应用成纤维细胞胶原网格体外三维培养模型观察重组核心蛋白多糖固相态时对转化生长因子β1刺激正常皮肤成纤维细胞的应答

Observation on the effect of solid-state recombinant human decorin on transforming growth factor beta 1 stimulating fibroblast of normal skin by using three-dimensional model of fibroblast populated collagen lattices cultured in vitro

目的:模拟体内核心蛋白多糖和转化生长因子β1接触方式,研究核心蛋白多糖在固相抑制转化生长因子β1刺激正常皮肤成纤维细胞的作用。方法:实验于2005-01/09在广州市创伤研究所完成。①实验成纤维细胞的组织来源:取材于广州市红十字会医院,患者自愿,正常皮肤为包皮。取新鲜包皮,修除表皮和皮下组织,加入含体积分数0.02的胎牛血清的DMEM培养,实验用第4～8代细胞。②采用醋酸法从鼠尾腱中提取I型胶原,使用浓度为2g/L,网格胶原终浓度为1g/L,成纤维细胞密度为1×109L-1。将胶原细胞混悬液按2mL/皿均匀加入直径35mm的培养皿内,37℃培养10min,混悬液便形成凝胶即成纤维细胞胶原网格,再加入相应的培养液。分为4组:对照组、核心蛋白多糖组、转化生长因子β1组、转化生长因子β1+核心蛋白多糖组。③观察成纤维细胞胶原网格的收缩,Westernblot法检测成纤维细胞胶原网格中成纤维细胞纤溶酶原激活物抑制物-1、α-平滑肌肌动蛋白的表达,反转录-聚合酶链反应检测其mRNA表达。结果:①成纤维细胞胶原网格收缩的变化:对照组成纤维细胞胶原网格培养12h,收缩到起始面积的(81.2±4.9)%,12～24h收缩加剧,在24h收缩到起始面积的(47.4±4.7)%,以后收缩减慢,在48h收缩到起始面积的(37.3±3.6)%,72h收缩到起始面积的(29.6±3.6)%,96h收缩到起始面积的(28.7±2.8)%,以后不再收缩;核心蛋白多糖组成纤维细胞胶原网格收缩到起始面积的百分比在12,24,48,72,96h均高于对照组(P<0.05);转化生长因子β1组成纤维细胞胶原网格收缩到起始面积的百分比在各时相点均低于对照组(P<0.01);转化生长因子β1+核心蛋白多糖组成纤维细胞胶原网格收缩到起始面积的百分比在各时相点均高于转化生长因子β1组(P<0.05),与对照组比较无显著差异(P>0.05)。②成纤维细胞胶原网格中细胞表达纤溶酶原激活物抑制物-1、α-平滑肌肌动蛋白的变化:核心蛋白多糖组纤溶酶原激活物抑制物-1、α-平滑肌肌动蛋白蛋白表达(1391.61±126.27,525.97±60.10),与对照组(1474.52±133.84,569.41±62.55)比较无显著差异(P>0.05),转化生长因子β1组纤溶酶原激活物抑制物-1、α-平滑肌肌动蛋白蛋白表达(2909.77±264.04,2725.46±150.54)显著高于对照组(P<0.01),转化生长因子β1+核心蛋白多糖组纤溶酶原激活物抑制物-1、α-平滑肌肌动蛋白蛋白表达(1775.25±161.09,926.34±51.16)显著低于转化生长因子β1组(P<0.05),且与对照组比较无显著差异(P>0.05)。③成纤维细胞胶原网格中细胞表达纤溶酶原激活物抑制物-1、α-平滑肌肌动蛋白mRNA的变化:核心蛋白多糖组纤溶酶原激活物抑制物-1、α-平滑肌肌动蛋白mRNA表达(0.19±0.05,0.07±0.02),与对照组(0.21±0.06,0.08±0.03)比较无显著差异(P>0.05),转化生长因子β1组纤溶酶原激活物抑制物-1、α-平滑肌肌动蛋白mRNA表达(0.86±0.15,0.36±0.10)显著高于对照组(P<0.01),转化生长因子β1+核心蛋白多糖组纤溶酶原激活物抑制物-1、α-平滑肌肌动蛋白mRNA表达(0.34±0.09,0.13±0.04)显著低于转化生长因子β1组(P<0.05),且与对照组比较无显著差异(P>0.05)。结论:重组核心蛋白多糖融入胶原凝胶,能显著抑制转化生长因子β1刺激正常皮肤成纤维细胞的作用,表明在体内核心蛋白多糖具有拮抗转化生长因子β1的作用。

