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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 observe the effects of basic fibroblast growth factor (bFGF) antisense oligonucleotide (AODN) on the proliferation and transformation of rat lung fibroblasts. METHODS: Adult rat lung fibroblasts were isolated and divided into 4 groups: control group (untransfected lung fibroblasts); TGF-β1 group (untreated lung fibroblasts + 5ng / ml TGF-β1 stimulation); TGF-β1 + AODN group (AODN lung fibroblasts transfected with 5ng / ml TGF-β1); TGF-β1 + RODN -β1 stimulation). Each set of 3 complex holes, and for cell slide, counting the number of lung fibroblasts, draw the growth curve. MTT colorimetric determination of lung fibroblast proliferation rate. Immunocytochemistry was used to detect myofibroblast-specific alpha-smooth muscle actin (α-SMA). The contents of bFGF, CTGF and type Ⅰ collagen in culture medium were detected by ELISA. The expression of Smad3 mRNA, Smad7 mRNA and type Ⅰ collagen mRNA in lung fibroblasts were analyzed by RT-PCR. The number of fibroblasts in TGF-β1 + RODN group was significantly higher than that in control group, TGF-β1 group and TGF-β1 + AODN group (P <0.05) The number of fibroblast proliferation was significantly lower than that of TGF-β1 group (P <0.05). The results of 2MTT showed that the optical density of lung fibroblasts in TGF-β1 + RODN group was significantly higher than that in control group, TGF-β1 group and TGF-β1 + AODN group (P <0.05) Cell optical density was significantly lower than TGF-β1 group (P <0.05). Immunocytochemical staining showed that the average optical density (IOD) of a-SMA staining was significantly increased in TGF-β1 + RODN group compared with control group, TGF-β1 group and TGF-β1 + AODN group P <0.05). The mean optical density (IOD) of a-SMA staining of TGF-β1 + AODN group was significantly lower than that of TGF-β1 group (P <0.05). The results of ELISA showed that the content of bFGF, CTGF and type Ⅰ collagen in TGF-β1 + RODN group was significantly higher than that in control group, TGF-β1 group and TGF-β1 + AODN group (P < 0.05). The levels of bFGF, CTGF and collagen Ⅰ in TGF-β1 + AODN group were significantly lower than those in TGF-β1 group (P <0.05). The results of 5RT-PCR showed that the mRNA expression of Smad3 and type Ⅰ collagen in lung fibroblasts of TGF-β1 + RODN group was significantly higher than that in control group, TGF-β1 group and TGF-β1 + AODN group (P <0.05). The expression of Smad3 mRNA and type Ⅰ collagen mRNA in lung fibroblasts of TGF-β1 + AODN group was significantly lower than that of TGF-β1 group (P <0.05); the expression of Smad7 mRNA in lung fibroblasts of TGF-β1 + RODN group was significantly lower than that of the control group TGF-β1 group and TGF-β1 + AODN group (P <0.05). The expression of Smad7 mRNA in lung fibroblasts of TGF-β1 + AODN group was significantly higher than that of TGF-β1 group (P <0.05). CONCLUSION: bFGF antisense oligonucleotide inhibits the proliferation, transformation and collagen synthesis of lung fibroblasts. The bFGF antisense oligonucleotide may play a role by down-regulating TGF-β1-Smad signaling pathway.