骨髓间充质干细胞抑制转化生长因子-&beta;1诱导的肺泡Ⅱ型上皮细胞间质化

王鑫 高俊玲* 赵曼曼 朱会兴 田艳霞 李冉 江小华 于雷 田景瑞 崔建忠

doi:10.16098/j.issn.0529-1356.2016.06.006

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解剖学报 ›› 2016, Vol. 47 ›› Issue (6) : 756-762. DOI: 10.16098/j.issn.0529-1356.2016.06.006

细胞和分子生物学

骨髓间充质干细胞抑制转化生长因子-β1诱导的肺泡Ⅱ型上皮细胞间质化

王鑫¹ 高俊玲^1,2,3* 赵曼曼¹ 朱会兴¹田艳霞¹ 李冉¹江小华¹ 于雷¹ 田景瑞¹ 崔建忠⁴

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Inhibition of bone marrow mesenchymal stem cells on epithelial mesenchymal transformation induced by transforming growth factor-β1

WANG Xin¹GAO Jun-ling ^1,2,3* ZHAO Man-man¹ ZHU Hui-xing¹TIAN Yan-xia¹ LI Ran¹JIANG Xiao-hua¹ YU Lei¹ TIAN Jing-rui¹CUI Jian-zhong⁴

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摘要

目的探讨骨髓间充质干细胞（BMSCs）是否通过抑制肺泡Ⅱ型上皮细胞向肌成纤维细胞的转化，进而发挥其抗纤维化的作用及其机制。方法培养大鼠肺泡Ⅱ型上皮细胞系RLE-6TN细胞，用5μg/L的TGF-β1诱导分化，并分为对照组、转化生长因子（TGF）-β1刺激组、BMSCs干预组。采用倒置相差显微镜观察RLE-6TN细胞的形态变化；免疫荧光法检测E-钙黏蛋白（E-cad）、α-平滑肌肌动蛋白（α-SMA）的定位；Western blotting法检测E-cad、α-SMA、p-Smad3、Snail1蛋白的表达。结果在TGF-β1的诱导下，肺泡Ⅱ型上皮细胞逐渐向肌成纤维细胞发生形态改变，由上皮细胞的鹅卵石状变成间充质细胞的梭形或纺锤形，且伴随着上皮标志物E-cad表达降低，而间充质细胞标志物α-SMA表达上调；给予BMSCs干预后，间充质化有所抑制，表现于细胞形态趋于上皮型，且上皮标志物E-cad表达上调，间质标志物α-SMA表达减少。与对照组相比，TGF-β1刺激组p-Smad3、 Snail1蛋白表达上调。给予BMSCs干预后p-Smad3、Snail1蛋白表达显著降低。结论 BMSCs可能通过阻断TGF-β1/Smad3信号转导通路，抑制肺泡Ⅱ型上皮细胞向肌成纤维细胞的转化，从而抑制上皮细胞-间充质转化的进程。

Abstract

Objective To explore whether bone marrow mesenchymal stem cells could inhibit epithelial-mesenchymal transition(EMT) of rat type Ⅱalveolar epithelial cells through inhibiting Smad3 phosphorylation, and play a role on anti pulmonary fibrosis. Methods Rat type Ⅱ alveolar epithelial cell line RLE-6TN cells were cultivated, induced by 5μg/L transforming growth factor(TGF)-β1 in vitro, and divided into normal control group. TGF-β1 induced group and BMSCs intervention group. Morphological changes of epithelial-mesenchymal transition in RLE-6TN cells were observed under an inverted phase contrast microscope. Immunofluorescence methods were used to determine the location of E-cadherin (E-cad) and α-smooth muscle actin（α-SMA). The expressions of E-cad, α-SMA, p-Smad3 and Snail1 were detected by Western blotting in each group. Results The process of EMT induced by TGF-β1 was accompanied by morphological alteration. RLE-6TN cells had an epithelial cobblestone-like morphology, a round or polygonal shape and exhibited very close cell-cell proximity, while after exposure to TGF-β1, the cells displayed spindle-shaped elongated fibroblast-like cells morphology. Concomitant with a down-regulation of the epithelial markers E-cad, the expression of interstitial cells markers α-SMA up-regulated. Similar morphological changes were noticeably suppressed in the BMSCs intervention group, and the BMSCs intervention group showed a reduction in TGF-β1-induced up-regulation of α-SMA, while a down-regulation of E-cad was observed. Compared with control group, the expression levels of p-Smad3 and Snail1 increased significantly after the stimulation of TGF-β1. After BMSCs intervention,the expressions of p-Smad3 and Snail1 significantly decrease compared to the TGF-β1 group. Conclusion BMSCs may block TGF-β1/Smad3 signal transduction pathway, and then inhibit epithelial mesenchymal transition of type Ⅱ alveolar epithelial cells.

导出引用

王鑫高俊玲赵曼曼朱会兴田艳霞李冉江小华于雷田景瑞崔建忠. 骨髓间充质干细胞抑制转化生长因子-β1诱导的肺泡Ⅱ型上皮细胞间质化[J]. 解剖学报. 2016, 47(6): 756-762 https://doi.org/10.16098/j.issn.0529-1356.2016.06.006

WANG Xin GAO Jun-ling ZHAO Man-man ZHU Hui-xing TIAN Yan-xia LI Ran JIANG Xiao-hua YU Lei TIAN Jing-rui CUI Jian-zhong. Inhibition of bone marrow mesenchymal stem cells on epithelial mesenchymal transformation induced by transforming growth factor-β1[J]. Acta Anatomica Sinica. 2016, 47(6): 756-762 https://doi.org/10.16098/j.issn.0529-1356.2016.06.006

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