發(fā)布時(shí)間：2018-02-01 07:16

  本文關(guān)鍵詞： 電刺激 胚胎干細(xì)胞 心肌細(xì)胞 共培養(yǎng) 細(xì)胞分化　出處：《重慶醫(yī)科大學(xué)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 目的探討仿生電刺激對(duì)離體心肌微環(huán)境誘導(dǎo)胚胎干細(xì)胞(ESCs)向心肌樣細(xì)胞分化的作用,為建立一套簡(jiǎn)單實(shí)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。收集昆明小鼠3.5d和4d胚齡的胚胎,以鼠間充質(zhì)干細(xì)胞為飼養(yǎng)層,4～5d后取隆起生長(zhǎng)的內(nèi)細(xì)胞團(tuán)分離后再培養(yǎng),觀察集落的生長(zhǎng)情況并通過(guò)堿性磷酸酶染色、OCT-4染色等對(duì)細(xì)胞集落進(jìn)行鑒定。然后取3～4代ESCs,按是否給予胚胎干細(xì)胞仿生電刺激及心肌微環(huán)境誘導(dǎo)分為對(duì)照組、電刺激組、心肌組、電刺激+心肌細(xì)胞條件培養(yǎng)液組、電刺激+心肌組,每組又以實(shí)驗(yàn)干預(yù)培養(yǎng)時(shí)間(5、7、14天)分為3個(gè)亞組(Ⅰ、Ⅱ、Ⅲ組)。分別于實(shí)驗(yàn)的5、7、14天觀察各組細(xì)胞生長(zhǎng)情況,行肌鈣蛋白T(cTnT)免疫熒光染色檢測(cè)。 結(jié)果(1)以間充質(zhì)干細(xì)胞作為飼養(yǎng)層細(xì)胞分離培養(yǎng)的昆明小鼠胚胎干細(xì)胞(ESCs)有其典型的形態(tài)學(xué)特征,對(duì)ESCs堿性磷酸酶進(jìn)行檢測(cè),ES細(xì)胞顯微鏡下為棕褐色;Oct-4染色陽(yáng)性。(2)電刺激組及對(duì)照組培養(yǎng)體系中始終未出現(xiàn)節(jié)律搏動(dòng)的心肌樣細(xì)胞,分化的細(xì)胞cTnT蛋白熒光染色均陰性;(3)心肌組、電刺激+心肌細(xì)胞條件培養(yǎng)液組、電刺激+心肌組均約7天左右出現(xiàn)節(jié)律搏動(dòng)的心肌樣細(xì)胞,觀察至14天時(shí),搏動(dòng)頻率約為30～50次/min。(4)電刺激+心肌細(xì)胞組和肌細(xì)胞、電刺激+心肌細(xì)胞條件培養(yǎng)液組相比,電刺激心肌細(xì)胞組中心肌細(xì)胞長(zhǎng)勢(shì)更好,ESCs分化而來(lái)的心肌樣細(xì)胞搏動(dòng)更快,更趨于一致性,記錄已搏動(dòng)和未搏動(dòng)ESCs誘導(dǎo)分化后的心肌樣細(xì)胞cTnT蛋白熒光染色陽(yáng)性率更高,約為40%,顯著高于心肌細(xì)胞組(28.7%)、電刺激+心肌細(xì)胞條件培養(yǎng)液組(17.1%)(P0.05)。 結(jié)論(1)以間充質(zhì)干細(xì)胞作為飼養(yǎng)層細(xì)胞分離培養(yǎng)昆明小鼠胚胎干細(xì)胞可行,得到的胚胎干細(xì)胞能保持特殊的形態(tài)和未分化狀態(tài)。(2)未經(jīng)建系操作的ESCs可被誘導(dǎo)分化為節(jié)律搏動(dòng)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定向分化中較強(qiáng)的誘導(dǎo)因素。(3)單獨(dú)仿生電刺激因素還不足以誘導(dǎo)胚胎干細(xì)胞向心肌細(xì)胞定向分化。(4)仿生電刺激可促進(jìn)心肌微環(huán)境誘導(dǎo)胚胎干細(xì)胞向心肌樣細(xì)胞分化,提高胚胎干細(xì)胞定向分化為心肌樣細(xì)胞的分化率,但是不能使胚胎干細(xì)胞向心肌樣細(xì)胞分化的時(shí)間點(diǎn)提前。
[Abstract]:Objective to investigate the effect of bionic electrical stimulation on the differentiation of embryonic stem cells (ESCs) into cardiomyoid cells induced by isolated myocardial microenvironment, and to provide a theoretical basis for the establishment of a simple and practical technique for embryonic stem cell transplantation. Methods the neonatal rat cardiomyocytes were isolated and cultured, and the nuclei of DAPI4 (6-dime-2-phenylindole) were used as cell markers. The embryos of Kunming mice were collected at 3. 5 and 4 days of embryo age. Mouse mesenchymal stem cells (MSCs) were used as feeding layer for 5 days. The inner cell clusters were isolated and cultured. The colony growth was observed and stained by alkaline phosphatase (ALP). OCT-4 staining was used to identify the cell colonies, and then three or four generations of ESCs were selected and divided into control group, electrical stimulation group and myocardial group according to whether embryonic stem cells were given bionic electrical stimulation and myocardial microenvironment induction. Electrical stimulation of cardiomyocyte conditioned medium group and electrical stimulation group were divided into three subgroups (鈪，

本文編號(hào)：1481277