【摘要】： 實(shí)驗(yàn)背景和研究目的 背景 心力衰竭是全世界范圍內(nèi)發(fā)生率最高的心臟疾病,其主要特征為正常心肌細(xì)胞的死亡而被無(wú)生理功能的纖維瘢痕組織所替代。因?yàn)槌审w心肌細(xì)胞幾乎無(wú)法再生,所以目前最有效的治療方案就是進(jìn)行心臟移植。近10年的體外及體內(nèi)的研究表明:對(duì)于晚期的心力衰竭進(jìn)行干細(xì)胞移植治療或許是另外一種有效的治療方案。胚胎干細(xì)胞(embryonic stem cells,ESCs)具有無(wú)限增殖能力及多向分化潛能,在體內(nèi)及體外環(huán)境中都可以分化成包括心肌細(xì)胞在內(nèi)的各種類(lèi)型的細(xì)胞。如何更加高效的誘導(dǎo)ESCs定向分化為心肌細(xì)胞引起了研究者們的極大興趣。 目的 本研究旨在優(yōu)化胚胎干細(xì)胞的擴(kuò)增、培養(yǎng)方法;觀察小鼠ESCs(CGR8系)能否在體外分化成具有功能性的心肌細(xì)胞;比較體外多種因素誘導(dǎo)ESCs的定向分化的效率,探討一種安全有效的途徑用于體外誘導(dǎo)ESCs向心肌細(xì)胞分化。 方法和結(jié)果 取乳鼠心肌組織按差速貼壁分離法,純化乳鼠的心肌細(xì)胞,收集心肌細(xì)胞培養(yǎng)液,以1 000 r/min離心5 min。取上清,與完全培養(yǎng)基按1∶1的比例混合制備乳鼠心肌細(xì)胞條件培養(yǎng)液。復(fù)蘇本實(shí)驗(yàn)室凍存的ESCs并擴(kuò)增培養(yǎng)。經(jīng)三步法形成擬胚體(embryoid bodies,EBs):(1)懸滴培養(yǎng)2d;(2)懸浮培養(yǎng)5d;(3)貼壁培養(yǎng):5d。按培養(yǎng)基添加的誘導(dǎo)劑的不同分為隨機(jī)等分四組:乳鼠CMs條件培養(yǎng)液誘導(dǎo)組;催產(chǎn)素(oxytocin,OT)誘導(dǎo)組;混合添加誘導(dǎo)組(乳鼠CMs條件培養(yǎng)液加OT);對(duì)照組(無(wú)任何添加)。 觀察ESCs分化后的搏動(dòng)性發(fā)現(xiàn)試驗(yàn)各組8d后肉眼可見(jiàn)EBs上出現(xiàn)節(jié)律性自發(fā)收縮的區(qū)域,隨著培養(yǎng)時(shí)間的延長(zhǎng),逐漸整個(gè)EBs出現(xiàn)一致性搏動(dòng),但對(duì)照組未觀察到此現(xiàn)象�；旌咸砑诱T導(dǎo)組細(xì)胞的搏動(dòng)率分別和OT誘導(dǎo)組和乳鼠CMs條件培養(yǎng)液誘導(dǎo)組比較差異均顯著(P0.01)。隨著心肌細(xì)胞的發(fā)育成熟,細(xì)胞的搏動(dòng)頻率也會(huì)逐漸下降。通過(guò)免疫組化染色觀察發(fā)現(xiàn)混合添加誘導(dǎo)組分化成CMs的效率明顯高于另外兩組。分別于各組跳動(dòng)的細(xì)胞團(tuán)使用下列藥物:異丙腎上腺、乙酰膽堿和維拉帕米。結(jié)果ESCs經(jīng)OT聯(lián)合CMs的條件培養(yǎng)液誘導(dǎo)后,在分化的早期對(duì)藥物的反應(yīng)性均顯著高于其他實(shí)驗(yàn)組;而分化晚期因各組CMs均逐漸發(fā)育成熟,三組細(xì)胞對(duì)于藥物的反應(yīng)性均有所增強(qiáng)。提示CMs的條件培養(yǎng)液可能有促進(jìn)上述3種受體成熟的作用。接著通過(guò)透射電鏡觀察到三組ESCs分化的CMs出現(xiàn)了類(lèi)似原代CMs的肌原纖維、肌節(jié)、Z線及縫隙連接,橋粒連接結(jié)構(gòu)。細(xì)胞基因表達(dá)的測(cè)定過(guò)程中發(fā)現(xiàn),作為ESCs標(biāo)志的OCT-4僅僅在細(xì)胞分化的早期表達(dá)。心肌特異性因子cTnT及ANF在三組細(xì)胞分化的全程均有表達(dá),但早期cTnT的表達(dá)量混合添加誘導(dǎo)組顯著高于乳鼠CMs條件培養(yǎng)液誘導(dǎo)組及OT誘導(dǎo)組(P0.05);分化中、晚期心肌特異性因子的表達(dá)量無(wú)明顯差異。這些都證實(shí)了乳鼠CMs條件培養(yǎng)液添加OT后誘導(dǎo)ESCs成CMs的高效性。 結(jié)論 小鼠ESCs(CGR8系)在體外的培養(yǎng)過(guò)程中可以被誘導(dǎo)分化成心肌細(xì)胞;ESCs誘導(dǎo)分化成的心肌細(xì)胞具有收縮功能、具有藥理反應(yīng)性并且可以表達(dá)心肌特異性結(jié)構(gòu)蛋白;與以前的研究結(jié)果相同,懸滴、懸浮、貼壁三步培養(yǎng)法是一種可靠的培養(yǎng)ESCs成EBs的方法;ESCs誘導(dǎo)而成的CMs隨著細(xì)胞發(fā)育的成熟,細(xì)胞的搏動(dòng)頻率也會(huì)逐漸下降,這個(gè)可能與成熟心肌細(xì)胞內(nèi)較少含有竇房結(jié)細(xì)胞有關(guān);在體外環(huán)境中OT、乳鼠CMs條件培養(yǎng)液、OT添加乳鼠CMs的條件培養(yǎng)液均具有誘導(dǎo)ESCs分化為CMs的作用,且后者的誘導(dǎo)更具有高效性和安全性。
[Abstract]:Experimental Background and Study Purpose Background Heart Failure is the highest incidence of heart disease worldwide, characterized by the absence of physiological functions for the death of normal cardiomyocytes The fibrous tissue is replaced. Because the body cardiomyocytes are almost impossible to regenerate, the most effective healers are currently The case is a heart transplant. In vitro and in vivo studies in nearly 10 years suggest stem cell transplantation for late heart failure may be another Embryonic stem cells (ESCs) have unlimited proliferation ability and multi-directional differentiation potential, and can differentiate into cardiomyocytes in vivo and in vitro. Various types of cells in the cells. How to induce ESCs to differentiate into cardiomyocytes more efficiently? Study The aim of this study was to optimize the amplification and culture of embryonic stem cells, and to observe whether mouse ESCs (CGR8 system) could differentiate into functional cardiac myocytes in vitro; External factors induce the efficiency of directional differentiation of ESCs and explore a safe and effective method pathway for body The method and the results of the method and the results of the method and the results of the method are as follows: the myocardial tissue of the milk mouse is separated by the differential adherence method to purify the heart muscle of the milk mouse; Cells were collected and cultured for 5 min at 1,000 r/ min. and mixing with the complete culture medium according to the ratio of 1: 1 to prepare the milk The rat cardiomyocytes conditioned medium were cultured. ESCs frozen in this laboratory were recovered and cultured. 