骨骼肌损伤修复过程中细胞去分化现象的初步研究

发布时间：2018-01-17 17:07

本文关键词：骨骼肌损伤修复过程中细胞去分化现象的初步研究　出处：《南方医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 再生医学领域中,因干细胞具有多分化潜能和自我更新能力的生物学特征,其在修复再生过程的作用,受到越来越多的关注。目前存在的问题是：获取人类的胚胎干细胞不仅对胚胎组织是种损伤性的操作,更是受到伦理道德规范的束缚。成熟细胞去分化在哺乳动物再生中的作用一直存在争议。在低等动物中,再生过程中细胞去分化是有机体长期进化过程中获得的一种保护性和适应性现象,可以控制并且有限增殖。在低等动物(如蝾螈)机体损伤修复过程中,其体内存在着强大的细胞去分化能力,这一机体组织再生的理论已经被广泛接受和普遍认可；由于哺乳动物的组织器官在损伤修复的再生过程中,很大程度上受其局部环境(如炎症细胞的作用)和机体整体环境(如免疫系统的破坏)因素的影响,一般认为人类等哺乳动物的细胞去分化能力是相对低下的。去分化在哺乳动物组织损伤修复中的作用已经逐步受到国内外学者的重视。然而,在组织细胞损伤修复过程中,去分化现象观察方法的建立,细胞去分化与凋亡之间的相互联系等问题都有待进一步深入研究。鉴于此,我们建立大鼠的肌肉损伤模型,观察肌肉损伤修复过程中组织形态学的变化,试图从形态学角度观察细胞去分化现象,并探讨细胞去分化与细胞凋亡之间可能存在的某种联系。是否存在某种途径使得成体细胞转化为类干细胞状态呢?Chen等人应用人工合成的化学物质"Reversine" (2-(4-morpholinoanilino)-6-cyclohexylaminopurine)成功诱导已分化成熟的细胞去分化返回到干细胞。Reversine能使C2C12细胞去分化至“类干细胞”。这些“类干细胞”在不同的条件下,又分别能被诱导成为成骨细胞和脂肪细胞。已经有学者开始利用分子生物学等技术去探索Reversine诱导去分化发生的作用机理。但关于Reversine在诱导去分化过程中对细胞增殖分化究竟是如何影响的,却鲜见报道。为此我们以体外培养的C2C12小鼠成肌细胞为研究对象,分析Reversine对C2C12成肌细胞生长的影响,以明确Reversine在诱导去分化过程中对成肌细胞状态和功能的作用。 1观察骨骼肌细胞损伤修复过程中骨骼肌细胞形态学变化； 2初步探讨Reversine对小鼠C2C12成肌细胞生长的影响。 1制作SD大鼠下肢腓肠肌肌肉损伤修复模型,术后27d在损伤处取材进行形态学观察。 1.1通过透射电镜对肌肉组织损伤修复细胞去分化现象进行观察； 1.2通过HE染色和免疫荧光染色观察骨骼肌细胞组织形态学变化。 2 C2C12小鼠成肌细胞系的体外培养,分组处理。 2.1将C2C12小鼠成肌细胞分三组：A组：正常对照组,B组：1μM Reversine处理12h组,C组：1μM Reversine处理24h组。上述三组用Annexin-V/PI双染法处理,并通过流式细胞技术检测三组C2C12细胞凋亡比例的差异； 2.2将C2C12小鼠成肌细胞分五组：A组：正常对照组,B组：单独1μM Reversine处理7d,C组：不加Reversine处理,单独成骨诱导7d,D组：1μM Reversine诱导12h+单独成骨诱导7d,E组：1μM Reversine诱导12h+1μM Reversine联合成骨诱导7d。上述五组光镜下观察细胞形态的变化,并通过逆转录聚合酶链反应(RT-PCR)法检测五组C2C12成肌细胞分化抑制因子(Id2),肌肉发生调节因子(Myogenin),生肌因子后结蛋白(Desmin) mRNA的表达。 1电镜观察：损伤后的肌纤维溶解,并出现原始幼稚的细胞； 2 HE染色观察：与正常组织细胞相比,损伤处的组织细胞排列紊乱,细胞核大量聚集,且由多核细胞变成了单核细胞； 3免疫荧光染色：正常骨骼肌AIF未见表达,损伤后的骨骼肌细胞中可见AIF明显表达于细胞核内； 4流式细胞术检测结果：与正常对照组相比,1μM Reversine处24h能引起C2C12细胞显著的凋亡,1μM Reversine处理12h的C2C12细胞未见显著的凋亡； 5 Reversine处理后细胞形态学观察结果：与正常对照组相比,单独Reversine处理组和1μM Reversine处理12h后再联合成骨诱导组的细胞增殖明显受到抑制,单独诱导组和1μM Reversine处理12h后再单独诱导组细胞逐渐增殖并融合； 6 Reversine处理后RT-PCR结果：与正常对照组相比,1μM Reversine 12h的培养条件下,Id2表达显著增强,且有统计学差异,表明对Reversine耐受细胞的细胞增殖能力显著增强；同时,1μM Reversine 12h的培养条件下,Myogenin和Desmin两者所表达的趋势基本一致,表达显著减弱,有统计学差异,表明对Reversine耐受细胞的细胞成肌分化能力显著减弱,细胞生长受到抑制。 1骨骼肌损伤修复过程中可能存在着细胞去分化的现象。 2 Reversine在诱导细胞去分化的过程中,能够显著的抑制成肌细胞的生长作用,特异性肌源性转录蛋白Myogenin和Desmin的表达也受到显著抑制。
[Abstract]:In the field of regenerative medicine , because stem cells have the biological characteristics of multi - differentiation potential and self - renewal ability , it is more and more concerned about the role of human embryonic stem cells in repairing regeneration process . The effect of Reversine on the growth of C2C12 myoblasts was studied by using the C2C12 mouse myoblasts cultured in vitro . 1 To observe the morphological changes of skeletal muscle cells during the repair of skeletal muscle cell injury ; 2 To investigate the effect of Reversine on the growth of C2C12 myoblasts in mice . 