摘要
目的探讨血清反应因子(serum response factor,SRF)在大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)分化为膀胱平滑肌细胞(smooth muscle cells,SMCs)中的作用和机制。方法贴壁法分离4周龄雌性SD大鼠的骨髓MSCs与膀胱SMCs,通过Transwell小室共培养诱导MSCs分化。收集MSCs,根据共培养不同时间点进行分组,通过实时荧光定量PCR和免疫荧光法检测共培养SRF MSCs中SMCs标志基因表达;通过染色质免疫共沉淀(chromatin immunoprecipitation assay,ChIP)定量PCR技术检测SMCs相关基因启动子区组蛋白乙酰化修饰、组蛋白去乙酰化酶(histone deacetylase,HDAC)和SRF的富集。丁酸钠(sodium butyrate,NaB)预处理MSCs后与SMCs共培养,实验分为正常共培养对照组和NaB预处理干预组,ChIP-qPCR对比两组间组蛋白修饰和SRF富集情况。结果共培养MSCs中,α-SMA、Calponin和SM-MHC基因表达量增加,在这些基因启动子区H3K9ace,H3ace和H4ace富集量增加(P<0.05),同时MSCs中HDAC1和HDAC2降低(P<0.05)。与对照组相比,NaB干预组SRF、H3K9ace和H4ace富集量增加(P<0.05)。结论 SRF可能在HDAC1和HDAC2调节下通过结合H3K9ace和H4ace控制SMCs标志基因表达,从而促进MSCs向SMCs的分化。
Objective To explore the role and mechanism of serum response factor(SRF) in differentiation of rat bone marrow mesenchymal stem cells(MSCs) into bladder smooth muscle cells(SMCs). Methods Rat MSCs and SMCs were isolated respectively from the bone marrow and bladder of 4-week-old female SD rats by adherent method, and then co-cultured together in a Transwell chamber. After the MSCs were harvested at different time points, real-time PCR and immunofluorescense assay were used to detect the expression of SMCs specific marker genes in co-cultured MSCs. Furthermore, the enrichment of histone acetylation modification, histone deacetylase(HDAC) and SRF in the promoter of these genes were tested by chromatin immunoprecipitation assay(ChIP) q-PCR in co-cultured MSCs. Finally, ChIP-qPCR was used to compare the histone acetylation modification and SRF enrichment in sodium butyrate(NaB) pretreatment group with control cells. Results The expression levels of α-SMA, Calponin and SM-MHC were increased in MSCs co-cultured with SMCs. H3 K9 ace, H3 ace and H4 ace in the promoter of these genes were enriched(P<0.05), and then, HDAC1 and HDAC2 in the induced MSCs were correspondingly decreased significantly(P<0.05). The enrichment of SRF in the NaB pretreatment group shared the same increasing tendency with H3 K9 ace and H4 ace. Conclusion SRF may bind to H3 K9 ace and H4 ace, through the regulation of HDAC1 and HDAC2 to control the expression of SMCs specific genes, and thus promote the differentiation from MSCs to SMCs.
作者
刘晶霞
梁志清
LIU Jingxia;LIANG Zhiqing(Department of Gynecology and Obstetrics,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Gynecology and Obstetrics,Chongqing General Hospital,Chongqing,401121,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2021年第4期303-310,共8页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81170552)。
关键词
骨髓间充质干细胞
血清反应因子
分化
组蛋白乙酰化
丁酸钠
bone mesenchymal stem cells
serum response factor
differentiation
histone acetylation
sodium butyrate
作者简介
通信作者:梁志清,E-mail:zhi.lzliang@gmail.com。
