芍药苷对增生性瘢痕成纤维细胞增殖及生物学行为的影响

发布时间：2018-05-09 13:39

本文选题：增生性瘢痕 + 转化生长因子β1　；参考：《暨南大学》2015年硕士论文

【摘要】：研究背景增生性瘢痕是深度烧伤常见的并发症,病理表现为大量的成纤维细胞增殖及细胞外基质(ECM)的异常沉积。转化生长因子β1(Transforming growth factorβ1,TGF-β1)是一种具有多种功能的细胞因子,通过细胞内信号转导发挥作用,在增生性瘢痕的形成过程中扮演重要角色:它一方面能调节成纤维细胞生长、分化、迁移凋亡;另一方面促进成纤维细胞合成、分泌胶原蛋白,抑制基质金属蛋白酶(MMPs)合成,减少胶原纤维降解。芍药苷(Paeoniflorin)是传统中药芍药(Paeonia lactiflora pall)干燥根中的提取物,也是芍药的主要单体活性成分。研究发现,芍药苷在肝、肾、肺中表现出抗纤维化的作用,其主要机制为减少组织内TGF-β1的含量,抑制细胞内TGF-β1的自分泌,使胶原等细胞外基质的合成明显减少。增生性瘢痕是皮肤创伤后发生在真皮的纤维增生性疾病,而芍药苷对增生性瘢痕的形成的影响,尚未见报道。目的探讨芍药苷对增生性瘢痕成纤维细胞增殖及其生物学行为的影响。方法收集增生性瘢痕组织及来自同一个体的正常皮肤组织,采用组织块法分离培养增生性瘢痕成纤维细胞和正常皮肤成纤维细胞,分别加入不同浓度(0,200,400,800μM)的芍药苷:(1)培养24h、48h、72h,MTS法检测细胞增殖速度;(2)培养48h,流式细胞仪检测细胞周期及凋亡;(3)培养48h,收集细胞培养上清液,酶联免疫吸附分析方法(ELISA)检测TGF-β1、I型胶原含量;(4)培养48h,蛋白质印迹法(Western Blot)检测细胞中α平滑肌肌动蛋白(α-SMA)的表达水平。结果(1)浓度为400μM、800μM的芍药苷能明显抑制各时相点(24h、48h、72h)增生性瘢痕及正常皮肤成纤维细胞的增殖。(2)200μM、400μM、800μM的芍药苷能明显增加两种成纤维细胞处于G2期的百分比,各组中未检测到细胞凋亡。(3)400μM、800μM的芍药苷能明显减少增生性瘢痕成纤维细胞培养上清液中TGF-β1含量,但不影响正常皮肤成纤维细胞培养上清液中TGF-β1含量。(4)200μM、400μM、800μM的芍药苷能明显减少增生性瘢痕及正常皮肤成纤维细胞上清液中I型胶原含量。(5)200μM、400μM、800μM的芍药苷能明显减少增生性瘢痕及正常皮肤成纤维细胞中α-SMA蛋白的表达。结论一定浓度的芍药苷可抑制增生性瘢痕及正常皮肤成纤维细胞的增殖,减少I型胶原的分泌及α-SMA的表达;芍药苷可以减少增生性瘢痕成纤维细胞TGF-β1的自分泌,但是对正常皮肤成纤维细胞TGF-β1的自分泌却没有明显的影响,提示芍药苷可能对于增生性瘢痕的形成有抑制作用。
[Abstract]:Background Hypertrophic scar is a common complication of deep burn. The pathological features of hypertrophic scar are proliferation of fibroblasts and abnormal deposition of extracellular matrix (ECM). Transforming growth factor 尾 1(Transforming growth factor 尾 1 (TGF- 尾 1) is a multifunctional cytokine that plays an important role in the process of hypertrophic scar formation through intracellular signal transduction: on the one hand, it can regulate the growth and differentiation of fibroblasts. Migration and apoptosis; on the other hand, promote fibroblast synthesis, secrete collagen, inhibit matrix metalloproteinase (MMPs) synthesis, reduce collagen degradation. Paeoniflorin (Paeoniflorin) is an extract from the dried roots of Paeonia lactiflora pallin, a traditional Chinese medicine, which is also the main monomer active component of paeoniflorin. It was found that paeoniflorin had anti-fibrosis effect in liver, kidney and lung. The main mechanism of paeoniflorin was to reduce the content of TGF- 尾 _ 1 in tissue, inhibit the autocrine of TGF- 尾 _ 1 and decrease the synthesis of extracellular matrix such as collagen. Hypertrophic scar is a fibrous hyperplastic disease occurring in the dermis after skin trauma, but the effect of paeoniflorin on the formation of hypertrophic scar has not been reported. Objective to investigate the effects of paeoniflorin on proliferation and biological behavior of hypertrophic scar fibroblasts. Methods the hypertrophic scar tissue and the normal skin tissue from the same body were collected. The fibroblasts of hypertrophic scar and normal skin were isolated and cultured by tissue block method. Paeoniflorin 1 of different concentrations (0 200 400 800 渭 M) was added to culture for 24 h, 48 h and 72 h, respectively. The cell proliferation rate was detected by MTS method. The cell cycle and apoptosis were detected by flow cytometry (FCM) for 48 h. The supernatants of cell culture were collected. The level of 伪 -SMAs was detected by Elisa and Western blotts were used to detect the expression of 伪 -SMAin in TGF- 尾 _ 1C _ I type I collagen (TGF- 尾 _ (1) I) cultured for 48 h. The expression of 伪 -SMAs was detected by Western blot assay (Western blot). The expression of 伪 -SMAs was detected by Western blot assay. Results Paeoniflorin at the concentration of 400 渭 M and 800 渭 M could significantly inhibit the proliferation of hypertrophic scar and normal skin fibroblasts at 24 h and 48 h / 72 h) Paeoniflorin at the concentration of 400 渭 M or 800 渭 M could significantly increase the percentage of the two fibroblasts in G2 phase. No apoptosis was detected in each group. Paeoniflorin (800 渭 M) could significantly reduce the content of TGF- 尾 1 in the culture supernatant of hypertrophic scar fibroblasts. However, Paeoniflorin of 200 渭 M, 400 渭 M, 800 渭 M of paeoniflorin in the supernatant of normal skin fibroblast culture could significantly reduce the content of collagen type I in the supernatant of hypertrophic scar and fibroblast supernatant of normal skin, and the content of paeoniflorin in the supernatant of normal skin fibroblast cell culture. 200 渭 M, 400 渭 M, 800 渭 M of paeoniflorin, could significantly reduce the proliferative ability of paeoniflorin. Expression of 伪 -SMA protein in fibroblasts of scar and normal skin. Conclusion Paeoniflorin can inhibit the proliferation of hypertrophic scar and normal skin fibroblasts, decrease the secretion of type I collagen and the expression of 伪 -SMA, and decrease the autocrine of TGF- 尾 1 in hypertrophic scar fibroblasts. However, there was no obvious effect on the autosecretion of TGF- 尾 1 in normal skin fibroblasts, suggesting that paeoniflorin might inhibit the formation of hypertrophic scar.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R622

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