【摘要】：羊水干細(xì)胞具有向三胚層細(xì)胞分化的能力,心肌細(xì)胞起源于中胚層,因此羊水干細(xì)胞也具有向心肌細(xì)胞分化的潛能,但目前的研究顯示羊水干細(xì)胞向節(jié)律性心肌細(xì)胞分化的效率還相對(duì)較低。豬在生理和生化等性狀上與人類(lèi)具有很多的相似處,因此研究豬羊水干細(xì)胞向心肌細(xì)胞分化具有重要的臨床應(yīng)用價(jià)值。 1、心肌細(xì)胞誘導(dǎo)分化研究 本研究以豬羊水干細(xì)胞為研究對(duì)象,用5-氮胞苷(5-aza)和維生素C(Vc)為誘導(dǎo)劑,對(duì)豬羊水干細(xì)胞形成的類(lèi)胚體(EBs)進(jìn)行誘導(dǎo)分化。應(yīng)用免疫熒光、RT-PCR、透射電鏡技術(shù)檢測(cè)跳動(dòng)細(xì)胞團(tuán)中心肌特異性標(biāo)記的表達(dá)情況。結(jié)果顯示,在豬羊水干細(xì)胞形成的類(lèi)胚體中加入心肌細(xì)胞誘導(dǎo)劑10 d后,有節(jié)律性跳動(dòng)的細(xì)胞團(tuán)出現(xiàn),采用0.1 mmol/L Vc加5μmol/L 5-aza聯(lián)合誘導(dǎo)組的誘導(dǎo)效率最高,出現(xiàn)約33%的跳動(dòng)細(xì)胞團(tuán)。免疫熒光結(jié)果顯示,跳動(dòng)心肌細(xì)胞團(tuán)表達(dá)細(xì)胞骨架蛋白α-actin和肌鈣蛋白Tnni3。RT-PCR檢測(cè)跳動(dòng)心肌細(xì)胞團(tuán)發(fā)現(xiàn),心肌細(xì)胞特異性標(biāo)記分子TbX5、Gata4、α-MHC、Tnni3均呈陽(yáng)性表達(dá)。借助透射電鏡觀察誘導(dǎo)后的跳動(dòng)樣細(xì)胞團(tuán),能清晰觀察到肌絲、糖原粒、糖原池等結(jié)構(gòu)。說(shuō)明5-氮胞苷和維生素C可以促進(jìn)豬羊水干細(xì)胞向心肌細(xì)胞的誘導(dǎo)分化,從而建立了一種有效誘導(dǎo)豬羊水干細(xì)胞向心肌細(xì)胞分化的方法。 2、Wnt/β-catenin信號(hào)通路在心肌細(xì)胞誘導(dǎo)分化中的作用 為了進(jìn)一步探索豬羊水干細(xì)胞向心肌細(xì)胞分化的機(jī)理,本研究用經(jīng)典Wnt信號(hào)通路的激活劑和抑制劑處理向心肌細(xì)胞分化的pAFS細(xì)胞,運(yùn)用RT-PCR、實(shí)時(shí)定量PCR、免疫熒光等檢測(cè)手段,研究了經(jīng)典Wnt信號(hào)通路在pAFS細(xì)胞向心肌細(xì)胞分化過(guò)程中的調(diào)控作用,結(jié)果顯示W(wǎng)nt/β-catenin信號(hào)通路在一定程度上能夠促進(jìn)pAFS向心肌細(xì)胞分化,對(duì)心肌細(xì)胞誘導(dǎo)分化的機(jī)制進(jìn)行了初步探索。
[Abstract]:Amniotic fluid stem cells have the ability to differentiate into triembryonic cells, and cardiomyocytes originate from mesoderm, so amniotic fluid stem cells also have the potential to differentiate into cardiomyocytes. However, current studies have shown that amniotic fluid stem cells can differentiate into rhythmic cardiomyocytes. Pigs have many similarities with human in physiological and biochemical traits, so it is of great clinical value to study the differentiation of pig amniotic fluid stem cells into cardiomyocytes. 1. In this study, pig amniotic fluid stem cells were used as the object of study. 5-azacytidine (5-aza) and vitamin C (Vc) were used as inducers to induce the differentiation of embryoid (EBs) formed by pig amniotic fluid stem cells. The expression of muscle specific markers in the center of beating cell mass was detected by immunofluorescence and RT-PCR, transmission electron microscope. The results showed that 10 days after adding cardiomyocyte inducer to the embryoid formed by pig amniotic fluid stem cells, the rhythmic beating cell mass appeared. The induction efficiency of 0.1 mmol/L Vc plus 5 渭 mol / L 5-aza was the highest, and about 33% of the beating cell mass appeared. The results of immunofluorescence showed that the expression of cytoskeleton protein 偽-actin and troponin Tnni3.RT-PCR were positive in beating cardiomyocytes. TbX5,Gata4, 偽-MHC,Tnni3, a specific marker molecule, was found to be positive in beating cardiomyocytes. With the help of transmission electron microscope, the structure of myofilament, glycogen, glycogen pool and so on could be clearly observed by observing the induced beating-like cell mass. It is suggested that 5-azacytidine and vitamin C can promote the differentiation of pig amniotic fluid stem cells into cardiomyocytes, thus establishing an effective method to induce the differentiation of pig amniotic fluid stem cells into cardiomyocytes. 2. The role of WNT / 尾-catenin signaling pathway in the differentiation of cardiomyocytes in order to further explore the mechanism of differentiation of pig amniotic fluid stem cells into cardiomyocytes, pAFS cells differentiated into cardiomyocytes were treated with classical Wnt signal pathway activators and inhibitors, and RT-PCR, real-time quantitative PCR, immunofluorescence was used. The regulation of classical Wnt signaling pathway in the differentiation of pAFS cells into cardiomyocytes was studied. the results showed that Wnt/ 尾-catenin signaling pathway could promote the differentiation of pAFS into cardiomyocytes to a certain extent, and the mechanism of induction and differentiation of cardiomyocytes was explored.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

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