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 张犇 牛淑冬 王玉阁 文丽波 梁晨 齐晓娟 李宇 雷蕾*

doi:10.16098/j.issn.0529-1356.2019.01.005

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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

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*

作者信息 +

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*

Author information +

文章历史 +

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。

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.

导出引用

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 https://doi.org/10.16098/j.issn.0529-1356.2019.01.005

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 https://doi.org/10.16098/j.issn.0529-1356.2019.01.005

参考文献

［1］ Johansen LK, Frees D, Aalbaek B, et al. A porcine model of acute, haematogenous, localized osteomyelitis due to Staphylococcus aureus: a pathomorphological study ［J］. APMIS, 2011, 119(4):111-118.

［2］ Kotoulas C, Panagiotou Ⅰ, Tsipas P, et al. Experimental studies in the bronchial circulation. Which is the ideal animal model ［J］. J Thorac Dis, 2014, 6 (10):1506-1512.

［3］ Wang L, Tao Y, Zhang XR, et al. Advances in the establishment of pig embryonic stem cells［J］.Progress in Veterinary Medicine, 2010, 31(11):86-91. (in Chinese)

王磊, 陶勇, 章孝荣,等. 猪胚胎干细胞建系研究进展［J］. 动物医学进展, 2010, 31(11):86-91.

［4］ Chakritbudsabong W, Sariya L, Pamonsupornvichit S, et al. Generation of a pig induced pluripotent stem cell (piPSC) line from embryonic fibroblasts by incorporating LIN28 to the four transcriptional factor-mediated reprogramming: VSMUi001-D ［J］.Stem Cell Res, 2017,24(5):21-24.

［5］ Fang YQ, Zhu ShH, Tang JM, et al. Research progress on animal iPS cells［J］.Progress in Veterinary Medicine, 2015, 36(12):145-149. (in Chinese)

方月琴, 朱少晖, 汤俊梅,等. 动物iPS细胞研究进展［J］. 动物医学进展, 2015, 36(12):145-149.

［6］ Xue BH, Liu ZhH. Porcine pluripotent stem cells: facts, challenges and hopes ［J］. Biotechnology Bulletin, 2015, 31(4):82-91. (in Chinese)

薛冰华, 刘忠华. 猪多能性干细胞研究进展与前瞻［J］. 生物技术通报, 2015, 31(4):82-91.

［7］ Biswas D, Peng J. Chemically induced reprogramming of somatic cells to pluripotent stem cells and neural cells ［J］. Int J Mol Sci, 2016, 17(2):218-226.

［8］ Fan WJ, Ma YCh, Chen XD, et al. Mouse induced pluripotent stem cells differentiate into neurons by retinoic acid ［J］. Chinese Journal of Neuroanatomy, 2015, 31(1):103-108. (in Chinese)

范文娟, 马永超, 陈旭东，等. 维甲酸诱导小鼠iPS细胞向神经细胞分化［J］. 神经解剖学杂志, 2015, 31(1):103-108.

［9］ Shan ZY, Shen JL, Li QM, et al. pCREB is involved in neural induction of mouse embryonic stem cells by RA. ［J］. Anat Rec, 2008, 291(5):519-526.

［10］Bilic J, Izpisua Belmonte JC. Concise review: induced pluripotent stem cells versus embryonic stem cells: close enough or yet too far apart ［J］. Stem Cells, 2012, 30(1):33-41.

［11］Li X, Shan ZhY, Wu YSh, et al. Generation of induced pluripotent stem cells from human foreskin fibroblasts ［J］. Acta Anatomica Sinica, 2012, 43(5): 601-607. (in Chinese)

李雪, 单智焱, 吴嫣爽,等. 人包皮成纤维细胞来源的多能性干细胞的建立［J］. 解剖学报, 2012, 43(5): 601-607.

［12］Robinton DA, Daley GQ. The promise of induced pluripotent stem cells in research and therapy ［J］. Nature, 2012, 481(10761): 295-305.

［13］Li Y, Zhang Q, Yin X, et al. Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules ［J］. Cell Res, 2011, 21(1):196-204.

［14］Alexander MA, Mikkelsen TS, Gu HC, et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells ［J］. Nature, 2008, 454(7205):766-770.

［15］Moon JH, Kim JH, Im HJ, et al. Proposed motor scoring system in a porcine model of Parkinson’s disease induced by chronic subcutaneous injection of MPTP ［J］. Exp Neurobiol, 2014, 23(3):258-265.