肌特异性miRNA在肌细胞分化中的作用及对肌营养不良的影响

发布时间：2018-04-10 18:45

本文选题：miRNA + 肌营养不良　；参考：《福建医科大学》2014年硕士论文

【摘要】：【目的】系统检测肌特异性miRNA在肌细胞增殖和分化成熟过程中的变化以及在肌营养不良病人中的表达，初步探讨miRNA与肌营养不良的关系。结合细胞学实验以及生物信息学网站，探讨miRNA过表达对成肌细胞分化及靶基因表达的影响，为进一步研究miRNA在肌营养不良发病过程中的作用奠定基础。【方法】 1.培养成肌细胞株C2C12，实时定量PCR(qRT-PCR)检测肌特异性miRNA（-1，-133a，-206）在肌细胞增殖期和分化过程中的表达。 2.结合临床表现、HE染色和肌酶组化、特殊染色方法，筛选肌营养不良病例。以非特异性改变肌组织为对照组，应用qRT-PCR检测肌营养不良病人肌特异性miRNA（-1，-133a，-206）的表达。 3.通过以上实验筛选出对成肌细胞分化及肌营养不良影响较为明显的肌特异性miRNA，应用生物信息学软件预测该miRNA可能作用的靶基因。 4.合成该miRNA的mimic，转染成肌细胞C2C12，利用MTT法检测过表达该miRNA对C2C12细胞生长增殖的影响，光学显微镜观察成肌细胞生长分化情况，同时qRT-PCR、Western blot进行靶基因表达检测。【结果】 1. miR-1，-133a，-206在增殖期的表达量较低，与增殖期比较，随着成肌细胞分化时间的延长，肌特异性miR-1，-133a，-206表达均显著升高（P值分别为0.0002、0.005、0.0001），其中miR-1表达升高最明显。 2.筛选8例肌营养不良标本，，与非特异性改变肌组织对比，miR-1，-133a，-206表达均升高，其中miR-206在肌营养不良病人的肌组织表达升高最显著（P=0.04）。 3.通过miRDB、miRNA.org、TargetScanHuman6.2、Pictar等生物信息学软件预测miR-206在肌细胞分化及肌营养不良发病过程的靶基因Pax7、Pax3、Cx43、Hmgb3。 4. C2C12细胞转染miR-206mimic后，qRT-PCR显示Pax7、Pax3、Cx43、Hmgb3表达明显下降。Western blot显示Pax7、Pax3、Cx43、Hmgb3蛋白表达下降。 5. C2C12细胞转染miR-206mimic后，光学显微镜观察细胞增殖受到明显抑制。与阴性对照（NC组）相比，MTT法检测显示C2C12抑制率为（21.35±0.0068）%（P=0.004）【结论】 1. miR-1、miR-133a和miR-206随着成肌细胞的分化成熟，表达水平逐步增高。 2.在肌特异性miRNA中，miR-206在肌营养不良的病理生理过程中发挥更为重要的作用。 3. miR-206通过调节多个靶基因的表达参与成肌细胞增殖和分化过程。
[Abstract]:[objective] to investigate the relationship between miRNA and myodystrophy by systematically detecting the changes of myo-specific miRNA in the process of myocyte proliferation and differentiation and maturation, as well as the expression of miRNA in patients with muscular dystrophy.Combined with cytological experiments and bioinformatics website, the effects of miRNA overexpression on myoblast differentiation and target gene expression were studied, which laid a foundation for further study of the role of miRNA in the pathogenesis of muscular dystrophy.[methods]1.The expression of myogenic cell line C2C12 was assayed by real-time quantitative PCRQRT-PCR. The expression of myo-specific miRNA-1r-133a- 206 was detected during the proliferation and differentiation of myocytes.2.Cases of muscular dystrophy were screened with HE staining and enzyme histochemistry and special staining.QRT-PCR was used to detect the expression of myospecific miRNA-1mRNA-133a- 206 in patients with muscular dystrophy.3.The specific miRNAs which had obvious effects on myoblast differentiation and muscular dystrophy were screened by the above experiments. The target gene of miRNA was predicted by bioinformatics software.4.The mimicic of the miRNA was synthesized and transfected into the myoblast C2C12. The effect of the miRNA expression on the growth and proliferation of C2C12 cells was detected by MTT assay. The growth and differentiation of the myoblasts were observed by optical microscope, and the target gene expression was detected by qRT-PCR- Western blot.[results]2.8 cases of muscular dystrophy were selected and compared with non-specific changes in muscle tissue, the expression of miR-1 + -133a- 206 was increased, and the expression of miR-206 in muscle tissue of patients with muscular dystrophy was the most significant (P < 0.04).3.Bioinformatics software such as miRDBN miRNA.orgScanHuman6.2Pictar was used to predict the target gene Pax7Pax3Cx43 (Hmgb3Hmgb3) of miR-206 in myocyte differentiation and the pathogenesis of muscular dystrophy.4.After C2C12 cells were transfected with miR-206mimic, the expression of Pax7, Pax3Cx43, Hmgb3 and Hmgb3 protein in Pax7, Pax3, Pax3Cx43, Cx43 and Hmgb3 were detected by Western blot.5.The proliferation of C2C12 cells was inhibited by optical microscope after transfection of C2C12 cells into miR-206mimic.The inhibition rate of C2C12 was 21. 35 卤0. 0068% (P < 0. 004).[conclusion]1. The expression of miR-1 miR-133a and miR-206 gradually increased with the differentiation and maturation of myoblasts.2.MiR-206 plays a more important role in the pathophysiological process of muscular dystrophy in myo-specific miRNA.3. MiR-206 participates in the proliferation and differentiation of myoblasts by regulating the expression of multiple target genes.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R746.2

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