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  本文選題：胚胎干細(xì)胞 + 心肌細(xì)胞��； 參考：《重慶醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的擬證實成熟心肌細(xì)胞對早期胚胎或胚胎干細(xì)胞(ESCs)定向分化存在特殊的誘導(dǎo)作用,為建立一套簡單實用的胚胎干細(xì)胞移植技術(shù)提供理論依據(jù)。 方法分離培養(yǎng)SD大鼠乳鼠心肌細(xì)胞,以DAPI(4’6-聯(lián)瞇-2-苯基吲哚)染核作為細(xì)胞標(biāo)記。分別應(yīng)用自然合籠及超排卵技術(shù)獲取昆明小鼠的早期胚胎(8～16細(xì)胞期胚)與心肌細(xì)胞及相關(guān)心肌微環(huán)境共培養(yǎng);取昆明小鼠(3.5～4天)的囊胚培養(yǎng)后分離出ESCs,以1到2代的小鼠ESCs細(xì)胞團(tuán)與心肌細(xì)胞及心肌成纖維細(xì)胞共培養(yǎng)。錄像動態(tài)觀察早期胚胎或ESCs向心肌細(xì)胞分化的情況;分別于共培養(yǎng)后3、7、14天行肌鈣蛋白T(cTnT)、α-肌動蛋白(α-Actinin)免疫熒光染色檢測。 結(jié)果(1)早期胚胎與心肌細(xì)胞或心肌成纖維細(xì)胞共培養(yǎng)時,可繼續(xù)發(fā)育分化,所分化出的細(xì)胞無cTnT表達(dá)。(2)1代或2代的ESCs細(xì)胞和/或細(xì)胞團(tuán)與心肌細(xì)胞共培養(yǎng)約7天左右出現(xiàn)節(jié)律搏動的心肌樣細(xì)胞;觀察至14天時,搏動頻率約為30～50次/min。最好實驗批次,可計數(shù)到1/4以上的ESCs細(xì)胞和/或ESCs細(xì)胞團(tuán)出現(xiàn)節(jié)律性搏動;ESCs細(xì)胞和/或細(xì)胞團(tuán)與心肌細(xì)胞共培養(yǎng)并添加0.6%的DMSO時,節(jié)律搏動的心肌樣細(xì)胞分化率未見顯著提高。(3)心肌細(xì)胞誘導(dǎo)組,記錄已搏動和未搏動ESCs誘導(dǎo)分化后的心肌樣細(xì)胞cTnT、α-Actin蛋白熒光染色陽性率為27%;ESCs與心肌成纖維細(xì)胞共培養(yǎng)后的成纖維細(xì)胞誘導(dǎo)組,分化的細(xì)胞cTnT、α-Actin蛋白熒光染色均陰性;0.6%DMSO誘導(dǎo)組中約3%的分化細(xì)胞cTnT、α-Actin蛋白熒光染色陽性;DMSO聯(lián)合心肌細(xì)胞誘導(dǎo)組,其結(jié)果與心肌細(xì)胞誘導(dǎo)組相似。 結(jié)論(1)心肌細(xì)胞和心肌成纖維細(xì)胞所提供的微環(huán)境有利于早期胚胎發(fā)育分化,分化出的細(xì)胞可以繼續(xù)發(fā)育增殖。但是,不能誘導(dǎo)出cTnT陽性表達(dá)的心肌樣細(xì)胞,考慮主要是滋養(yǎng)層細(xì)胞的分化影響了細(xì)胞間的直接接觸,導(dǎo)致誘導(dǎo)失敗。(2)未經(jīng)建系操作的ESCs可被誘導(dǎo)分化為節(jié)律搏動的心肌樣細(xì)胞;成熟心肌細(xì)胞與ESCs共培養(yǎng)狀態(tài)下,成熟心肌細(xì)胞可誘導(dǎo)ESCs向心肌細(xì)胞分化,這種微環(huán)境下ESCs向心肌細(xì)胞定向分化率顯著高于自然分化組,即成熟心肌細(xì)胞是誘導(dǎo)ESCs細(xì)胞定向分化中較強(qiáng)的誘導(dǎo)因素。
[Abstract]:Objective to prove that mature cardiomyocytes can induce the directional differentiation of early embryonic or embryonic stem cells (ESCs) and provide a theoretical basis for the establishment of a set of simple and practical techniques for embryonic stem cell transplantation. Methods the neonatal rat cardiomyocytes were isolated and cultured, and the nuclei of DAPI4 (6-diphenylin-2-phenylindole) were used as cell markers. Natural cage and superovulation were used to obtain the embryo of Kunming mouse. The blastocysts of Kunming mice were cultured for 4 days. The blastocysts were isolated and co-cultured with cardiomyocytes and cardiac fibroblasts in 1 to 2 passages of mouse ESCs cells. The differentiation of early embryos or ESCs into cardiomyocytes was observed dynamically by video recording, and the expression of troponin TnTnT, 偽 -actin (偽 -actin) was detected by immunofluorescence staining on the 7th and 14th days after co-culture. Results 1) when the early embryo was co-cultured with cardiomyocytes or myocardial