Smad交互蛋白1在增生性瘢痕组织中的表达及其对纤维化相关因子的影响

Expression of Smad interacting protein 1 in hyperplastic scar tissue and effects on fibrosis-related factors

目的研究Smad交互蛋白1（SIP1）在人增生性瘢痕组织中的表达及其对纤维化相关因子的影响。方法收集临床整形手术切取的9例患者增生性瘢痕标本及其自体正常皮肤标本。分离培养原代人增生性瘢痕成纤维细胞（HSFBs）,取第3-5代细胞用于实验。（1）免疫组织化学法检测增生性瘢痕组织标本及其自体正常皮肤组织标本中SIP1的表达情况。（2）真核表达质粒pcDNA3.1（＋）/SIP1转染HSFBs。按照随机数字表法将细胞分为6组：对照组;pcDNA3.1（＋）组（空载体组）;pcDNA3.1（＋）/SIP1组;对照＋TGF-β1组;pcDNA3.1（＋）＋TGF-β1组;pcDNA3.1（＋）/SIP1＋TGF-β1组。于转染后第48 h和72 h分别提取细胞总RNA和细胞总蛋白。应用实时荧光定量PCR法检测各组细胞α平滑肌肌动蛋白（α-SMA）及结缔组织生长因子（CTGF）的mRNA表达水平;采用Western-blotting检测α-SMA的蛋白表达水平。结果 （1）免疫组织化学染色结果显示：增生性瘢痕组织中SIP1表达明显低于自体正常皮肤组织。（2）pcDNA3.1（＋）/SIP1转染HSFBs后纤维化相关因子的表达：①α-SMA的mRNA和蛋白表达水平：与对照组和pcDNA3.1（＋）组相比,pcDNA3.1（＋）/SIP1组在mRNA和蛋白表达水平均显著下调,差异有统计学意义（P〈0.05）。当细胞给予TGF-β1刺激后,各组α-SMA表达均上调,但pcDNA3.1（＋）/SIP1＋TGF-β1组mRNA和蛋白表达水仍低于对照＋TGF-β1组,差异有统计学意义（P〈0.05）。②CTGF的mRNA表达水平：与对照组和pcDNA3.1（＋）组相比,pcDNA3.1（＋）/SIP1组在mRNA表达水平显著下调,差异有统计学意义（P〈0.05）。当细胞给予TGF-β1刺激后,各组CTGF表达均显著上调,但pcDNA3.1（＋）/SIP1＋TGF-β1组mRNA表达水仍低于对照＋TGF-β1组,差异有统计学意义（P〈0.05）。结论与正常皮肤组织相比较,SIP1在增生性瘢痕组织中低表达。SIP1基因转染HSFBs可降低纤维化相关因子α-SMA和CTGF的表达,有利于抑制瘢痕形成和减轻挛缩。

Objective To study the expression of Smad interacting protein 1（SIP1） in hyperplastic scar tissue and its effects on fibrosis-related factors. Methods Collection of clinical specimens that were remove from 9 patients of hypertrophic scars and autologous normal skin specimens. Primary human hypertrophic scars fibroblasts（HSFBs）were isolated and cultured in vitro. The 3rd to 5th generation cells were used in the experiments.（1）The expression levels of SIP1 in hypertrophic scars and autologous normal skin specimens were detected by immunohistochemistry.（2） pcDNA3. 1（ ＋） / SIP1 plasmid transfection HSFBs. HSFBs were divided into 6 groups according to a random number table cells. ① control group; ②pcDNA3. 1（ ＋） group（ empty vector group）; ③ pcDNA3. 1（ ＋） / SIP1 group;④ control ＋ TGF-β1group; ⑤ pcDNA3. 1（ ＋） ＋ TGF-β1 group; ⑥ pcDNA3. 1（ ＋） / SIP1 ＋ TGF-β1 group. After transfection for 48 h and 72 h,total RNA and proteins were extracted from the cells,respectively. The mRNA expression levels of α-SMA and CTGF were detected by real-time quantitative PCR. The protein expression levels of α-SMA were detected by Western blotting. Results（1）Immunohistochemical staining showed that expression of SIP1 in hypertrophic scar tissue was significantly reduced compared with autologous normal skin tissue.（2） The expression of fibrosis-related factors after pcDNA3. 1（ ＋） / SIP1 transfected into HSFBs. ① mRNA and protein expression levels of α-SMA showed that compared with the control group and pcDNA3. 1（ ＋） group,in pcDNA3. 1（ ＋） /SIP1 group,the α-SMA expression leves both at mRNA and protein were significantly reduced,the differences were statistically significant（ P〈0. 05）. After cells stimulate with TGF-β1,α-SMA expression in each group were raised,but the expression levels both mRNA and protein in pcDNA3. 1（ ＋） / SIP1 ＋ TGF-β1 group remained lower than the control ＋ TGF-β1 group and the differences were statistically significant（P〈0. 05）. ② CTGF mRNA expression levels showed that compared with the control group and pcDNA3. 1（ ＋） group,in pcDNA3. 1（ ＋） /SIP1 group,the mRNA expression levels of CTGF were significantly reduced,the differences were statistically significant（ P〈0. 05）. After cells stimulate with TGF-β1,CTGF expression in each group were raised,but the expression levels of mRNA in pcDNA3. 1（ ＋） / SIP1 ＋ TGF-β1 group remained lower than the control ＋ TGF-β1group and the differences were statistically significant（P〈0. 05）. Conclusions Compared to normal skin tissue,SIP1 expression in hypertrophic scar tissue were decreased. HSFBs transfected SIP1 gene can reduce the expression of fibrosis-related factors（ α-SMA and CTGF） that may be inhibit scars formation and attenuate their contracture.