BMSC共培养对人瘢痕疙瘩成纤维细胞DNMT1的影响

The Effects of Bone Marrow Derived Mesenchymal Stem Cells on the Expression of DNA Methyltransferase 1 in Keloid Fibroblasts

该实验探究人骨髓间充质干细胞(bone marrow derived mesenchymal stem cells, BMSC)能否通过旁分泌作用影响人瘢痕疙瘩成纤维细胞(keloid fibroblast, KF)中DNA甲基转移酶1(DNA methyltransferase 1, DNMT1)的表达,从而影响TGF-β/Smad信号通路。培养BMSC、KF及正常人皮肤成纤维细胞(normal human skin fibroblast, NHDF),使用0.4μm微孔孔径的悬挂式细胞培养皿构建BMSC与成纤维细胞之间非接触的细胞间接共培养模型。通过免疫荧光、激光共聚焦技术、Western blot和qPCR检测各组细胞内DNMT1的表达情况。Western blot和qPCR检测TGF-β/Smad信号通路相关分子TGF-β1、Smad7的表达。在成功构建共培养模型后,利用细胞免疫荧光和激光共聚焦技术, Western blot及qPCR检测各组细胞内DNMT1的表达情况,结果显示, DNMT1在KF中高表达而在NHDF中呈低表达,共培养组KF较非共培养组DNMT1的蛋白和m RNA水平表达明显降低(P<0.01)。利用Western blot, qPCR检测TGF-β/Smad信号通路相关分子的表达,结果显示较非共培养组,共培养组KF中TGF-β1在蛋白水平表达显著降低(P<0.01),抑制性蛋白Smad7在m RNA水平表达增高(P<0.05)。研究证实,人BMSC可能通过旁分泌作用抑制了瘢痕疙瘩成纤维细胞中DNMT1的表达,从而抑制TGF-β/Smad信号通路,使瘢痕疙瘩成纤维细胞的增殖趋于正常。

This article investigated the effects of bone marrow derived mesenchymal stem cells(BMSC) on the expression of DNA methyltransferase 1(DNMT1) and related factor expression changes of TGF-β/Smad signal transduction pathways in keloid fibroblasts(KF). BMSC and fibroblasts were cultured, the indirect coculture system using Transwell chamber with BMSC was established. The expression changes of DNMT1 were detected by immunofluorescence, Western blot and qPCR. Western blot assay was used to detect the protein expression of TGF-β1. qPCR was used to detect the expression of Smad7. Immunohistochemistry experiments showed that DNMT1 showed high expression in KF. Western blot and qPCR results showed that the expression of DNMT1 in co-cultured KF group was significantly reduced than that in the control group(P<0.01). The protein expression of TGF-β1 in co-cultured KF group was significantly reduced than that in the control group(P<0.01).Smad7 mRNA expression of co-culture KF group was significantly increased than the KF group(P<0.05). BMSC microenvironment could inhibit the expression of DNMT1 in co-cultured KF, thus inhibiting the KF by regulating TGF-β/Smad signal transduction pathway.