本文關(guān)鍵詞：骨骼肌損傷修復(fù)過(guò)程中細(xì)胞去分化現(xiàn)象的初步研究　出處：《南方醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 損傷修復(fù) 去分化 Reversine C2C12成肌細(xì)胞 凋亡 增殖 細(xì)胞分化

【摘要】： 再生醫(yī)學(xué)領(lǐng)域中,因干細(xì)胞具有多分化潛能和自我更新能力的生物學(xué)特征,其在修復(fù)再生過(guò)程的作用,受到越來(lái)越多的關(guān)注。目前存在的問(wèn)題是：獲取人類的胚胎干細(xì)胞不僅對(duì)胚胎組織是種損傷性的操作,更是受到倫理道德規(guī)范的束縛。成熟細(xì)胞去分化在哺乳動(dòng)物再生中的作用一直存在爭(zhēng)議。在低等動(dòng)物中,再生過(guò)程中細(xì)胞去分化是有機(jī)體長(zhǎng)期進(jìn)化過(guò)程中獲得的一種保護(hù)性和適應(yīng)性現(xiàn)象,可以控制并且有限增殖。在低等動(dòng)物(如蠑螈)機(jī)體損傷修復(fù)過(guò)程中,其體內(nèi)存在著強(qiáng)大的細(xì)胞去分化能力,這一機(jī)體組織再生的理論已經(jīng)被廣泛接受和普遍認(rèn)可；由于哺乳動(dòng)物的組織器官在損傷修復(fù)的再生過(guò)程中,很大程度上受其局部環(huán)境(如炎癥細(xì)胞的作用)和機(jī)體整體環(huán)境(如免疫系統(tǒng)的破壞)因素的影響,一般認(rèn)為人類等哺乳動(dòng)物的細(xì)胞去分化能力是相對(duì)低下的。去分化在哺乳動(dòng)物組織損傷修復(fù)中的作用已經(jīng)逐步受到國(guó)內(nèi)外學(xué)者的重視。然而,在組織細(xì)胞損傷修復(fù)過(guò)程中,去分化現(xiàn)象觀察方法的建立,細(xì)胞去分化與凋亡之間的相互聯(lián)系等問(wèn)題都有待進(jìn)一步深入研究。鑒于此,我們建立大鼠的肌肉損傷模型,觀察肌肉損傷修復(fù)過(guò)程中組織形態(tài)學(xué)的變化,試圖從形態(tài)學(xué)角度觀察細(xì)胞去分化現(xiàn)象,并探討細(xì)胞去分化與細(xì)胞凋亡之間可能存在的某種聯(lián)系。 是否存在某種途徑使得成體細(xì)胞轉(zhuǎn)化為類干細(xì)胞狀態(tài)呢?Chen等人應(yīng)用人工合成的化學(xué)物質(zhì)"Reversine" (2-(4-morpholinoanilino)-6-cyclohexylaminopurine)成功誘導(dǎo)已分化成熟的細(xì)胞去分化返回到干細(xì)胞。Reversine能使C2C12細(xì)胞去分化至“類干細(xì)胞”。這些“類干細(xì)胞”在不同的條件下,又分別能被誘導(dǎo)成為成骨細(xì)胞和脂肪細(xì)胞。已經(jīng)有學(xué)者開(kāi)始利用分子生物學(xué)等技術(shù)去探索Reversine誘導(dǎo)去分化發(fā)生的作用機(jī)理。但關(guān)于Reversine在誘導(dǎo)去分化過(guò)程中對(duì)細(xì)胞增殖分化究竟是如何影響的,卻鮮見(jiàn)報(bào)道。為此我們以體外培養(yǎng)的C2C12小鼠成肌細(xì)胞為研究對(duì)象,分析Reversine對(duì)C2C12成肌細(xì)胞生長(zhǎng)的影響,以明確Reversine在誘導(dǎo)去分化過(guò)程中對(duì)成肌細(xì)胞狀態(tài)和功能的作用。 1觀察骨骼肌細(xì)胞損傷修復(fù)過(guò)程中骨骼肌細(xì)胞形態(tài)學(xué)變化； 2初步探討Reversine對(duì)小鼠C2C12成肌細(xì)胞生長(zhǎng)的影響。 1制作SD大鼠下肢腓腸肌肌肉損傷修復(fù)模型,術(shù)后27d在損傷處取材進(jìn)行形態(tài)學(xué)觀察。 1.1通過(guò)透射電鏡對(duì)肌肉組織損傷修復(fù)細(xì)胞去分化現(xiàn)象進(jìn)行觀察； 1.2通過(guò)HE染色和免疫熒光染色觀察骨骼肌細(xì)胞組織形態(tài)學(xué)變化。 