuclei and cytoplasm were distinguished clearly. The melanocytes and cytoplasm of melanocytes could be seen and melanin granules were distributed in the cells. Melanocytes appeared at the junction of epidermis and dermis and were also distributed around hair follicles. HE staining showed that melanin content near hair papilla was higher in Datong yak hair follicles than in other parts around hair follicles. The melanocytes in the epidermis tend to form vacuoles, showing irregular morphology, and their nuclei are relatively large. A small amount of melanin granules were found in the basal layer of the epidermis of Tianzhu white yak, but there were no melanin granules in and around the hair follicle. In Datong yak, a large number of melanin granules were distributed in the basal layer of epidermis and around the hair follicle. White coat color is the melanocyte of hair follicle of Tianzhu white yak can not produce melanin granules, resulting in the absence of melanin granules in hair fibers. (3) B16 cells were used as biological model in this study. The expression of target gene MC1R was inhibited by shRNA. The content of melanin granules in the cells was determined. The morphology of the cells transfected with shRNA recombinant plasmid was not changed and the activity of the cells was not affected. The experiment was divided into three groups: the shRNA recombinant plasmid group which specifically inhibited the target gene, the negative control group containing invalid shRNA recombinant plasmid and the blank control group. After transfection of specific shRNA recombinant plasmid, the content of melanin granules in B16 cells in the experimental group was significantly lower than that in the blank control group and the negative control group. The protein levels of the blank control and negative control were 7.5 times and 6.83 times of that of the experimental group, respectively, and the inhibition rate was 11.21 times and 10.19 times higher than that of the control and negative control, respectively. The expression level in the experimental group was significantly lower than that in the other two groups (P0.01), and the inhibitory rate was over 91%. Melanocytes and a small amount of melanin granules were distributed in the hair follicles of Tianzhu white yak, and the expression of MC1R gene played an important role in the formation of melanin granules.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S823.85

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 Junsei Taira;Eito Tsuchida;Masatsugu Uehara;Natsuko Ohhama;Wakana Ohmine;Takayuki Ogi;;The leaf extract of Mallotus japonicus and its major active constituent, rutin,suppressed on melanin production in murine B16F1 melanoma[J];Asian Pacific Journal of Tropical Biomedicine;2015年10期

2 鄭嫩珠;辛清武;朱志明;繆中緯;李麗;劉鳳輝;黃勤樓;;白絨烏雞MITF基因的cDNA克隆、表達(dá)及其對(duì)黑色素沉積的影響[J];中國農(nóng)業(yè)科學(xué);2015年18期

3 任航行;王高富;陸健;李杰;蔣婧;劉良佳;周鵬;;不同膚色山羊皮膚組織形態(tài)學(xué)及黑色素生成相關(guān)基因的表達(dá)分析[J];畜牧獸醫(yī)學(xué)報(bào);2015年09期

4 敖U_燕;胡玲玲;羅師紅;趙永超;王振;劉若余;;不同毛色貴州水牛MC1R、TYR基因SNP研究[J];中國牛業(yè)科學(xué);2015年04期

5 韓吉龍;岳耀敬;裴杰;郭婷婷;劉建斌;郭健;孫曉萍;馮瑞林;楊博輝;;藏羊刺鼠信號(hào)蛋白基因(Agouti)的多態(tài)性及不同顏色被毛皮膚組織中Agouti與小眼畸形相關(guān)轉(zhuǎn)錄因子基因(MITF)表達(dá)的定量分析[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2013年03期

6 趙光;楊文信;;黑色素細(xì)胞的研究近況[J];西南軍醫(yī);2013年02期

7 王巍;胡紅霞;孫向軍;牛翠娟;;錦鯉酪氨酸酶基因序列分析及其在不同錦鯉品系不同組織中的表達(dá)[J];水產(chǎn)學(xué)報(bào);2012年11期

8 朱亮;蔡月琴;屠玨;陳民利;;不同品種實(shí)驗(yàn)兔虹膜酪氨酸酶基因序列和表達(dá)量的變化[J];中國比較醫(yī)學(xué)雜志;2012年08期

9 任玉紅;楊剛;范瑞文;朱芷葳;董常生;;黑素皮質(zhì)素受體1(MC1R)在不同毛色羊駝皮膚組織中的表達(dá)與定位研究[J];畜牧獸醫(yī)學(xué)報(bào);2012年07期

10 張瑞娜;范瑞文;程志學(xué);田雪;劉佳;高磊;馬崢;董常生;;CDK5對(duì)羊駝皮膚黑色素細(xì)胞TYR和MITF mRNA表達(dá)的調(diào)節(jié)[J];畜牧獸醫(yī)學(xué)報(bào);2011年12期

相關(guān)碩士學(xué)位論文 前3條

1 韓吉龍;藏羊毛色四個(gè)候選基因多態(tài)性及在不同顏色被毛皮膚組織中表達(dá)的定量研究[D];中國農(nóng)業(yè)科學(xué)院;2013年

2 吳偉偉;山羊Agouti基因5’UTR變異及ASIP蛋白表達(dá)定位研究[D];河北農(nóng)業(yè)大學(xué);2012年

3 許華偉;朝鮮鵪鶉MC1R基因克隆及其在不同羽色個(gè)體中表達(dá)水平的研究[D];河南科技大學(xué);2012年

，

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