miR-21在鸭胚胎期胸肌发育中的功能研究
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摘要
旨在通过研究鸭miR-21的分子特征、时空表达模式以及对鸭骨骼肌卫星细胞的增殖与分化的影响,为探索骨骼肌发育机制提供支撑。本研究选取180枚(98±5)g鸭蛋,相同条件孵化。从孵化第11天到第27天每天取3枚鸭蛋(分别表示为:E11、E12、E13……E26、E27),无菌状态下逐只采集鸭胚胸肌样品。E27时另取3只鸭胚逐只采集腿肌、心、肝、肾、肌胃、小肠、腹脂和皮脂。混合酶消化法和差速贴壁法分离和纯化骨骼肌卫星细胞,骨骼肌卫星细胞增殖、分化时期分别过表达和干扰miR-21表达,利用荧光定量、蛋白杂交、EdU检测及流式细胞术等检测miR-21对鸭骨骼肌卫星细胞增殖和分化的影响。qRT-PCR结果显示,miR-21的表达随着孵化时间的增加而增加,E19天时到达最高,然后逐渐降低;E27时,miR-21在肝中的表达量最高。在增殖的鸭骨骼肌卫星细胞中过表达miR-21,荧光定量PCR检测CDK2和CyclinD1基因的mRNA表达量分别显著(P<0.05)和极显著增加(P<0.01),EdU检测阳性细胞数目显著增加(P<0.05),流式细胞仪检测发现G2期细胞数目极显著降低(P<0.01)、S期细胞数量显著增加(P<0.05)。在增殖的骨骼肌卫星细胞中,干扰miR-21表达,所得结果相反。在分化的鸭骨骼肌卫星细胞中过表达miR-21,qRT-PCR检测MyoG和MyHC基因的mRNA表达量均极显著增加(P<0.01);蛋白杂交(WB)检测结果显示,MyoG蛋白表达显著增加(P<0.05),MyHC蛋白表达极显著增加(P<0.01)。免疫荧光检测发现,MyHC阳性细胞数和10核以上的肌管数都极显著增加(P<0.01)。在分化的鸭骨骼肌卫星细胞中,干扰miR-21的表达,所得结果相反。结果表明,miR-21具有促进成肌细胞鸭骨骼肌卫星细胞增殖和分化的作用。
The molecular characteristics,the spatiotemporal expression patterns and the function of miR-21 during the proliferation and differentiation of duck skeletal muscle satellite cells were studied to provide support for exploring the mechanism of skeletal muscle development.One hundred and eighty eggs with the weight of(98±5)g were hatched under the same conditions.The embryo breast muscle samples were collected from 3 hatched ducks from 11 th day to 27 th day of hatching(E11,E12,E13……E26,E27)under sterile condition,respectively.The leg muscle,heart,liver,kidney,stomach,small intestine,abdominal fat and sebum were sampled from hatched eggs on E27 under sterile condition.The skeletal muscle satellite cells were isolated and purified by mixed enzyme digestion and differential adherence methods.The role of miR-21 was explored by fluorescence quantitative analysis,protein hybridization,EdU detection and flow cytometry during the proliferation and differentiation of duck skeletal muscle satellite cells transfected with Agomir and Antagomir of miR-21.The results of qRT-PCR showed that the expression of miR-21 increased gradually with the increase of incubation age,reached the highest level on E19,and then decreased gradually.In addition,the highest level of miR-21 expression was found in liver on E27.miR-21 was overexpressed in the proliferating duck skeletal muscle satellite cells,the mRNA expression levels of CDK2 and CyclinD1 significantly(P<0.05)and extremely significantly increased(P<0.01),respectively.The number of positive cells detected by EdU was significantly increased(P<0.05).The number of G2 phase cells was extremely significantly decreased(P<0.01),and the number of S phase cells was significantly increased(P<0.05)by flow cytometry analysis.When the expression of miR-21 was suppressed in the proliferating duck skeletal muscle satellite cells,the results were contrary to that of miR-21 overexpression in the proliferating duck skeletal muscle satellite cells.When the miR-21 was overexpressed in the differentiated duck skeletal muscle satellite cells,the mRNA expression of MyoGand MyHC were extremely significantly increased(P<0.01)by qRT-PCR.The protein expression of MyoG was siginificantly increased(P<0.05),and the protein expression of MyHC was extremely significantly increased(P<0.01)by Western blot(WB)analysis.Both the number of MyHC positive cells and the number of myotubes containing more than 10 nuclei were extremely significantly increased(P<0.01)detected by immunofluorescence assay.The results were contrary to that of miR-21 was suppressed in the differentiated duck skeletal muscle satellite cells.The results indicate that miR-21 can promote the proliferation and differentiation of skeletal muscle satellite cells of myoblasts of ducks.
The molecular characteristics,the spatiotemporal expression patterns and the function of miR-21 during the proliferation and differentiation of duck skeletal muscle satellite cells were studied to provide support for exploring the mechanism of skeletal muscle development.One hundred and eighty eggs with the weight of(98±5)g were hatched under the same conditions.The embryo breast muscle samples were collected from 3 hatched ducks from 11 th day to 27 th day of hatching(E11,E12,E13……E26,E27)under sterile condition,respectively.The leg muscle,heart,liver,kidney,stomach,small intestine,abdominal fat and sebum were sampled from hatched eggs on E27 under sterile condition.The skeletal muscle satellite cells were isolated and purified by mixed enzyme digestion and differential adherence methods.The role of miR-21 was explored by fluorescence quantitative analysis,protein hybridization,EdU detection and flow cytometry during the proliferation and differentiation of duck skeletal muscle satellite cells transfected with Agomir and Antagomir of miR-21.The results of qRT-PCR showed that the expression of miR-21 increased gradually with the increase of incubation age,reached the highest level on E19,and then decreased gradually.In addition,the highest level of miR-21 expression was found in liver on E27.miR-21 was overexpressed in the proliferating duck skeletal muscle satellite cells,the mRNA expression levels of CDK2 and CyclinD1 significantly(P<0.05)and extremely significantly increased(P<0.01),respectively.The number of positive cells detected by EdU was significantly increased(P<0.05).The number of G2 phase cells was extremely significantly decreased(P<0.01),and the number of S phase cells was significantly increased(P<0.05)by flow cytometry analysis.When the expression of miR-21 was suppressed in the proliferating duck skeletal muscle satellite cells,the results were contrary to that of miR-21 overexpression in the proliferating duck skeletal muscle satellite cells.When the miR-21 was overexpressed in the differentiated duck skeletal muscle satellite cells,the mRNA expression of MyoGand MyHC were extremely significantly increased(P<0.01)by qRT-PCR.The protein expression of MyoG was siginificantly increased(P<0.05),and the protein expression of MyHC was extremely significantly increased(P<0.01)by Western blot(WB)analysis.Both the number of MyHC positive cells and the number of myotubes containing more than 10 nuclei were extremely significantly increased(P<0.01)detected by immunofluorescence assay.The results were contrary to that of miR-21 was suppressed in the differentiated duck skeletal muscle satellite cells.The results indicate that miR-21 can promote the proliferation and differentiation of skeletal muscle satellite cells of myoblasts of ducks.
引文
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