过表达JHDM2A对猪iPSCs诱导效率的影响

doi:10.16431/j.cnki.1671-7236.2021.12.009

摘要/Abstract

摘要： 研究旨在探讨组蛋白赖氨酸去甲基化酶(JHDM2A)对猪成纤维细胞诱导为多能干细胞效率的影响,并对其内在的分子机制进行探究。以猪成纤维细胞为材料,采用多西环素(DOX)诱导的慢病毒生产诱导多能干细胞(iPSCs)体系,在此基础上过表达JHDM2A,通过碱性磷酸酶染色和绘制诱导时间轴检测其对诱导效率的影响;普通PCR技术检测克隆的多能性;免疫荧光检测多能因子的蛋白表达;实时荧光定量PCR检测JHDM2A在形成克隆后以及克隆分化过程中对多能因子、组蛋白甲基化相关基因表达的影响。结果表明,JHDM2A过表达组细胞在第3天发生形态变化,第8天形成iPSCs克隆,相比于对照组分别提前了1和2 d。碱性磷酸酶染色结果显示,与对照组相比,JHDM2A过表达组克隆形态明显改善,染色着色更深,诱导效率提高8倍。普通PCR结果显示,JHDM2A过表达组iPSCs、对照组iPSCs以及猪成纤维细胞(PFF)均表达内源性Oct4、Sox2、Klf4、c-Myc、Nanog,且iPSCs的Oct4表达量高于PFF。免疫荧光结果显示,JHDM2A过表达组iPSCs表达Oct4、Sox2、Stat3、JHDM2A,弱表达SSEA1、SSEA4。实时荧光定量PCR结果显示,与对照组和猪成纤维细胞相比,JHDM2A过表达组iPSCs的Sox2、Klf4、c-Myc、Nanog、Oct4、Tcl1的表达显著升高(P<0.05),Tfcp2l1和Zfp57的表达显著降低(P<0.05);JHDM2A过表达组P5代iPSCs培养基去除DOX后,随着代数的增加,Sox2、Nanog的表达逐渐降低,Klf4、c-Myc的表达先升高后降低,Oct4、Tcl1、Tfcp2l1、Zfp57的表达先降低后升高。以上结果表明,过表达JHDM2A通过促进组蛋白去甲基化提高猪成纤维细胞的诱导效率。

关键词: 猪; JHDM2A; iPSCs; 诱导效率

Abstract: The purpose of this study was to investigate the effect of histone demethylase (JHDM2A) on porcine fibroblast induced pluripotent stem cell efficiency, and to explore its intrinsic molecular mechanism.Porcine fibroblasts were used as materials, doxycycline (DOX)-inducible lentiviral vectors were used to produce iPSCs, and JHDM2A was overexpressed on this basis.The effect of JHDM2A on induction efficiency was detected by alkaline phosphatase (AP) staining and induction time axis.The pluripotency of the iPSCs was determined by PCR.The protein expression of the pluripotent factors was detected by immunofluorescence.The effects of JHDM2A on the expression of pluripotent factors and histone methylation related genes after clonies formation and during clonies differentiation were detected by Real-time quantitative PCR.The results showed that the cells of the JHDM2A overexpressed group deformed on the 3rd day and formed iPSCs clonies on the 8th day, which were 1 and 2 d earlier than those of control group.The results of AP staining showed that compared with the control group, the morphology of iPSCs in the JHDM2A overexpressed group was significantly improved, the AP staining was deeper, and the induction efficiency was increased by 8 times.Conventional PCR results showed that endogenous Oct4, Sox2, Klf4, c-Myc and Nanog were expressed in JHDM2A overexpressed group, control group and porcine fibroblasts (PFF), and the expression of Oct4 in iPSCs was higher than that in PFF.Immunofluorescence results showed that Oct4, Sox2, Stat3 and JHDM2A were expressed in iPSCs of JHDM2A overexpressed group, while SSEA1 and SSEA4 were weakly expressed.Real-time quantitative PCR results showed that compared with control group and PFF, the expressions of iPSCs genes Sox2, Klf4, c-Myc, Nanog, Oct4 and Tcl1 in JHDM2A overexpressed group were significantly increased (P<0.05), and the expressions of Tfcp2l1 and Zfp57 were significantly decreased (P<0.05).After DOX was removed from P5 iPSCs culture medium in the JHDM2A overexpressed group, with the increase of generation, the expressions of Sox2 and Nanog gradually decreased, the expressions of Klf4 and c-Myc first increased and then decreased, and the expressions of Oct4, Tcl1, Tfcp2l1 and Zfp57 first decreased and then increased.These results suggested that overexpression of JHDM2A could improve the induction efficiency of porcine fibroblasts by promoting histone demethylation.

Key words: pigs; JHDM2A; iPSCs; induction efficiency

中图分类号:

S828

任宣, 宋延霞, 孙乐, 黄时海, 石德顺, 李湘萍. 过表达JHDM2A对猪iPSCs诱导效率的影响[J]. 中国畜牧兽医, 2021, 48(12): 4412-4421.

REN Xuan, SONG Yanxia, SUN Le, HUANG Shihai, SHI Deshun, LI Xiangping. Effect of Overexpression of JHDM2A on iPSCs Induction Efficiency in Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4412-4421.

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