1) suspension culture 2d; (2) suspension culture 5d; (3) adherent culture: 5d. The different groups of inducers added according to culture medium were randomly divided into four groups: milk rat CMs conditioned medium induction group; oxytocin (OT) induction group; mixed addition Induction group (CMMs conditioned medium with OT); control group (without any addition). Observation of the beat after ESCs differentiation showed that there was a rhythmic spontaneous contraction in the EBs after eight days of test. There was a consistent beat of the whole EBs, but this phenomenon was not observed in the control group. The beat rate of the mixed addition-induced group cells was Compared with OT-induced group and CMs condition culture medium-induced group, the difference was significant. (P 0.01). With the development of cardiac myocytes, the frequency of beating of cells gradually decreased. The results showed that the efficiency of the mixed addition induced component formation was significantly higher than that in the other two groups. Results ESCs were significantly higher in the early stage of differentiation than in other experimental groups. Due to the mature development of CMs in each group, three groups of cells respond to the drug. The results suggested that CMs conditioned medium might promote the maturation of the above three receptors, and then the CM of three groups of ESCs were observed by transmission electron microscope. S is similar to primary CMs of myofibrils, muscle segments, Z-rays and gap junctions, bridging structures, and cellular gene expression. The results showed that OCT-4, which was an ESCs marker, was only expressed early in the differentiation of cells. In T-induced group (P0.05), there was no significant difference in the expression of specific factor of late cardiac muscle in differentiation. It's all. Conclusion In vitro culture, ESCs (CR8) can be induced to differentiate into cardiomyocytes. The contractile function has pharmacological reactivity and can express the myocardial specific structural protein; the three-step culture method is the same as the previous research results; the suspension, suspension and adherent three-step culture method is a reliable method for culturing the ESCs into ESCs; and the Cs induced by the ESCs are mature and the cells are developed with the development of the cells. The frequency of beating may also decrease gradually, which may be related to the smaller cells in mature myocardial cells; in vitro, OT, CMCs conditioned medium, OT may be added to the mice.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R329

【共引文獻(xiàn)】

相關(guān)期刊論文 前3條

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相關(guān)博士學(xué)位論文 前2條

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相關(guān)碩士學(xué)位論文 前6條

1 錢(qián)海燕;誘導(dǎo)胚胎干細(xì)胞分化為心肌細(xì)胞的實(shí)驗(yàn)研究[D];武漢大學(xué);2004年

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5 李元敏;重組人促紅細(xì)胞生成素對(duì)瓣膜置換術(shù)患者心肌保護(hù)作用研究[D];蘭州大學(xué);2007年

6 陳素華;心鈉素對(duì)小鼠胚胎干細(xì)胞分化的心肌細(xì)胞L型鈣通道的調(diào)控[D];山東師范大學(xué);2007年

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