本文選題：miRNA + 肌營養(yǎng)不良��； 參考：《福建醫(yī)科大學(xué)》2014年碩士論文

【摘要】：【目的】系統(tǒng)檢測肌特異性miRNA在肌細(xì)胞增殖和分化成熟過程中的變化以及在肌營養(yǎng)不良病人中的表達(dá)，初步探討miRNA與肌營養(yǎng)不良的關(guān)系。結(jié)合細(xì)胞學(xué)實(shí)驗(yàn)以及生物信息學(xué)網(wǎng)站，探討miRNA過表達(dá)對成肌細(xì)胞分化及靶基因表達(dá)的影響，為進(jìn)一步研究miRNA在肌營養(yǎng)不良發(fā)病過程中的作用奠定基礎(chǔ)。 【方法】 1.培養(yǎng)成肌細(xì)胞株C2C12，實(shí)時(shí)定量PCR(qRT-PCR)檢測肌特異性miRNA（-1，-133a，-206）在肌細(xì)胞增殖期和分化過程中的表達(dá)。 2.結(jié)合臨床表現(xiàn)、HE染色和肌酶組化、特殊染色方法，篩選肌營養(yǎng)不良病例。以非特異性改變肌組織為對照組，應(yīng)用qRT-PCR檢測肌營養(yǎng)不良病人肌特異性miRNA（-1，-133a，-206）的表達(dá)。 3.通過以上實(shí)驗(yàn)篩選出對成肌細(xì)胞分化及肌營養(yǎng)不良影響較為明顯的肌特異性miRNA，應(yīng)用生物信息學(xué)軟件預(yù)測該miRNA可能作用的靶基因。 4.合成該miRNA的mimic，轉(zhuǎn)染成肌細(xì)胞C2C12，利用MTT法檢測過表達(dá)該miRNA對C2C12細(xì)胞生長增殖的影響，光學(xué)顯微鏡觀察成肌細(xì)胞生長分化情況，同時(shí)qRT-PCR、Western blot進(jìn)行靶基因表達(dá)檢測。 【結(jié)果】 1. miR-1，-133a，-206在增殖期的表達(dá)量較低，與增殖期比較，隨著成肌細(xì)胞分化時(shí)間的延長，肌特異性miR-1，-133a，-206表達(dá)均顯著升高（P值分別為0.0002、0.005、0.0001），其中miR-1表達(dá)升高最明顯。 2.篩選8例肌營養(yǎng)不良標(biāo)本，，與非特異性改變肌組織對比，miR-1，-133a，-206表達(dá)均升高，其中miR-206在肌營養(yǎng)不良病人的肌組織表達(dá)升高最顯著（P=0.04）。 3.通過miRDB、miRNA.org、TargetScanHuman6.2、Pictar等生物信息學(xué)軟件預(yù)測miR-206在肌細(xì)胞分化及肌營養(yǎng)不良發(fā)病過程的靶基因Pax7、Pax3、Cx43、Hmgb3。 4. C2C12細(xì)胞轉(zhuǎn)染miR-206mimic后，qRT-PCR顯示Pax7、Pax3、Cx43、Hmgb3表達(dá)明顯下降。Western blot顯示Pax7、Pax3、Cx43、Hmgb3蛋白表達(dá)下降。 5. C2C12細(xì)胞轉(zhuǎn)染miR-206mimic后，光學(xué)顯微鏡觀察細(xì)胞增殖受到明顯抑制。與陰性對照（NC組）相比，MTT法檢測顯示C2C12抑制率為（21.35±0.0068）%（P=0.004） 【結(jié)論】 1. miR-1、miR-133a和miR-206隨著成肌細(xì)胞的分化成熟，表達(dá)水平逐步增高。 2.在肌特異性miRNA中，miR-206在肌營養(yǎng)不良的病理生理過程中發(fā)揮更為重要的作用。 3. miR-206通過調(diào)節(jié)多個(gè)靶基因的表達(dá)參與成肌細(xì)胞增殖和分化過程。
[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 鹵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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R746.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 鄭江;鄧忠良;;pax7對肌肉形成作用的研究進(jìn)展[J];重慶醫(yī)科大學(xué)學(xué)報(bào);2007年06期

2 王淑輝;張成;尚延昌;于美娟;張雅妮;馮善偉;李美山;;Micro-dsytrophin基因修飾的骨髓間充質(zhì)干細(xì)胞移植后mdx鼠腓腸肌病理改變及micro-dystrophin的表達(dá)[J];中國神經(jīng)免疫學(xué)和神經(jīng)病學(xué)雜志;2008年06期

本文編號：1732445