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组蛋白乙酰化修饰在骨髓间充质干细胞向平滑肌细胞分化中的作用
作者:杨婧 王延洲 梁志清 
单位:550001 贵阳 贵州省人民医院妇产科(杨婧) 400038 重庆 陆军军医大学(原第三军医大学)西南医院妇产科(王延洲、梁志清) 
关键词:间充质干细胞 膀胱平滑肌细胞 共同培养技术 乙酰化作用 免疫共沉淀 
分类号:R322.62;R329.2;R394.2
出版年,卷(期):页码:2019,44(5):369-374
摘要:

 [摘要]  目的  探讨骨髓间充质干细胞(BMSCs)在平滑肌微环境中发生分化后平滑肌标志性基因乙酰化水平的变化,以及组蛋白乙酰化修饰在干细胞分化中的作用及机制。方法  体外培养BMSCs和膀胱平滑肌细胞(BSMCs),选择同批次的第3BMSCs,将与BSMCs共培养3dBMSCs作为实验组,未经共培养的BMSCs作为对照组,采用RT-PCR检测两组BMSCs中平滑肌α肌动蛋白(α-SMA)、钙调节蛋白(calponin)、平滑肌肌球蛋白重链(SM-MHC)的表达丰度。采用条件为80%20次、0.5 s8个循环的超声破碎实验组及对照组BMSCsDNA,以H3K9抗体结合特定乙酰化位点, 采用免疫共沉淀技术(ChIP)沉淀BMSCs组蛋白乙酰化位点基因,接头PCR扩增所获基因,Real-time PCR检测所获基因中3种目的基因(α-SMACalponinSM-MHC)的表达水平。结果  RT-PCR检测结果显示,实验组BMSCs中的平滑肌标志性基因(α-SMACalponinSM-MHC)mRNA表达水平较对照组明显增高[分别为0.176±0.003 vs. 0.070±0.0020.079±0.002 vs. 0.051±0.0030.091±0.004 vs. 0.034±0.001],差异均有统计学意义(P<0.01)。分光光度计检测ChIP 后获得的DNA,结果表明H3K9乙酰化抗体沉淀所获DNA浓度高于IgG抗体,且实验组高于对照组(P<0.05)Real-time PCR分析BMSCs分化前后目的基因组蛋白H3K9乙酰化水平,结果显示,BMSCs分化后,实验组的平滑肌标志性基因α-SMAcalponinSM-MHCmRNA转录水平明显高于对照组[分别为9.26±5.03 vs. 1.01±0.052.33±0.65 vs. 0.99±0.052.63±0.37 vs. 1.00±0.03],差异均有统计学意义(P>0.05)结论  在平滑肌微环境中,BSMCs特定位点H3K9乙酰化程度的增加可促进BMSCsBSMCs的分化。

 [Abstract]  Objective  To study the changes of acetylation level of smooth muscle marker gene after the differentiation of bone marrow mesenchymal stem cells (BMSCs) in smooth muscle microenvironment, and explore the role and mechanism of histone acetylation modification in the differentiation of stem cells. Methods  BMSCs and bladder smooth muscle cells (BSMCs) were cultured in vitro. The third generation BMSCs of the same batch were selected, and BMSCs co-cultured with BSMCs for 3 days were set as the experimental group and the BMSCs without co-culture as the control group. RT-PCR was performed to compare the expression abundance between the two groups of α-smooth muscle actin (α-SMA), calponin and smooth muscle myosin heavy chain (SM-MHC) in BMSCs. Ultrasound of power 80%, 20 times, 0.5 s and 8 cycles were used to break the BMSCs DNA of both experimental and control group. Antibody H3K9 was used to bind to the specific acetylation sites. The acetylation site genes of histone in BMSCs were precipitated by chromatin immunoprecipitation (ChIP). The genes obtained was amplified by adaptor PCR. The expression levels of the three kinds of target genes (α-SMA, calponin, SM-MHC) were detected by Real-time PCR. Results  RT-PCR showed that the expression levels of mRNA of smooth muscle marker genes (α-SMA, calponin, SM-MHC) in BMSCs were significantly higher in experimental group than in control group (0.176±0.003 vs. 0.070±0.002; 0.079±0.002 vs. 0.051±0.003 and 0.091±0.004 vs. 0.034±0.001, respectively) with significant differences. Test results of spectrophotometer showed that the amount of DNA obtained by precipitation of H3K9 acetylated antibody was higher than that of IgG antibody, and was higher in experimental group than in control group (P<0.05). Real-time PCR used to analyze the acetylation level of H3K9 before and after the differentiation of BMSCs showed that the mRNA transcription levels of smooth muscle marker genes (α-SMA, calponin, SM-MHC) were significantly higher in experimental group than in the control group (9.26±5.03 vs. 1.01±0.05, 2.33±0.65 vs. 0.99±0.05, 2.63±0.37 vs. 1.00±0.03, respectively) after BMSCs differentiation with statistically significant differences. Conclusion  The increase of acetylation level of H3K9 specific site of BSMCs in smooth muscle microenvironment promotes the differentiation of BMSCs into BSMCs.

基金项目:
国家自然科学基金青年科学基金项目(30801234)
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