AIM： To mimic the contact pattern between decorin and TGF-β1 in vivo, and investigate the antagonistic effect of recombinant human decorin on transforming growth factor beta 1 （TGF-β1） stimulating normal skin fibreblasts. METHODS： This study was conducted in Guangzhou Institute of Trauma from January to September 2005. ①Source of fibreblast tissue： Fibreblast was established as a primary cell line from normal foreskin tissue donated voluntarily by the patients who received the treatment in the Guangzhou Red Cress Hospital. Fresh foreskin was chosen, and epidermal and subcutaneous tissue were removed. Then, DMEM containing 0.02 volume fraction of fetal bovine serum was added and the cells of the 4^th to 8^th generations were used for the experiment. ②Type I collagen was extracted from the tendon of the rats tail by using acetic acid method with concentration of 2 g/L The final concentration of collagen lattices was 1 g/L. The cell density of fibreblast was 1 ×10^9 L^-1. Suspension of collagen cells was added into the Petri dish with the diameter of 35 mm at 2 mL/Petri dish, and cultured at 37 ℃ for 10 minutes. The suspension gelated , which was fibreblast collagen lattices , then corresponding culture medium was added. They were divided into 4 groups： control group, deeorin group, TGF-β1 group, TGF-β1 ＋decorin group. ③ Contraction of fibroblast collagen lattice was observed. Expression of plasminogen activated inhibitor-1 （PAI-1） and α-smoothmuscleactin （α-SMA） in fibreblasts populated collagen lattices （FPCL） were detected by Western blot, and expressions of PAI-1 and α-SMA mRNA were examined by RT-PCR. RESULTS： ①Change of contraction of FPCL： Contraction of FPCL in the control group reached （81.2±4.9）% of the initial area after 12-bout culture. Contraction accelerated especially between 12 and 24 hours, reached just （47,4 ±4.7）% of the initial area by 24 hours. It then slowed down. It reached （37.3±3.6）% of the initial area by 48 hours, （29.6±3.6）% by 72 hours and （28,7±2.8）% by 96 hours, then no further contraction occurred. The percent of contraction of FPCL reaching to the initial area was higher at hours 12, 24, 48, 72 and 96 in the decorin group than in the control group （P 〈 0.05）; The percent of contraction of FPCL reaching to the initial area was lower at each time points in the TGF-β1 group than in the control group （P 〈 0.01）; The percent of contraction of FPCL reaching to the initial area was higher at each time point in the TGF-β1 ＋ decorin group than in the TGF-β1 group （P 〈 0.05）, without significant difference as compared with control group （P 〉 0.05）. ②Change of PAI-1 and α-SMA expressed in the FPCL： There was no significant difference of the expn±sion of PAI-1 and α-SMA between decorin group （1 391.61±126.27,525.97±60.10） andcontrol group （1 474.52±133.84,569.41±62.55）（P 〉 0.05）. The expression of PAI-1 and α-SMA was significantly higher in the TGF-β1 group （2 909.77±264.04,2 725.46±150.54）than in the control group, （P 〈 0.01 ）, and the expression of PAI-1 and α-SMA was significantly lower in the TGF-β1＋ decorin group （1775.25±161.09,926.34±51.16）than in the TGF- β1 group （P 〈 0.05）, without significant difference as compared with control group （P 〉 0.05）. ±） Change of PAI-1 mRNA and α-SMA mRNA expressed in the FPCL： there was no significant difference of the expression of PAI-1 and α-SMA mRNA in the decorin group （0.19±0.05, 0.07±0.02）than in the control group （0.21±0.06,0.08±0.03）（P 〉 0.05）. The expression of PAI-1 and α-SMA mRNA was significantly higher in the TGF-β1 group （0.86±0.15,0.36±0.10） than in the control group （P 〈 0.01 ）. The expression of PAl-1 and α-SMA mRNA was significantly lower in the TGF-β1＋ decorin group than in the TGF-β1 group （P 〈0.05）,without significant difference as compared with control group （P 〉 0.05）. CONCLUSION： When recombinant human decorin is blended in collagen lattices, the contraction of FI±L and expressions of PAI-1 and α-SMA in fibroblasts stimulated by TGF-β1 are suppressed, indicating that decorin may be effective in neutralizing TGF-β1 in vivo.