TGF-β_1在低氧诱导肺动脉内皮细胞向平滑肌样细胞转分化中的作用

The Role of Transforming Growth Factor β_1 in Hypoxia Inducing Transdifferentiation of Pulmonary Artery Endothelial Cells to Smooth Muscle-like Cells

目的研究低氧条件下肺动脉内皮细胞(PAEC)向平滑肌样细胞的转分化及转化生长因子β1(TGF-β1)在此转分化过程中的作用。方法将经免疫磁珠法(用血小板内皮细胞粘附分子1做免疫分选标记)纯化后的原代肺动脉内皮细胞分别在常氧(含21%O2、5%CO2、74%N2)和低氧(含1%O2、5%CO2、94%N2)条件下培养1、4、7 d,用核酸干扰法(RNAi)阻断TGF-β1的表达,用逆转录-聚合酶链式反应法(RT-PCR)和Western blot分别检测各组细胞TGF-β1mRNA和蛋白的表达,并用免疫细胞化学法检测平滑肌细胞标志蛋白α-平滑肌肌动蛋白(α-SM-actin)的表达,结合形态学观察从而判定有无平滑肌样细胞的转分化。结果低氧可增加肺动脉内皮细胞TGF-β1mRNA和蛋白的表达(P<0.05),RNAi沉默TGF-β1基因表达后可明显降低其mRNA和蛋白含量(P<0.05),随着低氧培养时间的延长,肺动脉内皮细胞转分化为平滑肌样细胞的比率逐渐增加(P<0.05);阻断TGFβ-1表达后,平滑肌样细胞的转化率明显降低(P<0.05)。结论肺动脉内皮中存在具有转分化为平滑肌样细胞潜能的细胞;低氧可明显促进转分化,TGF-β1在此转分化过程中起重要作用。

Objective To investigate the transdifferentiation of pulmonary artery endothelium cells to smooth muscle cells under hypoxia and the role of transforming growth factor β1 （TGF-β1 in this process. Methods Endothelial cells from pulmonary artery of pigs were purified by immunomagnetic （Dynabeads） purification techniques （based on PECAM-1 positive expression for sorting marker）. Cells were divided into normoxia group （cell culture box containing 21 % O2, 5 % CO2 and 74 % N2 ） and hypoxia group （cell culture box containing 1 % O2, 5 % CO2 and 94 % N2 ）. Two groups were cultured for one, 4 and 7 days respectively. RNA interference （RNAi） was used to block the expression of TGF-β1 , reverse transcription-polymerase chain reaction （RT-PCR） and Western blot to detect the expression of TGF-β1 mRNA and protein, then using the immunocytochemistry to detect the expression of smooth muscle cells specific marker α-SM-actin together with morphological observation to determine whether the endothelial cells transdifferentiated to the smooth muscle-like cells or not. Results The expression level of TGF-β1 mRNA and protein was higher in hypoxia groups than that in nomoxia groups （P〈0.05）. RNAi silencing the expression of TGF-β1 gene could markedly attenuate the expression of TGF-β1 mRNA and protein （P〈0. 05）. With the prolongation of hypoxic time, the ratio of transdifferentiated smooth muscle-like cells to pulmonary arterial endothelial cells was gradually increased （P〈0.05） , blocking the expression of TGF-β1 gene, however, could significantly decrease the ratio of transdifferentiation （P〈0. 05）. Conclusion Pulmonary arterial endothelial cells colony contains those cells which possess the transdifferentiation potential to smooth muscle-like cells. Hypoxia greatly attributes to the transdifferentiation, and TGF-β1 plays an important role in this process.