Effects of transforming growth factor β 1 receptor inhibitor SD-208 on human hypertrophic scar
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摘要: 目的 探讨TGF-β1受体抑制剂SD-208对人增生性瘢痕的作用及其机制。 方法 取废弃人增生性瘢痕组织,分离瘢痕Fb,并进行传代培养,采用第5代细胞进行以下实验。(1)按照随机数字表法(分组方法下同)将细胞分为空白对照组及0.5、1.0、3.0、5.0 μmol/L SD-208组,每组6孔。空白对照组加入1 μL PBS,后4组分别加入1 μL 0.5、1.0、3.0、5.0 μmol/L SD-208,培养12 h,细胞计数试剂盒8及酶标仪检测细胞增殖活性(以吸光度值表示)。根据细胞增殖活性结果选取适宜物质的量浓度的SD-208用于后续实验。(2)另取细胞,分为空白对照组及1、3 μmol/L SD-208组,同(1)处理,每组8孔,Transwell法检测迁移细胞数。(3)另取细胞,同(2)分组处理,罗丹明-鬼笔环肽染色,观察细胞微丝形态。(4)另取细胞,同(2)分组处理,蛋白质印迹法检测TGF-β1蛋白表达。(5)取48只BALB/c裸鼠,分为生理盐水组24只与1 μmol/L SD-208组24只,于背部做长度为1 cm的纵行切口,埋置人增生性瘢痕组织块。伤后7 d,生理盐水组与1 μmol/L SD-208组裸鼠瘢痕内分别多点注射0.05 mL生理盐水及1 μmol/L SD-208,每日注射1次。于首次给药后0(当日)、2、4、6、8、10、12、14、16、18、20 d,取8只裸鼠,电子秤称量体质量;用游标卡尺测量瘢痕面积,计算剩余瘢痕面积百分比。于首次给药后1、2、3周,取出人增生性瘢痕组织块,免疫组织化学染色法检测TGF-β1蛋白表达。对数据行单因素方差分析、两独立样本
t 检验。 结果 (1)空白对照组及0.5、1.0、3.0、5.0 μmol/L SD-208组细胞增殖活性分别为1.00±0.03、0.90±0.08、0.68±0.11、0.54±0.04、0.42±0.09,0.5、1.0、3.0、5.0 μmol/L SD-208组细胞增殖活性均明显低于空白对照组(t 值为2.9~22.1,P <0.05或P <0.01)。(2)1、3 μmol/L SD-208组迁移细胞数均明显少于空白对照组(t 值分别为6.5、6.4,P 值均小于0.01)。(3)与空白对照组比较,1、3 μmol/L SD-208组细胞的微丝荧光强度减弱,伪足伸出减少。(4)空白对照组及1、3 μmol/L SD-208组细胞TGF-β1蛋白表达量分别为1.00±0.08、0.80±0.08、0.61±0.05,1、3 μmol/L SD-208组细胞TGF-β1蛋白表达量均明显低于空白对照组(t 值分别为4.0、9.2,P 值均小于0.01)。(5)生理盐水组、1 μmol/L SD-208组裸鼠各时相点体质量相近(t 值为0.2~1.1,P 值均大于0.05)。2组裸鼠剩余瘢痕面积百分比随给药时间延长降低,首次给药后6~20 d,1 μmol/L SD-208组裸鼠剩余瘢痕面积百分比明显小于生理盐水组(t 值为1.8~15.9,P <0.05或P <0.01)。首次给药后1、2、3周,2组裸鼠体内人增生性瘢痕TGF-β1蛋白表达量均呈下降趋势,且1 μmol/L SD-208组裸鼠体内人增生性瘢痕TGF-β1蛋白表达量明显低于生理盐水组(t 值为6.2~19.1,P 值均小于0.01)。 结论 SD-208对人增生性瘢痕有明显的抑制作用,其作用机制与抑制内源性TGF-β1蛋白表达有关。Abstract: Objective To investigate the effects of transforming growth factor β1 (TGF-β1) receptor inhibitor SD-208 on human hypertrophic scar and its mechanisms. Methods Scar fibroblasts were isolated from deprecated human hypertrophic scar tissue and then sub-cultured. Cells of the fifth passage were used in the following experiments. (1) Cells were divided into blank control group (BC) and 0.5, 1.0, 3.0, and 5.0 μmol/L SD-208 groups according to the random number table (the same grouping method below), with 6 wells in each group. Cells in group BC were added with 1 μL phosphate buffer solution, while cells in the latter four groups were added with 0.5, 1.0, 3.0, and 5.0 μmol/L SD-208, respectively. After being cultured for 12 hours, the proliferation activity of cells was detected by cell counting kit 8 and microplate reader (denoted as absorbance value). Suitable amount of substance concentration of SD-208 according to the results of proliferation activity of cells was chosen for the following experiments. (2) Another batch of cells were divided into group BC and 1, 3 μmol/L SD-208 groups and treated as in (1), with 8 wells in each group. The number of migration cells was detected by transwell method. (3) Another batch of cells were grouped and treated as in (2), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of cells were grouped and treated as in (2), and the protein expression of TGF-β1 was assessed with Western blotting. (5) Forty-eight BALB/c nude mice were divided into normal