2 C2C12小鼠成肌細(xì)胞系的體外培養(yǎng),分組處理。 2.1將C2C12小鼠成肌細(xì)胞分三組：A組：正常對(duì)照組,B組：1μM Reversine處理12h組,C組：1μM Reversine處理24h組。上述三組用Annexin-V/PI雙染法處理,并通過(guò)流式細(xì)胞技術(shù)檢測(cè)三組C2C12細(xì)胞凋亡比例的差異； 2.2將C2C12小鼠成肌細(xì)胞分五組：A組：正常對(duì)照組,B組：?jiǎn)为?dú)1μM Reversine處理7d,C組：不加Reversine處理,單獨(dú)成骨誘導(dǎo)7d,D組：1μM Reversine誘導(dǎo)12h+單獨(dú)成骨誘導(dǎo)7d,E組：1μM Reversine誘導(dǎo)12h+1μM Reversine聯(lián)合成骨誘導(dǎo)7d。上述五組光鏡下觀察細(xì)胞形態(tài)的變化,并通過(guò)逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)法檢測(cè)五組C2C12成肌細(xì)胞分化抑制因子(Id2),肌肉發(fā)生調(diào)節(jié)因子(Myogenin),生肌因子后結(jié)蛋白(Desmin) mRNA的表達(dá)。 1電鏡觀察：損傷后的肌纖維溶解,并出現(xiàn)原始幼稚的細(xì)胞； 2 HE染色觀察：與正常組織細(xì)胞相比,損傷處的組織細(xì)胞排列紊亂,細(xì)胞核大量聚集,且由多核細(xì)胞變成了單核細(xì)胞； 3免疫熒光染色：正常骨骼肌AIF未見(jiàn)表達(dá),損傷后的骨骼肌細(xì)胞中可見(jiàn)AIF明顯表達(dá)于細(xì)胞核內(nèi)； 4流式細(xì)胞術(shù)檢測(cè)結(jié)果：與正常對(duì)照組相比,1μM Reversine處24h能引起C2C12細(xì)胞顯著的凋亡,1μM Reversine處理12h的C2C12細(xì)胞未見(jiàn)顯著的凋亡； 5 Reversine處理后細(xì)胞形態(tài)學(xué)觀察結(jié)果：與正常對(duì)照組相比,單獨(dú)Reversine處理組和1μM Reversine處理12h后再聯(lián)合成骨誘導(dǎo)組的細(xì)胞增殖明顯受到抑制,單獨(dú)誘導(dǎo)組和1μM Reversine處理12h后再單獨(dú)誘導(dǎo)組細(xì)胞逐漸增殖并融合； 6 Reversine處理后RT-PCR結(jié)果：與正常對(duì)照組相比,1μM Reversine 12h的培養(yǎng)條件下,Id2表達(dá)顯著增強(qiáng),且有統(tǒng)計(jì)學(xué)差異,表明對(duì)Reversine耐受細(xì)胞的細(xì)胞增殖能力顯著增強(qiáng)；同時(shí),1μM Reversine 12h的培養(yǎng)條件下,Myogenin和Desmin兩者所表達(dá)的趨勢(shì)基本一致,表達(dá)顯著減弱,有統(tǒng)計(jì)學(xué)差異,表明對(duì)Reversine耐受細(xì)胞的細(xì)胞成肌分化能力顯著減弱,細(xì)胞生長(zhǎng)受到抑制。 1骨骼肌損傷修復(fù)過(guò)程中可能存在著細(xì)胞去分化的現(xiàn)象。 2 Reversine在誘導(dǎo)細(xì)胞去分化的過(guò)程中,能夠顯著的抑制成肌細(xì)胞的生長(zhǎng)作用,特異性肌源性轉(zhuǎn)錄蛋白Myogenin和Desmin的表達(dá)也受到顯著抑制。
[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 .

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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相關(guān)期刊論文 前4條

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本文編號(hào)：1437150