ATRA 诱导小鼠间充质干细胞棕色成脂分化的分子机制

doi:10.3969/j.issn.1673-8640.2019.05.015

摘要/Abstract

摘要：

目的研究全反式维甲酸（ATRA）或骨形态发生蛋白7（BMP7）诱导小鼠间充质干细胞定向分化为成熟棕色脂肪细胞的作用及机制。方法采用ATRA或BMP7对小鼠间充质干细胞C3H10T1/2进行体外诱导,使其向棕色脂肪细胞定向分化,比较二者诱导分化棕色成脂效果;采用油红O染色鉴定棕色脂肪细胞成脂能力,线粒体染色检测棕色脂肪细胞富集的线粒体,免疫印迹法检测脂肪分化相关基因、棕色脂肪标志基因表达,采用实时荧光定量聚合酶链反应（PCR）检测BMP7、线粒体相关基因及棕色脂肪细胞分化抑制因子Necdin、Wnt10a及Pref1的表达情况。结果 ATRA诱导C3H10T1/2细胞向棕色脂肪细胞分化的最适浓度为1~2 μmol/ L。采用1 μmol/L ATRA预处理3 d（ATAR组）后,与对照组[采用二甲基亚砜（DMSO）处理]比较,ATRA组线粒体相关基因CyctoC、Tfam及Nrf1的mRNA相对表达量明显升高（P<0.05）,棕色脂肪细胞分化抑制因子Necdin和其下游靶基因Pref1、Wnt10a的mRNA相对表达量明显降低（P<0.05）,BMP7 mRNA相对表达量明显升高（P<0.05）。不同浓度的ATRA均可使BMP7蛋白表达量升高,且呈剂量效应。采用1 μmol/L ATRA预处理0、24和48 h,BMP7蛋白表达量逐渐升高,Necdin蛋白表达量逐渐降低。ATRA与BMP7的诱导效果一致。结论 ATRA具有诱导小鼠间充质干细胞C3H10T1/2分化为棕色脂肪细胞的能力,其机制可能是通过调控BMP7,诱导棕色前体脂肪细胞定向分化。

关键词: 全反式维甲酸, 骨形态发生蛋白7, 棕色脂肪细胞, 定向分化

Abstract:

Objective To investigate the effect and mechanism of all-trans retinoic acid （ATRA） or bone morphogenetic protein 7 （BMP7） on the development of brown adipocytes in C3H10T1/2 mesenchymal stem cells. Methods C3H10T1/2 mesenchymal stem cells were pretreated with ATRA or BMP7,followed by an adipogenic cocktail. To evaluate the adipogenesis,oil red O staining was used to identify brown adipogenic capacity. Mitochondrial track staining was used to analyze mitochondrial biogenesis in mature brown adipocytes. The expressions of BMP7,brown adipogenic inhibitors （Necdin,Wnt 10a and Pref 1） and brown adipocyte-related gene were determined by real-time fluorescence quantitation polymerase chain reaction （PCR）and western blotting. Results The optimal concentration of ATRA in promoting C3H10T1/2 brown adipogenesis was 1-2 μmol/ L. After pretreatment for 3 d with 1 μmol/L ATRA （ATRA group）,the mitochondrial biogenesis gene mRNA expressions of CyctoC,Tfam and Nrf1 were increased （P<0.05）,the mRNA expressions of brown adipogenic inhibitor Necdin and its downstream target genes （Pref1 and Wnt10a） were decreased （P<0.05）,and the mRNA expression of BMP7 gene was increased （P<0.05）,compared with control group treated by dimethyl sulfoxide （DMSO）. Different concentrations of ATRA can induce BMP7 protein expression in a dose-response manner. After the pretreatment with 1 μmol/L ATRA for 0,24 and 48 h,BMP7 protein expression was increased,and Necdin protein expression was decreased. The differentiation effect of ATRA and BMP7 was comparable. Conclusions ATRA promotes brown adipogenesis of C3H10T1/2 mesenchymal stem cells via the activation of BMP7 and reduces negative modulators of brown preadipocyte differentiation.

Key words: All-trans retinoic acid, Bone morphogenetic protein 7, Brown adipocyte, Directional differentiation

中图分类号:

R446.1

梁璆荔, 程金科, 左勇. ATRA 诱导小鼠间充质干细胞棕色成脂分化的分子机制[J]. 检验医学, 2019, 34(5): 451-456.

LIANG Qiuli, CHENG Jinke, ZUO Yong. ATRA for promoting brown adipogenesis in C3H10T1/2 mesenchymal stem cells[J]. Laboratory Medicine, 2019, 34(5): 451-456.

图/表 8

表1 引物序列

基因	上游引物（5'→3'）	下游引物（5'→3'）
Rps18	CATGCAGAACCCACGACAGTA	CCTCACGCAGCTTGTTGTCTA
Pparγ	GTGCCAGTTTCGATCCGTAGA	GGCCAGCATCGTGTAGATGA
PGC1α	CCCTGCCATTGTTAAGACC	TGCTGCTGTTCCTGTTTTC
PRDM16	CAGCACGGTGAAGCCATTC	GCGTGCATCCGCTTGTG
Ucp1	ACTGCCACACCTCCAGTCATT	CTTTGCCTCACTCAGGATTGG
Cidea	TGCTCTTCTGTATCGCCCAGT	GCCGTGTTAAGGAATCTGCTG
Necdin	CACTTCCTCTGCTGGTCTCC	ATCGCTGTCCTGCATCTCAC
Wnt10a	CCACTCCGACCTGGTCTACTTTG	TGCTGCTCTTATTGCACAGGC
Pref1	AGTACGAATGCTCCTGCACAC	CTGGCCCTCATCATCCAC
BMP7	ACACAGCTTCGTGGCGCTCT	ATGGACAGGATCTCCCGCTG
CyctoC	GCTACCCATGGTCTCATCGT	CATCATCATTAGGGCCATCC
Nrf1	CAACAGGGAAGAAACGGAAA	GCACCACATTCTCCAAAGGT
Tfam	GTCCATAGGCACCGTATTGC	CCCATGCTGGAAAAACACTT

图1 诱导C3H10T1/2细胞向棕色脂肪细胞分化过程示意图

图2 ATRA诱导小鼠间充质干细胞棕色成脂分化注：（a）油红O染色（×100）;（b）线粒体染色（×200）

图3 棕色脂肪细胞标志蛋白表达

图4 ATRA诱导后线粒体相关基因的表达mRNA相对表达量注：（a）CyctoC mRNA相对表达量;（b）Tfam mRNA相对表达量;（c）Nrf1 mRNA相对表达量;与对照组比较,*P<0.05

图5 ATRA诱导后棕色脂肪细胞分化抑制因子的表达mRNA相对表达量注：（a）Necdin mRNA相对表达量;（b）Pref1 mRNA相对表达量;（c）Wnt10a mRNA相对表达量;与对照组比较,*P<0.05

图6 ATRA处理后BMP7的表达BMP7 mRNA相对表达量注：（a）BMP7 mRNA相对表达量;与对照组比较,*P<0.05;（b）BMP7蛋白表达;（c）BMP7和Necdin蛋白表达

图7 ATRA和BMP7对C3H10T1/2诱导效果比较注：（a）油红O染色（×100）;（b）线粒体染色（×100）;（c）棕色脂肪细胞标志蛋白的表达;（d）棕色脂肪细胞标志基因mRNA的相对表达量;对照组;ATRA组;BMP7组;与对照组比较,*为P<0.05;**为P<0.01

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