低氧诱导因子-1α基因Asn_(803)位点在人骨髓间充质干细胞分化成心肌细胞中的作用  

The effects of Asn_(803) 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]广州医科大学附属广州市第一人民医院心血管内科,510180

出　　处：《广东医学》2014年第1期25-28,共4页Guangdong Medical Journal

基　　金：广东省自然科学基金资助项目(编号:S2011040004458);广州市科技计划重点项目(编号:2012J4100035);广州市医药卫生科技项目(编号:20121A011006)

摘　　要：目的研究低氧诱导因子-1α(HIF-1α)基因Asn803位点在人骨髓间充质干细胞(hMSCs)分化成心肌细胞中的作用。方法将重组腺病毒Ad-LacZ、Ad-HIF-1αnative、Ad-HIF-1α564、Ad-HIF-1α564/402及AdHIF-1α564/803分别以最佳转染复数转染体外5-氮胞苷(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(cTnI)含量,Western blot测定HIF-1α及血管内皮生长因子(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1 h,后用心肌细胞诱导液继续培养11 h,接着用RT-PCR检测细胞中VEGF mRNA表达水平的变化。结果 hMSCs经5-aza诱导6周后,对照组、Ad-LacZ组、Ad-HIF-1αnative组、Ad-HIF-1α564组、Ad-HIF-1α564/402组及Ad-HIF-1α564/803组cTnI值分别为0.690±0.019、0.699±0.007、0.736±0.007、0.748±0.008、0.771±0.008及0.814±0.012(P<0.05),其中以Ad-HIF-1α564/803组cTnI表达水平最高(P<0.05)。Western blot结果显示:与Ad-LacZ组相比,Ad-HIF-1αnative组、Ad-HIF-1α564组、Ad-HIF-1α564/402组、Ad-HIF-1α564/803组HIF-1α及VEGF蛋白均明显升高(P<0.05),其中以Ad-HIF-1α564/402组HIF-1α蛋白表达水平最高(P<0.05),以Ad-HIF-1α564/803组VEGF蛋白表达水平最高(P<0.05)。RT-PCR检测结果显示: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Asn803位点在HIF-1α促进hMSCs向心肌细胞分化中起关键作用。Objective To study the effects Asn803 of hypoxia - inducible factor - 1α （ HIF - 1α） on the differentiation of human mesenchymal stem cells （hMSCs） into cardiomyocyte induced by 5 - azacytidine in vitro. Methods Ad - LacZ, Ad - HIF - 1αnative, Ad - HIF - 1%64, Ad - HIF -1α64/402 and Ad - HIF - 1 oL564/803 were transfected to 5 - aza- cytidine - induced hMSCs at the optimal multiplicity of infection （MOI）. The transfected cells were harvested 6 weeks after the transfection, and the expression of cTnI was measured by ELISA, as well as HIF - 1 α, VEGF by Western blot analysis; or were pre -treated with 120 μmol/L curcumin for 1 h, cultured for another 11 h, and subsequently harvested for RT - PCR analysis. Results The expression of cTnI were 0. 690 ± 0. 019, 0. 699 ± 0. 007, 0. 736 ± 0. 007, 0. 748 ± 0. 008, 0. 771 ±0. 008 and 0. 814 ± 0. 012 in control group, Ad - LacZ group, Ad - HIF - 1αnative group, Ad - HIF - 1 cq64 group, Ad - HIF - 1α564/402 group and Ad - HIF - 1α564/503 group, respectively, with significant differences among them, among which the eTnI expression was the significantly highest in Ad - HIF - 1 cq64/803 group （ P 〈 0. 05 ）. Mean- while, significant differences in expression of HIF -1α and VEGF were revealed among Ad -HIF - 1αnative, Ad - HIF - 1α564, Ad - HIF - 1α564/402 and Ad - HIF - 1%64/803 groups （P 〈 0. 05 ）, among which the significantly highest expression of HIF - 1 α and VEGF were observed in Ad - HIF - 1α 564/402 and Ad - HIF - 1α564/803 groups, respectively （P 〈 0. 05 ）. Simultaneously, compared with the VEGF mRNA expression in control group, it was reduced by （50. 9 ± 4.6） % in curcumin suppression group. However, the VEGF mRNA in Ad - HIF - 1（x564/803 group was still significantly higher than Ad - HIF - 1%64 group （ P 〈 0. 05 ）. Conclusion The Asn803 of HIF - 1α plays a key role in the differentiation of hMSCs to cardiomyocytes.

关 键 词：低氧诱导因子1Α Asn803位点 人骨髓间充质干细胞 心肌细胞 

分 类 号：R[医药卫生]