武晓慧,徐玉乔,张丰,郜松,魏佳昶,许久航,李青,苏兴利Vol.41(4):534-541
摘要：【目的】探索胎鼠棕色脂肪细胞分化过程中组蛋白H3赖氨酸残基位点H3k4，H3k9，H3k27，H3k36的22种去甲基化酶的调控作用，为棕色脂肪细胞分化研究提供理论基础。【方法】收集怀孕时间为E13.5~E19.5d的小鼠胚胎，每个时间点至少取3只胎鼠。取肩胛间区棕色脂肪，进行HE染色，镜下观察棕色脂肪细胞的分化情况。Realtime RT-qPCR法检测随着小鼠胚胎的发育成熟，棕色脂肪标志基因Ucp1、Cidea、Prdml6及脂肪细胞分化基因Pparγ的表达情况和棕色脂肪中组蛋白H3的22种赖氨酸去甲基化酶的基因表达情况。Westen blot法检测棕色脂肪组织中Ucp1的蛋白表达。【结果】最早在E14.5 d的胎鼠背部肩胛间区观察到棕色脂肪组织；E18.5d胎鼠棕色脂肪细胞中出现光镜下可见的脂滴。与E15.5 d相比，Ucp1、Cidea、Prdm16及Pparγ的表达升高（P<0.05）；Ucp1的蛋白表达增加（P<0.05）。与E15.5d相比，H3k4的去甲基化酶中，Jarid1a、Jarid1b、Jarid1d、Lsd1基因表达减少（P＜0.05），Jarid1c表达差异无统计学意义（P＞0.05）。在H3k9的去甲基化酶中，Jmjd2a、Jmjd2b、Jmjd2c、Phf2、Jmjd1c、Jhdm2a基因表达减少（P＜0.05）；Phf8、Jhdm2b、Jmjd2d基因表达增加（P＜0.05），MINA表达差异无统计学意义（P＞0.05）。在H3k36的去甲基化酶中，Jmjd4、Jmjd5、Jhdm1b表达减少（P＜0.05），Jhdm1a表达增加（P＜0.05）。在H3k27的去甲基化酶中，Jmjd3、Utx、Jhdm1d表达增加（P＜0.05）。【结论】在小鼠胚胎期棕色脂肪细胞的分化过程中，H3k4和H3k36去甲基化酶主要表现为基因表达下调，H3k9和H3k27去甲基化酶部分基因表达上调，这些去甲基化酶构成以转录激活和转录延长为主要效应的复杂调控网络。
关键词：棕色脂肪细胞；分化；组蛋白H3；去甲基化酶
Abstract:【Objective】To explore the effects of 22 demethylases of histone H3k4，H3k9，H3k27 and H3k36 on the differentiation of fetal mice brown adipocytes，so as to provide a theoretical basis for the study of differentiation of brown adipocytes.【Methods】We collected the embryos from pregnant mice at stages E13.5 to E19.5，with no less than 3 fetal mice obtained at each time point. Then we isolated interscapular brown adipose tissues（BAT）for HE staining and observed the differentiation of brown adipocytes under a microscope. Realtime RT-qPCR was used to test the expression of BAT markers such as Ucp1，Cidea and Prdm16，adipocyte differentiation related gene Pparγ and 22 lysine demethylases of histone H3 in BAT. Ucp1 protein expression in BAT was detected by Western blot.【Results】BAT were first observed in the interscapular area of fetal mice at E14.5. Lipid droplets were observed under light microscope in the brown adipocytes of fetal mice at E18.5. Compared with those in E15.5 embryos，in E16.5 to E19.5 embryos，the expression of Ucp1，Cidea，Prdm16 and Pparγ increased（P<0.05）and Ucp1 protein expression increased（P<0.05）. Of H3k4 demethylases，Ja⁃rid1a，Jarid1b，Jarid1d and Lsd1 showed decrease in expression（P< 0.05）；Jarid1c expression had no statistical significance（P>0.05）. Of H3k9 demethylases，Jmjd2a，Jmjd2b，Jmjd2c，Phf2，Jmjd1c and Jhdm2a showed decrease in expression（P< 0.05）；Phf8，Jhdm2b and Jmjd2d showed increase in expression（P<0.05）；MINA expression had no statistical difference（P>0.05）. Of H3k36 demethylases，Jmjd4，Jmjd5 and Jhdm1b showed decrease in expression（P<0.05）；Jhdm1a showed increase in expression（P<0.05）. Of H3k27 demethylases，Jmjd3，Utx and Jhdm1d showed increase in expression（P<0.05）.【Conclusion】In the process of brown adipocytes differentiation，expressions of H3k4 and H3k36 demethylases were mainly downregulated and those of some H3k9 and H3k27 demethylases were upregulated. These demethylases formed a complex regulatory network with transcriptional activation and transcriptional elongation as the main effects.
Key words：brown adipocytes;differentiation;histone H3;demethylase
编辑：王清波
初审：余菁
复审：孙慧兰
审核发布：徐杰
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