1 To make a model of muscle injury repair in the lower extremity of SD rat , and observe the morphological observation at the injury site after operation . 1.1 To observe the effect of transmission electron microscope on the cell dedifferentiation in the repair of muscle tissue injury ; 1.2 The morphological changes of skeletal muscle cells were observed by HE staining and immunofluorescence staining . 2 . In vitro culture and grouping treatment of myoblasts of C2C12 mice . 2.1 The C2C12 mouse myoblasts were divided into three groups : group A : normal control group , group B : 1渭M Reversine treated 12h group , group C : 1渭M Reversine treated 24h group . The three groups were treated with annexin - V / PI double staining method , and the difference of apoptosis ratio of three groups of C2C12 cells was detected by flow cytometry . 2.2 The C2C12 mouse myoblasts were divided into five groups : group A : normal control group , group B : 1 渭M Reversine treated for 7 days , group C : 1 渭M Reversine for 12 h + 1 渭M Reversine for 7 days , E group : 1 渭M Reversine for 12h + 1渭M Reversine for 7 days . 1 . Electron microscopic observation : The muscle fibers after injury were dissolved , and the original immature cells appeared . 2 HE staining observation : Compared with normal tissue cells , the tissue cells in the lesions were disordered , the nuclei were aggregated , and the multinucleated cells became monocytes . 3 . Immunofluorescence staining : No expression was found in normal skeletal muscle , but in skeletal muscle cells after injury , it was clearly expressed in the nucleus . No significant apoptosis was observed in C2C12 cells treated with 1 渭M Reversine at 1 渭M Reversine at 1 渭M Reversine . After 12 h treatment with Reversine alone and 1 渭M Reversine for 12 h , the cell proliferation was inhibited , and the cells were treated with Reversine alone for 12 h . The results of RT - PCR after 6 Reversine treatment showed that the expression of Id2 was significantly increased under the culture conditions of 1 渭M Reversine 12h compared with the normal control group , and there was significant difference in the expression of both Myogenin and Desmin under the culture conditions of 1 渭M Reversine 12h , which showed a significant decrease in the expression of Myogenin and Desmin . There may be a phenomenon of cell de - differentiation in the repair of skeletal muscle injury . 2 Reversine can obviously inhibit the growth of myoblasts in the process of inducing cell dedifferentiation , and the expression of specific myogenic transcription protein Myogenin and Desmin is also significantly inhibited .

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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