fibroblasts, it could continue to develop and differentiate. There was no cTnT expression in the differentiated cells. The ESCs cells and / or cell clusters of the first or second passage were co-cultured with cardiomyocytes for about 7 days, and the beating frequency was about 30 ~ 50 beats / min at the 14th day. The best experiment batch can be counted when more than 1 / 4 of ESCs cells and / or ESCs cell clusters have rhythmic pulsating ESCs cells and / or cell clusters co-cultured with cardiomyocytes and added 0.6% DMSO. The differentiation rate of cardiomyoid cells in rhythmic pulsatile group was not significantly increased. The positive rate of cTnT, 偽 -actin protein fluorescence staining in cardiac myoid cells induced by pulsatile and non-pulsatile ESCs was 27% in the fibroblast induction group after co-culture with myocardial fibroblast. CTnT, 偽 -Actin protein fluorescent staining of differentiated cells was negative. About 3% of differentiated cells in DMSO group were positive for cTnT, 偽 -Actin protein fluorescence staining and combined with cardiomyocyte induction. The results were similar to those in cardiomyocyte induction group. Conclusion 1) the microenvironment provided by cardiac myocytes and myocardial fibroblasts is conducive to early embryonic development and differentiation, and the differentiated cells can continue to develop and proliferate. However, cardiomyoid cells with cTnT positive expression could not be induced. It was considered that the differentiation of trophoblast cells affected the direct contact between cells. ESCs can be induced to differentiate into rhythmically beating cardiomyocytes, and mature cardiomyocytes can induce ESCs to differentiate into cardiomyocytes when co-cultured with ESCs. In this microenvironment, the differentiation rate of ESCs into cardiomyocytes was significantly higher than that of natural differentiation group, that is, mature cardiomyocytes were the strong inducing factors in inducing the directional differentiation of ESCs cells.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 周穎,黃學(xué)鋒,林金菊,鄭菊芬,葉碧綠,趙軍招;體外受精-胚胎移植中胚胎形態(tài)和發(fā)育時期與受孕結(jié)果的相關(guān)性[J];生殖與避孕;2001年06期

2 劉蘭英,楊琨,朱振宇,劉玉川,余細(xì)勇,銀巍,湯建,馬澗泉,顧軍;胚胎干細(xì)胞體外分化為心肌細(xì)胞誘導(dǎo)因素的探討[J];中國病理生理雜志;2000年05期

3 李大強(qiáng),王智彪,白晉,趙頡,胡凱,王媛,杜永洪;小鼠囊胚與不同侵襲轉(zhuǎn)移潛能肝癌細(xì)胞系共培養(yǎng)模型的建立及生物學(xué)行為觀察[J];中國病理生理雜志;2004年01期

4 王秀麗,王常勇,虞星炬,郭希民,段翠密,趙云山,董靈芝;體外誘導(dǎo)小鼠胚胎干細(xì)胞分化為心肌細(xì)胞的初步研究[J];中國病理生理雜志;2005年03期

5 袁巖,陳連鳳,張抒揚(yáng),吳煒,陳浩,嚴(yán)曉偉;心肌細(xì)胞裂解液對骨髓間充質(zhì)干細(xì)胞向心肌細(xì)胞分化誘導(dǎo)作用的研究[J];中華心血管病雜志;2005年02期

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