bFGF反义寡核苷酸对大鼠肺成纤维细胞增殖信号通路的影响

Role of Antisense Oligonucleotides Basic Fibroblast Growth Factor on the Signaling Pathway of TGF-β_1-Induced Pulmonary Fibroblasts Proliferation

目的:观察碱性成纤维细胞生长因子(bFGF)反义寡核苷酸(AODN)对大鼠肺成纤维细胞增殖、转化信号通路的影响。方法:分离成年大鼠肺成纤维细胞,分为4组:对照组(未转染肺成纤维细胞);TGF-β1组(未转染肺成纤维细胞+5ng/ml TGF-β1刺激);TGF-β1+AODN组(转染AODN肺成纤维细胞+5ng/ml TGF-β1刺激);TGF-β1+RODN(正义寡核苷酸)组(转染RODN肺成纤维细胞+5ng/ml TGF-β1刺激)。每组设3复孔,并作细胞爬片,镜下计数肺成纤维细胞数量,绘制生长曲线。MTT比色法测定肺成纤维细胞增殖率。免疫细胞化学方法检测肌成纤维细胞特异性标志物α-平滑肌肌动蛋白(α-SMA)。通过ELISA法检测培养液中bFGF、CTGF及Ⅰ型胶原蛋白含量。用RT-PCR法分析肺成纤维细胞Smad3 mRNA、Smad7 mRNA、Ⅰ型胶原mRNA表达水平。结果:1细胞计数显示:TGF-β1+RODN组各时段成纤维细胞的数目均较对照组、TGF-β1组、TGF-β1+AODN组明显增多(P<0.05);TGF-β1+AODN组中成纤维细胞增殖数比TGF-β1组明显减低(P<0.05)。2MTT结果显示:TGF-β1+RODN组肺成纤维细胞光密度值明显高于对照组、TGF-β1组、TGF-β1+AODN组(P<0.05);TGF-β1+AODN组中肺成纤维细胞光密度值明显比TGF-β1组减低(P<0.05)。3免疫细胞化学染色结果显示:TGF-β1+RODN组肺成纤维细胞与对照组、TGF-β1组、TGF-β1+AODN组比较,α-SMA染色的平均光密度值(IOD)明显增加(P<0.05);TGF-β1+AODN组中肺成纤维细胞α-SMA染色的平均光密度值(IOD)比TGF-β1组明显减低(P<0.05)。4 ELISA法检测结果显示:在TGF-β1+RODN组肺成纤维细胞培养液中bFGF、CTGF及Ⅰ型胶原蛋白含量明显高于对照组、TGF-β1组、TGF-β1+AODN组(P<0.05);TGF-β1+AODN组肺成纤维细胞培养液中bFGF、CTGF及Ⅰ型胶原蛋白含量比TGF-β1组明显减低(P<0.05)。5RT-PCR法检测结果显示:在TGF-β1+RODN组肺成纤维细胞Smad3mRNA及Ⅰ型胶原蛋白mRNA表达明显高于对照组、TGF-β1组、TGF-β1+AODN组(P<0.05);TGF-β1+AODN组肺成纤维细胞Smad3mRNA及Ⅰ型胶原蛋白mRNA表达明显比TGF-β1组明显减低(P<0.05);TGF-β1+RODN组肺成纤维细胞Smad7mRNA表达明显低于对照组、TGF-β1组、TGF-β1+AODN组(P<0.05);TGF-β1+AODN组肺成纤维细胞Smad7 mRNA表达比TGF-β1组明显增加(P<0.05)。结论:bFGF反义寡核苷酸可抑制肺成纤维细胞的增殖、转化及胶原合成,bFGF反义寡核苷酸可能通过下调TGF-β1-Smad信号通路来发挥作用。

Objective：To investigate the role of antisense oligonucleotides basic fibroblast growth factoron the signaling pathway of TGF-β1-induced pulmonary fibroblasts proliferation.Methods：Fibroblasts were separated from adult rat lung and cultured invitro.Cells were divided into four groups：1control group：lung fibroblast cells cultured of normal rat;2 The TGF-β1 group：lung fibroblast cells cultured of normal rat with 5ng/ml TGF-β1;3The TGF-β1＋AODN group：the cells were transfected with AODN and cultured with 5ng/ml TGF-β1;4The TGF-β1＋RODN group：the cells were transfected with RODN and cultured with 5ng/ml TGF-β1.Lung fibroblast cells were counted everyday under microscope to set up cell growth curve.Lung fibroblast cells＇ proliferation was measured with MTT colorimetric method and cell counts.Immunohistochemistry methods was used to detectα-SMA,ELISA was used to determine the concentrations of bFGF,CTGF,and TypeⅠ collagen in culture supernatants.RT-PCR was utilized to detect mRNA expression of Smad3,Smad7 and TypeⅠcollagen in pulmonary fibroblasts.Results：The data suggested that RODN of bFGF increased proliferation rates of pulmonary fibroblasts（P0.05）,enhanced the expression of bFGF,CTGF,α-SMA and type Ⅰ collagen（P0.05）,however,AODN of bFGF had the opposite effects.Moreover,the expression of Smad3 markedly increased（P0.05）and Smad7 significantly decreased（P0.05）in the TGF-β1＋RODN group.On the contrary,the expression of Smad3 markedly decreased（P0.05）and Smad7 significantly increased（P0.05）in the TGF-β1＋AODN group.Conclusion：Antisense oligonucleotides basic fibroblast growth factor inhibits the proliferation,differentiation and collagen synthesis of pulmonary fibroblasts.The possible mechanism is related to regulation of TGF-β1-Smad signaling pathway.