摘要
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1×10^7L^-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。
AIM： How to use embryonic stem cells to differentiate into cardiomyocytes in vitro and then transplant so it can possess a mature cardiac function？ P19 embryonal carcinoma cells were induced by different drugs to establish the model of differentiation into cardiomyocytes. METHODS： The experiment was conducted at the Laboratory of Department of Biomolecular Engineering, Tokyo Institute of Technology from September 2006 to March 2007. （1）P19 cells was provided by Institute of Development, Aging and Cancer, Tohoku University. Fusion protein of the N-cadherin extracellular domain and the IgG Fc domain （N-cad-Fc） was synthesized by this laboratory. （2）P19 cells were seeded at a density of 1×10^7 L^-1 cells exposed to different concentrations （1, 5, 10, 50, 100 nmol/L） of retinoic acid or dimethyl sulfoxide （DMSO） （0.5%, 0.75%, 1%, 1.25%, 1.5%） for 4 days to induce aggregates, separately. The aggregated masses were cultured on 24-well plates, which were coated with 0.1% of gelatin, 2.5 mg/L N-cad-Fc, 10 mg/L N-cad-Fc, separately for 10 days. （3）Beating cells under different induction conditions and different matrixes were observed. Cardiomyocyte specific protein cardiac Troponin T was examined by immunofiuorescence staining. The expression of cardiac-specific genes such as cardiac actin and gata-4 were examined by reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： （1）Spontaneously contracting cardiomyocytes could be seen when induced by 1, 5, 10 nmol/L of retinoic acid or 0.5%, 0.75%, 1% of DMSO. P19 cells were aggregated in suspension culture for 20 days, and the beating masses were observed 44.6%, 71.3%, 70.1% when the aggregates were transferred to 0.1% gelatin, 2.5 mg/L N-cad-Fc, 10 mg/L N-cad-Fc-coated dishes, separately. （2）Cardiomyocyte specific protein cardiac Troponin T could be seen after P19 cells were induced by drugs. （3）The aggregates cultured on N-cad-Fc-coated dishes compared with gelatin-coated dishes, the expression of cardiac-specific genes cardiac actin and gata-4 were much higher, but the expression on 10 mg/L N-cad-Fc-coated dishes was slightly low than on 2.5 mg/L N-cad-Fc-coated. （4） Monolayer P19 cells cultured on dishes coated with 0.1% gelatin, 2.5 mg/L N-cad-Fc, 10 mg/L N-cad-Fc in the presence of DMSO, the cells still as epithelioid after 14 days, no beating cells could be seen and no expression of cardiac-specific genes could be examined by RT-PCR CONCLUSION： （1）DMSO （0.5%- 1%） or retinoic acid （1-10 nmol/L） can induce P 19 cells to differentiate into cardiomyocytes. The expression of Troponin T could be observed before cells beating. （2）As a model matrix for cell-cell adhesion, 2.5 mg/L N-cad-Fc could cause more effective differentiation. （3）P19 cells differentiated into cardiomyocytes depend on the interaction of drug and cell aggregation. Intercellular adhesion molecule is advantage for cell differentiation.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2277-2280,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30400361)~~
