解剖学报 ›› 2019, Vol. 50 ›› Issue (1): 24-28.doi: 10.16098/j.issn.0529-1356.2019.01.005

• 神经生物学 • 上一篇    下一篇

优化建立猪多能性细胞及向神经谱系细胞特异性分化

李雪1 张犇2 牛淑冬1 王玉阁1 文丽波1 梁晨3 齐晓娟1 李宇4 雷蕾5*   

  1. 1.齐齐哈尔医学院生理学教研室,黑龙江 齐齐哈尔 161000; 2.齐齐哈尔市第一医院心内科,黑龙江 齐齐哈尔 161000; 3.齐齐哈尔医学院第三附属医院对外协作科,黑龙江 齐齐哈尔 161000;  4.齐齐哈尔医学院机能实验学教研室,黑龙江 齐齐哈尔 161000; 5.哈尔滨医科大学组织学与胚胎学教研室,黑龙江 哈尔滨 150081
  • 收稿日期:2018-04-17 修回日期:2018-07-23 出版日期:2019-02-06 发布日期:2019-04-18
  • 通讯作者: 雷蕾 E-mail:lixue_0720@163.com

Optimization of establishment of pig pluripotent cells and directly differentiation into neural lineage cells

LI Xue1 ZHANG Ben2 NIU Shu-dong1 WANG Yu-ge1 WEN Li-bo1 LIANG Chen3 QI Xiao-juan1 LI Yu4 LEI Lei 5*   

  1. 1.Department of physiology, Qiqihar Medical College, Heilongjiang  Qigihar 161000,China; 2.Department of Cardiology, Qiqihar First Hospital, Heilongjiang Qigihar 161000,China; 3.Department of External Cooperation Senction, the Third Affiliated Hospital of Qiqihar Medical College, Heilongjiang  Qigihar 161000, China;  4.Department of Functional Experiment, Qiqihar Medical College, Heilongjiang Qigihar 161000, China; 5.Department of Histology and Embryology, Harbin Medical University, Heilongjiang Harbin 150081, China
  • Received:2018-04-17 Revised:2018-07-23 Online:2019-02-06 Published:2019-04-18
  • Contact: LEI Lei E-mail:lixue_0720@163.com

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Ⅲ β-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。

关键词: 胚胎成纤维细胞, 重编程, 诱导多能性细胞, 神经分化, 维甲酸, 免疫荧光,

Abstract:

Objective To generate the induced pig pluripotent cells (iPPCs) using the optimized induced pluripotent stem cell(iPSc) technology, and to discuss the method of directly differentiate into neural lineage cells. Methods we generated the iPPCs using the classical iPS 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 iPS 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.

Key words:  Embryonic fibroblast, Reprogramming, Induced pluripotent cell, Neural differentiation, Retinoic acid, Immunofluorescence, Pig