【摘要】 目的探讨建立猪多能性细胞(iPPCs)的优化方案,并探求其向神经谱系细胞特异性分化的方法。方法利用经典的Yamanaka方法,联合应用组蛋白乙酰化酶抑制剂丙戊酸(VPA)、甲基转移酶抑制剂5-氮-2’-脱氧胞苷(5-AZA)及Oct4病毒的重复感染,优化重编程方案,诱导巴马小型猪胚胎成纤维细胞为诱导的iPPCs。通过Real-time PCR检测重编程过程中多能性基因的分子表达。通过维甲酸(RA)及细胞外基质(ECM)的联合培养,诱导iPPCs向神经谱系细胞特异性分化,免疫荧光细胞化学方法检测神经特异性标记物表达。结果应用优化方案,将猪胚胎成纤维细胞重编程为iPPCs。Real-time PCR显示,VPA和Oct4病毒的重复感染可显著促进重编程过程中多潜能基因的表达。5-AZA未显著提高多潜能基因的表达。RA及ECM的联合培养可诱导iPPCs向神经谱系细胞分化,并表达神经特异性标志基因神经元类型Ⅲβ-微管蛋白(Tuj1)和胶质纤维酸性蛋白(GFAP)。结论在利用优化方案建立猪多能性细胞的基础上,将其向神经谱系细胞特异性分化,对人类神经性疾病的细胞替代治疗具有重要意义。更多还原

【Abstract】 Objective To generate the induced pig pluripotent cells(iPPCs) using the optimized induced pluripotent stem cell(i PSc) technology,and to discuss the method of directly differentiate into neural lineage cells.Methods we generated the iPPCs using the classical i PS technology in combination with valproic acid(VPA) and the methyltransferase inhibitor 5-aza-2’-deoxycytidine(5-AZA) with Oct4 retrovirus infected repetitively.Real-time PCR analysis of the expression of pluripotent related genes,immunofluorescent detection of neuron specific marker after using retinoic acid(RA)and extracellular matrix induction.Results By using this optimizing i PS technology,we successfully established a reasonable method to generate iPPCs.Real-time PCR result indicated VPA treatment significantly increased the expression of pluripotent genes,which was the same as repeated infection with Oct4 retrovirus.In addition,the iPPCs might directly differentiate into neural lineage cells after being induced with the retinoic acid and extracellular matrix.Conclusion We establishes a reasonable method to generate pig pluripotent cells,which may be a new donor cell source for human neural disease therapy.更多还原