HIF-1α基因转录活性位点在人骨髓间充质干细胞分化成心肌细胞中的作用  

Effects of Transcriptional Activity Site of Hypoxia-inducible Factor 1α on Differentiation of Human Mesenchymal Stem Cells to Cardiomyocytes

作　　者：刘城[1] 吴平生[1] 王月刚[1] 裴静娴[1] 赖艳娴[1] 

机构地区：[1]广州市第一人民医院心血管内科,广东广州510180

出　　处：《湖北民族学院学报（医学版）》2013年第2期1-5,共5页Journal of Hubei Minzu University(Medical Edition)

基　　金：国家自然科学基金项目(81100235)

摘　　要：目的研究HIF-1α(hypoxia inducible factor-1α,HIF-1α)基因转录活性位点—Asn803位点在人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)分化成心肌细胞中的作用。方法重组腺病毒Ad-LacZ、Ad-HIF-1αnative、Ad-HIF-1α564、Ad-HIF-1α564/402及Ad-HIF-1α564/803分别以最佳转染复数转染体外5-氮胞苷(5-azacyti-dine,5-aza)诱导的hMSCs,于诱导培养后第6周:(1)提取细胞浆蛋白,应用酶联免疫分析检测心肌肌钙蛋白I(cT-nI)含量,Western blot测定HIF-1α及VEGF蛋白表达水平;(2)更换含有120μmol/L姜黄素的心肌细胞诱导液培养1h,后用心肌细胞诱导液继续培养11h,接着用RT-PCR检测细胞中VEGF mRNA表达水平的变化。结果经5-aza诱导hMSC s第6周后,与Ad-LacZ组、Ad-HIF-1αnative组、Ad-HIF-1α564组及Ad-HIF-1α564/402组相比,Ad-HIF-1α564/803组cTnI、VEGF表达水平最高,与前述其它各组相比差异有显著性(P值均<0.05)。经120μmol/L姜黄素抑制后VEGF mRNA表达水平较未抑制组平均减少了(50.9±4.6)%。然而,在CBP/p300活性受同等抑制条件下,Pro564Asn803双突变型HIF-1α基因组VEGF mRNA表达水平虽然较未抑制组减少了约40%,但仍显著高于Pro564单突变型HIF-1α基因组(P<0.05)。结论 HIF-1α基因转录活性位点(Asn803)在HIF-1α促进hMSCs向心肌细胞分化中起关键作用。Objective To study the effects of transcriptional activity site （Asneo3） of hypoxia-in- ducible factor 1α during human mesenchymal stem cells differentiate into cardiomyocyte in- duced by 5-azacytidine in vitro. Methods To transfect 5-azacytidine induced hMSCs with Ad- La cZ, Ad- H IF-1α native, Ad- H IF-1α564, Ad- H IF- loL564/402 a n d Ad- H IF- 1 oL564/8o3 at the optimal m u Iti- plicity of infection （MOI）,cells were harvested at 6 weeks after the transfection, and then to detect the expression of cTnl by ELISA as well as HIF-1α,VEGF by western blot analysis; or were pre-treated with 120 μmol/L curcumin for 1 h, and cultured for another 11 hours, cells were then harvested sequentially for RT-PCR analysis.Results In contrast, there were more significant differences in expression of cTnl among the control group（0.700±0.013）, Ad-HIF- 1αnative（0.729＋0.004） ,Ad-HIF-1α64（0.741±0.008） ,Ad-HIF-1α64/4o2（0.764±0.009） and Ad-HIF-1α564/803（0.804±0.021） （P〈0.05）,and the cTnl expression level transfected by Ad-HIF-1α564/803 was significantly higher as compared with all other experimental group（all P〈0.05）. Mean- while,there were also significant differences in expression of HIF-1α and VEGF between Ad- HIF-1α native,Ad-HIF-1α564,Ad-HIF-l1α564/402 and Ad-HIF-1α564/803（P〈O.05）, and the expression level of HIF-1α was the highest in Ad-HIF-1α564/402（all P〈0.05）, but the significant higher ex- pression of VEGF were in Ad- HIF-1α564/803 （ all P〈0.05, respectively）. Simultaneously, com- pared with the VEGF mRNA expression level in the control group, the VEGF mRNA expression level in suppression group （ inhibited by 120μmol/L curcumin） decreased （50.9±4.6） % in av- erage. However, the VEGF mRNA level of Ad-HIF-1α564/803 was still significant higher than the Ad-HIF-1α564（P〈0.05） .Conclusion The transcriptional activity site （ Asns03 ） of hypoxia-inducible factor 1α plays pivotal roles in differentiation of human mesenchymal stem c

关 键 词：低氧诱导因子1Α 转录活性位点 人骨髓间充质干细胞 心肌细胞 

分 类 号：R542.2[医药卫生—心血管疾病]