【摘要】：【目的】系統(tǒng)檢測mi RNA在Dysferlin肌病中的表達,初步探討mi RNA與Dysferlin肌病的關系;結合靶基因預測及細胞學實驗,探討mi R-708 mimic對成肌細胞的影響�！痉椒ā�1.結合臨床表現(xiàn)、HE染色和肌酶特殊染色、免疫組化等方法,篩選Dysferlin肌病病例為實驗組,非特異性改變肌組織為對照組,取部分組織行mi RNA芯片分析。2.靶基因預測網(wǎng)站預測作用于DYSF基因的mi RNA,結合mi RNA芯片結果,選擇與dysferlin肌病關系密切的mi RNA,Taqman探針實時定量PCR(RT-PCR)檢測其在人肌肉組織及小鼠成肌細胞C2C12增殖和分化過程中的表達。3.合成mi R-708 mimic,轉(zhuǎn)染成肌細胞C2C12,光學顯微鏡下觀察成肌細胞生長分化情況,利用CCK-8檢測mi R-708 mimic對C2C12細胞生長增殖的影響,用流式細胞術檢測轉(zhuǎn)染mi R-708 mimic后的增殖期C2C12細胞周期和凋亡情況,RT-PCR、Western blot檢測肌細胞增殖分化相關基因的表達。【結果】1.免疫組化顯示:dysferlin蛋白在正常肌細胞的肌膜上呈線性表達,而在Dysferlin肌病組織中,dysferlin蛋白表達明顯減弱或缺失。光鏡下Dysferlin肌病組織病理學改變:肌纖維呈輕-重度圓形萎縮伴多量肥大肌纖維形成,散在肌纖維見鑲邊空泡,個別肌纖維溶解壞死,內(nèi)核纖維明顯增多,間質(zhì)纖維脂肪組織增生。2.mi RNA芯片結果顯示:Dysferlin肌病組織,116個mi RNA表達上調(diào),176個mi RNA表達下調(diào),部分mi RNA表達水平異常與肌病時細胞內(nèi)各種酶活性的調(diào)節(jié)、細胞代謝的調(diào)控及MAPK、Wnt信號通路等病理過程相關。結合芯片結果與靶基因預測結果,選擇與DYSF基因關系密切的3個mi R(-122、-346、-708)。其中mi R-122、-346下調(diào),mi R-708上調(diào)。3.篩選9例Dysferlin肌病標本進行mi RNA檢測,與非特異性改變肌組織對比,mi R-122、-346、-708表達均升高(P值分別為0.04,0.04,0.03),其中mi R-708在Dysferlin肌病患者肌組織的表達模式與芯片結果一致。4.檢測mi R-122、-346、-708在C2C12成肌細胞生長分化過程中的表達,顯示增殖期mi R-122、-346、-708表達量較低,分化早期mi R-122、-346、-708均升高,但隨著C2C12分化時間的延長,mi R-122表達水平基本不變,mi R-346、-708表達下調(diào),其中mi R-708下調(diào)更明顯,達到增殖期水平。5.C2C12細胞轉(zhuǎn)染mi R-708 mimic后,光學顯微鏡下可見成肌細胞增殖受到明顯抑制。CCK-8檢測顯示mimic組C2C12抑制率為(15.40±0.0052)%,(P=0.004),明顯高于NC組(2.87±0.0034)%;流式細胞術結果顯示:轉(zhuǎn)染mi R-708 mimic后,C2C12細胞周期變化明顯,S期明顯縮短(20.87%),G1期明顯延長(68.64%),NC組分別為45.66%、42.69%,而細胞凋亡指數(shù)mimic組與NC組無顯著差異;RT-PCR及Western blot結果顯示:與陰性對照(NC組)相比,轉(zhuǎn)染mi R-708mimic后MYOD、MYOG、MHC、DYSF蛋白表達水平無明顯變化�！窘Y論】1.Dysferlin肌病可引起多種mi RNA表達模式的改變,這些mi RNA可能通過肌動蛋白骨架形成的調(diào)控、細胞內(nèi)各種酶活性的調(diào)節(jié)、細胞代謝的調(diào)控及MAPK、Wnt信號通路的調(diào)節(jié)等環(huán)節(jié)參與Dysferlin肌病病理生理過程。2.Mi R-708可能是成肌細胞增殖的調(diào)控分子,但與成肌細胞分化和凋亡無明顯相關,其在Dysferlin肌病中的作用機制有待于進一步探討。
[Abstract]:[Objective] To detect the expression of MI RNA in Dysferlin myopathy and explore the relationship between MI RNA and Dysferlin myopathy, and to explore the effect of MI R-708 mimic on myoblasts by target gene prediction and cytological experiments. [Methods] 1. To screen Dysferlin myopathy by combining clinical manifestations, HE staining, enzyme specific staining and immunohistochemistry. The patients were the experimental group and the non-specific muscle tissues were the control group. Some tissues were taken for microarray analysis. 2. The target gene prediction website predicted the MI RNA of DYSF gene, and combined with the results of microarray, selected the MI RNA closely related to dysferlin myopathy. Taqman probe real-time quantitative PCR (RT-PCR) was used to detect the MI RNA in human muscle tissues and mice. Expression of C2C12 in myocytes during proliferation and differentiation Immunohistochemistry showed that dysferlin protein was linearly expressed on the sarcolemma of normal myocytes, while in Dysferlin myopathy, the expression of dysferlin protein was significantly decreased or deleted. Mild to severe round atrophy accompanied by a large number of hypertrophic muscle fibers, scattered muscle fibers with rimmed vacuoles, individual muscle fibers dissolved and necrosis, the number of nuclear fibers increased significantly, interstitial fibrous adipose tissue proliferation. 