优化建立猪多能性细胞及向神经谱系细胞特异性分化

doi:10.16098/j.issn.0529-1356.2019.01.005

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

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.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-dong¹WANG Yu-ge¹ WEN Li-bo¹ LIANG Chen³ QI Xiao-juan¹ LI Yu⁴ LEI Lei ^5*

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/Abstract

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, 猪

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

李雪张犇牛淑冬王玉阁文丽波梁晨齐晓娟李宇雷蕾. 优化建立猪多能性细胞及向神经谱系细胞特异性分化[J]. 解剖学报, 2019, 50(1): 24-28.

LI Xue ZHANG Ben NIU Shu-dong WANG Yu-ge WEN Li-bo LIANG Chen QI Xiao-juan LI Yu LEI Lei. Optimization of establishment of pig pluripotent cells and directly differentiation into neural lineage cells[J]. Acta Anatomica Sinica, 2019, 50(1): 24-28.

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优化建立猪多能性细胞及向神经谱系细胞特异性分化

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

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