结缔组织生长因子、转化生长因子β1真核表达载体的构建及对硬皮病的FB凋亡的影响

摘要

Objective To build the eukaryotic expression mode of connective tissue growth factor (CTGF) and transforming growth factor-β1 (TGF-β1), and to observe the apoptosis of CTFF, TGF-β1 gene which was transfected into the fibroblasts (FB) of sclerodermatous patient and normal skin. Methods The cells were grouped into normal, sclerosis, CTGF and TGF-β1 groups. LipofectamineTM 200CTGF and TGF-β1DNA plasmid were transfected into the FB that were cultivated by generations. The expression of green fluorescin was observed under the fluorescence microscope. The apoptosis of CTFF, TGF-β1 gene, then transfection ratio were computed, the apoptosis of FB in every groups were detected by flow cytometer. Results The eukaryotic expression vector was successfully built. Stable green fluorescent gene of CTGF, TGF-β1 which had been transfected was obtained. The transfection ratio of CTGF and TGF-β1 group was (85.7±2.1)%,(66.9±3.3)% respectively. The result showed that the apoptosis ratio in normal group was higher than those in other groups. However, the apoptosis ratio was descended in the sclerosis group and transfection group. Among them, CTGF group was the lowest (P<0.05). Conclusion The gene of CTGF and TGF-β1 could be successfully transfected into the FB cells by LipofectamineTM 2000. And the cell apoptosis could be inhibited by CTGF and TGF-β1.%目的 构建结缔组织生长因子(connective tissue growth factor,CTGF)、转化生长因子β1 (transforming growth factor-β1,TGF-β1)真核表达载体,并观察CTGF、TGF-β1基因转染硬皮病患者皮损培养的成纤维细胞凋亡的影响.方法 采用阳离子脂质体LipofectamineTM 2000 CTGF、TGF-β1DNA质粒转染至传代培养的硬皮病FB中,用倒置荧光显微镜观察转染后绿色荧光蛋白基因的表达,计算转染率,用流式细胞术检测CTGF、TGF-β1DNA质粒分组转染的各组成纤维细胞的凋亡情况.结果 实验成功构建了CTGF、TGF-β1真核表达载体,荧光显微镜下观察到转染后的CTGF、TGF-β1基因发出的稳定的绿色荧光的信号,转染率为(85.7±2.1)％、(66.9±3.3)％,与正常组比较差异有统计学意义(P＜0.05).流式细胞仪法显示正常组细胞凋亡较高(6.52％),而硬皮病和各转染组细胞凋亡率降低,尤以CTGF转染硬皮病组最低(P＜0.05).结论 阳离子脂质体LipofectamineTM 2000可有效介导真核表达质粒转染CTGF、TGF-β1,且CTGF、TGF-β1能抑制成纤维细胞的凋亡及促进细胞增殖的作用.