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目的:如何在体外将胚胎干细胞培育转化为大量的心肌细胞并进行移植,使之成为具有成熟心肌细胞功能?实验采用不同诱导剂对P19胚胎干细胞进行诱导,拟构建体外定向心肌细胞分化模型。方法:实验于2006-09/2007-03在日本东京工业大学生体分子机能工学研究室完成。①材料:P19细胞由日本东北大学加龄医学研究所提供。钙粘素融合蛋白N-cad-Fc由本研究室合成。②实验方法:P19细胞按1×107L-1密度接种进行悬浮培养,分别以终浓度1,5,10,50,100nmol/L视黄酸及0.5%、0.75%、1%、1.25%、1.5%二甲基亚砜各自诱导4d,将形成的细胞聚集体分别接种于预铺有0.1%明胶、2.5mg/LN-cad-Fc、10mg/LN-cad-Fc基质的24孔板中培养10d。③实验评估:观察不同诱导条件及不同基质上的细胞跳动情况,免疫荧光染色检测心肌特异性蛋白TroponinT的表达,RT-PCR法检测心肌细胞标志性基因cardiacactin、gata-4的表达。结果:①P19细胞经1,5,10nmol/L视黄酸或0.5%、0.75%、1%二甲基亚砜诱导后,均出现可自主节律跳动的细胞。P19细胞聚集体悬浮培养至20d,预铺有0.1%明胶、2.5mg/LN-cad-Fc及10mg/LN-cad-Fc基质的培养板上节律跳动细胞团的出现率分别为44.6%、71.3%和70.1%。②药物诱导后,P19细胞心肌特异性蛋白TroponinT呈阳性表达。③与传统预铺有明胶的培养板比较,在预铺有N-cad-Fc的培养板上心肌标志性基因cardiacactin、gata-4的表达均明显升高,但10mg/LN-cad-Fc的表达量略低于2.5mg/LN-cad-Fc。④分别在上述不同基质上单层培养的P19细胞,二甲基亚砜诱导14d后均仍呈上皮样细胞形态,未见跳动细胞出现,RT-PCR检测无心肌标志性基因表达。结论:①使用0.5%~1%二甲基亚砜或1~10nmol/L视黄酸可成功诱导P19细胞心肌分化,心肌特异性蛋白TroponinT的表达早于心肌跳动的出现。②作为一种基质模型来模拟细胞间黏附,2.5mg/LN-cad-Fc是较适宜P19细胞心肌分化的条件。③P19细胞心肌分化有赖于药物诱导和细胞成团的共同作用,细胞间黏附分子对其分化有促进作用。
OBJECTIVE: To study how to transform embryonic stem cells into a large number of cardiomyocytes in vitro and transplant them into mature cardiomyocytes. In this experiment, different inducing agents were used to induce P19 embryonic stem cells and to construct in vitro cardiomyocyte differentiation model. METHODS: The experiment was performed at the Molecular Engineering Laboratory of Tokyo Polytechnic University from September 2006 to March 2007. ① Materials: P19 cells were provided by Institute of Aging Medicine, Tohoku University. Cadherin fusion protein N-cad-Fc was synthesized by our laboratory. ② Experimental methods: P19 cells were inoculated in 1 × 107 L-1 density medium and cultured in suspension at concentrations of 1, 5, 10, 50 and 100 nmol / L retinoic acid and 0.5%, 0.75%, 1%, 1.25% and 1.5% Dimethylsulfoxide was induced for 4 days. The formed cell aggregates were inoculated into 24-well plates pre-plated with 0.1% gelatin, 2.5 mg / LN-cad-Fc and 10 mg / LN-cad- ③ Experimental evaluation: We observed the different conditions and cell stripping on different matrices. The expression of myocardial specific protein TroponinT was detected by immunofluorescence staining. The expression of cardiacactin and gata-4, a marker gene of cardiomyocytes, was detected by RT-PCR. Results: ① P19 cells were induced by 1,5,10 nmol / L retinoic acid or 0.5%, 0.75%, 1% dimethylsulfoxide. P19 cell aggregates were cultured in suspension for 20 days. The incidences of rhythmic beating cell clusters on the plates pre-plated with 0.1% gelatin, 2.5 mg / LN-cad-Fc and 10 mg / LN-cad-Fc were 44.6%, 71.3 % And 70.1%. ② After drug induction, P19 cells were positive for TroponinT. ③ Compared with the traditional gelatin-coated plate, the expression of cardiacactin and gata-4 in the N-cad-Fc-plated plate was significantly increased, but the expression of gata-4 The expression level was slightly lower than 2.5 mg / LN-cad-Fc. ④ The P19 cells cultured in different layers on the above substrates showed epithelioid cell morphology 14 days after dimethyl sulfoxide induction, no beating cells appeared, and no myocardial gene expression was detected by RT-PCR. Conclusion: ① The myocardial differentiation of P19 cells can be induced successfully by using 0.5% ~ 1% dimethyl sulfoxide or 1 ~ 10 nmol / L retinoic acid. The expression of Troponin T is earlier than the appearance of myocardial beating. ② As a matrix model to simulate the intercellular adhesion, 2.5mg / LN-cad-Fc is more suitable for P19 cell cardiac differentiation conditions. ③ P19 cell differentiation depends on the drug-induced cell division and the synergy of cells, intercellular adhesion molecules promote their differentiation.