Effects of blocking two sites of transforming growth factor-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts
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摘要: 目的 探讨阻断TGF-β/Smads通路的双位点对人皮肤Fb生成瘢痕相关蛋白的影响。 方法 将携带可溶性TGF-β受体Ⅱ(sTβRⅡ)、突变型Smad 4––Smad 4ΔM4基因的2种慢病毒载体按最适感染复数(MOI)50分别转染人皮肤Fb细胞株人包皮Fb 1(HFF-1)细胞,蛋白质印迹法测定2种转染细胞的sTβRⅡ、Smad 4ΔM4蛋白表达情况,并与未转染细胞进行对比。采用随机数字表法将HFF-1细胞分为6组,每组6皿,每皿1×104个。共转染组,将2种病毒按MOI=50、1∶1混合共转染细胞后用5 ng/mL TGF-β1刺激72 h;sTβRⅡ组,慢病毒sTβRⅡ取MOI=50转染细胞,余处理同前;Smad 4ΔM4组,慢病毒Smad 4ΔM4取MOI=50转染细胞,余处理同前;阴性病毒对照组,空载体病毒转染细胞,余处理同前;阳性对照组,仅用5 ng/mL TGF-β1刺激72 h;空白对照组,常规培养细胞,不作任何其他处理。刺激结束后,采用蛋白质印迹法、实时荧光定量RT-PCR法分别检测各组细胞纤维连接蛋白的蛋白和mRNA表达,分别采用ELISA法和Sircol法检测各组细胞培养上清液中结缔组织生长因子(CTGF)及总胶原蛋白表达量。对数据行单因素方差分析及SNK-
q 检验。 结果 (1)慢病毒sTβRⅡ转染的HFF-1细胞sTβRⅡ蛋白表达明显高于未转染的HFF-1细胞,慢病毒Smad 4ΔM4转染的HFF-1细胞Smad 4ΔM4蛋白表达亦明显高于未转染的HFF-1细胞。经验证,2种慢病毒转染的细胞均能有效表达相应的目的蛋白。(2)6组细胞纤维连接蛋白的蛋白及mRNA表达量总体均差异明显(F 值分别为53.536和24.365,P 值均小于0.001)。阳性对照组细胞纤维连接蛋白的蛋白与mRNA表达量分别为1.60±0.18、1.99±0.40,与阴性病毒对照组的1.60±0.15、1.94±0.28相近(q 值分别为0.091和0.419,P 值均大于0.05),但高于其余4组(q 值为5.245~18.228,P 值均小于0.05);共转染组细胞纤维连接蛋白的蛋白与mRNA表达量分别为0.60±0.05、0.70±0.11,明显低于sTβRⅡ组的0.89±0.13、1.24±0.17和Smad 4ΔM4组的0.91±0.14、1.28±0.19(q 值为3.964~4.294,P 值均小于0.05)。(3)6组细胞培养上清液中CTGF及总胶原蛋白表达量总体均差异明显(F 值分别为107.680和38.347,P 值均小于0.001)。阳性对照组细胞培养上清液中CTGF及总胶原蛋白表达量与阴性病毒对照组相近(q 值分别为1.106和0.491,P 值均大于0.05),但明显高于其余4组(q 值为6.414~26.420,P 值均小于0.05);共转染组细胞培养上清液中CTGF及总胶原蛋白表达量均明显低于sTβRⅡ组和Smad 4ΔM4组(q 值为3.424~7.143,P 值均小于0.05)。 结论 在皮肤Fb中,阻断TGF-β/Smads通路双位点在抑制TGF-β1导致的瘢痕相关蛋白上调方面优于阻断单个位点。Abstract: Objective To explore the effects of blocking two sites of TGF-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts. Methods Two lentivirus vectors encoding soluble TGF-β receptor Ⅱ (sTβRⅡ) and mutant Smad 4–Smad 4ΔM4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50. The protein expressions of sTβRⅡ and Smad 4ΔM4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells. The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1×104 cells per dish. Co-transfection group, transfected with the two previous lentivirus vectors, mixed with the ratio of 1∶1 and MOI of 50, and then stimulated with 5 ng/mL TGF-β1 for 72 h; sTβRⅡ group, transfected with lenti-sTβRⅡ with MOI of 50, with the other treatment as above; Smad 4ΔM4 group, transfected with lenti-Smad 4ΔM4 with MOI of 50, with the other treatment as above; negative virus group, transfected with empty lentivirus vector, with the other treatment as above; positive control group, stimulated with 5 ng/mL TGF-β1 for 72 h; and blank control group, conventionally cultured without any other treatment. After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group. ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group. Data were processed with one-way analysis of variance and SNK-q test. Results (1) HFF-1 cells transfected with lenti-sTβRⅡ and HFF-1 cells transfected with lenti-Smad 4ΔM4 respectively expressed higher levels of sTβRⅡ protein and Smad 4ΔM4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins. (2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (withF values respectively 53.536 and 24.365,P values below 0.001). The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60±0.18 and 1.99±0.40) were similar with those of negative virus group (respectively 1.60±0.15 and 1.94±0.28, withq values respectively 0.091 and 0.419,P values above 0.05), and they were significantly higher than those of the rest 4 groups (withq values from 5.245 to 18.228,P values below 0.05). The protein and mRNA expressions of fibronectin in cells of co-transfection group (respectively 0.60±0.05 and 0.70±0.11) were significantly lower than those of sTβRⅡ group (respectively 0.89±0.13 and 1.24±0.17) and Smad 4ΔM4 group (respectively 0.91±0.14 and 1.28±0.19, withq values from 3.964 to 4.294,P values below 0.05). (3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (withF values respectively 107.680 and 38.347,P values below 0.001). The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (withq values respectively 1.106 and 0.491,P values above 0.05), and they were significantly higher than those of the rest 4 groups (withq values from 6.414 to 26.420,P values below 0.05). The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sTβRⅡ group and Smad 4ΔM4 group (withq values from 3.424 to 7.143,P values below 0.05). Conclusions In human skin fibroblasts, blockage of two sites of TGF-β/Smads signaling can reduce the expression of scar related proteins which are up-regulated by TGF-β1 to a greater extent than that of blocking one single site.
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