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

【摘要】： 目的:探討完整成熟心肌細(xì)胞或心肌細(xì)胞裂解物對胚胎干細(xì)胞(embryonic stem cells,ESCs)定向分化為心肌細(xì)胞的誘導(dǎo)作用及強(qiáng)度。 方法:收集昆明小鼠3.5d和4d胚齡的囊胚,分別將其培養(yǎng)在小鼠胚胎成纖維細(xì)胞飼養(yǎng)層上,4～5d后取隆起生長的內(nèi)細(xì)胞團(tuán)分離后再培養(yǎng),觀察集落的生長情況并通過堿性磷酸酶染色、OCT-4染色等對細(xì)胞集落進(jìn)行鑒定。然后取3～4代ESCs,先將ESCs懸浮培養(yǎng)2～3d形成擬胚體(embryoid bodies,EBs),再用大鼠心肌細(xì)胞等誘導(dǎo)因素對其誘導(dǎo)培養(yǎng)。共分5組進(jìn)行定向誘導(dǎo)實(shí)驗,即心肌細(xì)胞誘導(dǎo)組、心肌細(xì)胞條件培養(yǎng)液誘導(dǎo)組、心肌細(xì)胞裂解液誘導(dǎo)組、胚胎干細(xì)胞分化培養(yǎng)液誘導(dǎo)組和對照組。ESCs分化為心肌細(xì)胞的結(jié)果判斷,用相差顯微鏡觀察EBs搏動情況,用免疫細(xì)胞熒光技術(shù)檢測心肌細(xì)胞特異性肌鈣蛋白T(TnT)、а-肌動蛋白(а-Actin)的表達(dá)。 結(jié)果:①采用0.1%胰蛋白酶-0.02%EDTA室溫作用2-4分鐘并輔以機(jī)械作用,分離克隆ESCs效果較好。②心肌細(xì)胞誘導(dǎo)組:誘導(dǎo)分化培養(yǎng)第3天起有EBs自發(fā)性、有節(jié)律跳動,12d時有93%的EBs出現(xiàn)節(jié)律性收縮。已分化的ESCs特異性肌鈣蛋白T、а-Actin陽性表達(dá)率56.5±13.44%,顯著高于其它各組(P0.05)。③心肌細(xì)胞條件培養(yǎng)液誘導(dǎo)組:12d時有8%的EBs出現(xiàn)節(jié)律性收縮。已分化的ESCs肌鈣蛋白T、а-Actin陽性表達(dá)率5.2±2.01%,顯著低于心肌細(xì)胞誘導(dǎo)組(P0.05),與對照組比較差異無顯著性(P0.05)。④心肌細(xì)胞裂解液誘導(dǎo)組:12d時有36%的EBs出現(xiàn)節(jié)律性收縮。已分化的ESCs特異性肌鈣蛋白T、а-Actin陽性表達(dá)率8.2±2.45%,顯著低于心肌細(xì)胞誘導(dǎo)組(P0.05),顯著高于對照組(P0.05)。⑤胚胎干細(xì)胞分化培養(yǎng)液誘導(dǎo)組:12d時有7%的EBs出現(xiàn)節(jié)律性收縮。已分化的ESCs特異性肌鈣蛋白T、а-Actin陽性表達(dá)率5.08±1.38%,顯著低于心肌細(xì)胞誘導(dǎo)組(P0.05),與對照組比較差異無顯著性(P0.05)。 結(jié)論:①采用4d胚齡的囊胚和0.1%胰蛋白酶-0.02%EDTA室溫作用2-4分鐘并輔以機(jī)械作用的消化方法有助于提高昆明小鼠ES細(xì)胞建株能力。②心肌細(xì)胞和心肌細(xì)胞裂解液均可誘導(dǎo)ESCs向心肌細(xì)胞定向分化,且心肌細(xì)胞的誘導(dǎo)作用強(qiáng)于心肌細(xì)胞裂解液。
[Abstract]:Aim: to investigate the induction effect and intensity of intact mature cardiomyocytes or cardiomyocyte lysates on differentiation of embryonic stem cells (embryonic stem cells) into cardiomyocytes. Methods: the blastocysts of Kunming mice were collected for 3.5 days and 4 days old respectively. The blastocysts were cultured on the feeder layer of mouse embryonic fibroblasts for 4 days. The colony growth was observed and the colony was identified by alkaline phosphatase staining and OCT-4 staining. ESCs suspension culture was carried out for 2 days to form embryoid bodies-ebs, and then was induced by rat cardiomyocytes. Five groups were divided into five groups: cardiomyocyte induction group, cardiomyocyte conditioned medium group, cardiomyocyte lysis medium induction group, embryonic stem cell differentiation culture medium induction group and control group. ESCs differentiated into cardiomyocytes. The EBs pulsation was observed by phase contrast microscope, and the expression of TnTnTnTnTnTnTnTnTX, actin was detected by immunocytofluorescence technique. Results using 0.1% trypsin and 0.02TA at room temperature for 2 to 4 minutes, the cloned ESCs was isolated and cloned. 2 the cardiomyocyte induction group. 2. In the cardiomyocyte induction group, there was spontaneous EBs from the third day after induced differentiation and culture. At 12 days, 93% of EBs showed rhythmic contraction. The positive rate of differentiated ESCs specific troponin T and tactin was 56.5 鹵13.44, which was significantly higher than that of the other groups (P 0.05n.3). 8% of EBs showed rhythmic contraction at 12 days in the conditioned medium group. The positive expression rate of ESCs troponin Tand activity was 5.2 鹵2.01, which was significantly lower than that in cardiomyocyte induction group (P 0.05), but there was no significant difference between the two groups. 36% of EBs showed rhythmic contraction at 12 days after being induced by lytic solution of cardiomyocytes. The positive expression rate of ESCs specific troponin Tand activity was 8.2 鹵2.45, which was significantly lower than that in cardiomyocyte induction group (P 0.05), and was significantly higher than that in control group (P 0.05.5). 7% of EBs showed rhythmic contraction at 12 days in the control group induced by P0.055 embryonic stem cell differentiation medium. The positive expression rate of ESCs specific troponin Tand activity was 5.08 鹵1.38, which was significantly lower than that in cardiomyocyte induced group (P 0.05), and there was no significant difference compared with the control group (P 0.05). Conclusion the digestion of the blastocyst with 4 d embryo age and 0.1% trypsin-0.02TA at room temperature for 2 to 4 minutes, supplemented by mechanical action, is helpful to improve the ability of mouse es cells to build 2 cardiomyocytes and cardiomyocyte lytic fluid. All of them could induce ESCs to differentiate into cardiomyocytes. The inductive effect of myocardial cells was stronger than that of myocardial cell lysate.
【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

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