2. MiRNA microarray results showed that in Dysferlin myopathy, 116 mi RNA expression was up-regulated, 176 mi RNA expression was down-regulated, and some mi RNA expression levels were down-regulated. Abnormalities were associated with the regulation of intracellular enzymes, cellular metabolism, MAPK and Wnt signaling pathways in myopathy. Three miRs (-122, -346, -708) closely related to DYSF gene were selected by combining the results of microarray and target gene prediction. Among them, mi R-122, -346 were down-regulated, and MI R-708 was up-regulated. 3. Nine Dysferlin myopathy specimens were screened. MiR-122, -346, and-708 expressions were increased in non-specific muscle tissues (P values were 0.04, 0.04, 0.03 respectively). The expression pattern of MI R-708 in muscle tissues of Dysferlin myopathy patients was consistent with that of microarray. 4. The expression of MI R-122, -346, and-708 in C2C12 myoblasts during proliferation and differentiation was detected. 2, -346, -708 expression was low, and the expression of MI R-122, -346, -708 increased in the early stage of differentiation. However, with the prolongation of differentiation time of C2C12, the expression of MI R-122 remained unchanged, and the expression of MI R-346, -708 was down-regulated. MiR-708 was down-regulated more significantly, reaching the proliferative stage level. CCK-8 assay showed that the inhibition rate of C2C12 in mimic group was (15.40 +0.0052)%, (P = 0.004), which was significantly higher than that in NC group (2.87 +0.0034)%. Flow cytometry showed that the cell cycle of C2C12 was significantly changed, S phase was significantly shortened (20.87%), G1 phase was significantly prolonged (68.64%), NC group was 45.66%, and apoptosis index was 42.69%. The expression of MYOD, MYOG, MHC and DYSF was not significantly changed after transfection of MI R-708 Mi mic compared with the negative control group (NC group). [Conclusion] 1. Dysferlin myopathy can induce changes in expression patterns of multiple mi RNA, which may be regulated by actin cytoskeleton formation. Mi R-708 may be involved in the pathophysiological process of Dysferlin myopathy. 2. Mi R-708 may be a regulator of myoblast proliferation, but it is not related to myoblast differentiation and apoptosis. Its mechanism in Dysferlin myopathy remains to be further explored. Please.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R685

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本文編號：2248354