saline group (NS) and 1 μmol/L SD-208 group, and one longitudinal incision with length of 1 cm was made on their back. Then human hypertrophic scar tissue was embedded into the incision. On post injury day 7, multipoint injection of NS in a volume of 0.05 mL was performed in wounds of rats in group NS, while rats in 1 μmol/L SD-208 group were given 0.05 mL 1 μmol/L SD-208, once a day. On the day 0 (the same day), 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 post first time of injection, the weight of 8 nude mice was weighed by electronic scale, and scar area was measured by vernier caliper and the ratio of rest scar area was calculated. (6) In week 1, 2, and 3 post first time of injection, the protein expression of TGF-β1 of human hypertrophic scar tissue was assessed with Western blotting. Data were processed with one-way analysis of variance and two independent-samplet test. Results (1) The proliferation activity of cells in group BC, 0.5, 1.0, 3.0, and 5.0 μmol/L SD-208 groups was respectively 1.00±0.03, 0.90±0.08, 0.68±0.11, 0.54±0.04, and 0.42±0.09, and the proliferation activity of cells in 0.5, 1.0, 3.0, and 5.0 μmol/L SD-208 groups was significantly lower than that in group BC (witht values from 2.9 to 22.1,P <0.05 orP <0.01). (2) The number of migration cells in 1, 3 μmol/L SD-208 groups was significantly less than that in group BC (witht values respectively 6.5 and 6.4,P values below 0.01). (3) Compared with that in group BC, fluorescence intensity of microfilaments of cells in 1, 3 μmol/L SD-208 groups was attenuated, and the pseudopod extended less. (4) The protein expressions of TGF-β1 of cells in group BC and 1, 3 μmol/L SD-208 groups were respectively 1.00±0.08, 0.80±0.08, and 0.61±0.05, and the protein expressions of TGF-β1 of cells in 1, 3 μmol/L SD-208 groups were significantly lower than those in group BC (witht values respectively 4.0 and 9.2,P values below 0.01). (5) The weights of nude mice in group NS and 1 μmol/L SD-208 group were similar on each time day (witht values from 0.2 to 1.1,P values above 0.05). The ratios of rest scar area of nude mice in two groups were decreased along with the injection time, and the ratios of rest scar area of nude mice in 1 μmol/L SD-208 group were significantly less than those in group NS from the day 6 to 20 post first time of injection (witht values from 1.8 to 15.9,P <0.05 orP <0.01). In week 1, 2, and 3 post first time of injection, the protein expressions of TGF-β1 of human hypertrophic scar tissue in nude mice in two groups showed a tendency of decrease, and the protein expressions of TGF-β1 of human hypertrophic scar tissue in nude mice in 1 μmol/L SD-208 group were significantly lower than those in group NS (witht values from 6.2 to 19.1,P values below 0.01). Conclusions SD-208 has significant inhibition effect on human hypertrophic scars, and the mechanism is correlated to the inhibition of protein expression of endogenous TGF-β1.-
Key words:
- Cicatrix /
- Transforming growth factor beta1 /
- Cell proliferation /
- Cell movement /
- Microfilaments /
- SD-208
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