创面负压治疗对糖尿病足创面肉芽组织肌成纤维细胞影响的研究
发布时间：2018-08-07 12:59
【摘要】：研究背景:糖尿病足是糖尿病严重慢性并发症之一,糖尿病足创面不同于一般的外科创面,一旦出现,常常迁延不愈,如何促进糖尿病足创面愈合已成为临床上的难点和热点问题。创面负压治疗(negative pressure wound therapy, NPWT )可促进创面的愈合,近些年来,其在糖尿病足创面上的应用逐渐增多。临床随机对照试验和荟萃分析显示,NPWT可加速糖尿病足创面的愈合,但是其促进糖尿病足创面愈合的具体机制尚不十分明确,有待深入研究。成纤维细胞是肉芽组织的主要成份细胞,其在创面愈合过程中逐渐向肌成纤维细胞转化,肌成纤维细胞与成纤维细胞的主要区别是前者表达a-平滑肌肌动蛋白(a-smooth muscle actin, a-SMA),这种特点使肌成纤维细胞在功能上具有收缩的作用,并且与成纤维细胞相比,肌成纤维细胞能具有分泌较大量胶原纤维的作用,因此,肌成纤维细胞在创面修复过程中起着更为重要的作用。转化生长因子β1 (Transforming growth factor β1, TGFβ1)是促进肌成纤维细胞形成的主要细胞生长因子,EDA+纤连蛋白(extradomainAfibronectin,EDA+FN)是促进肌成纤维细胞形成的主要细胞外基质。本研究通过对糖尿病足创面进行NPWT和常规换药治疗,观察创面面积的变化及新生肉芽组织生成情况,观察肌成纤维细胞相关蛋白a-SMA、TGFβ1及EDA+FN在糖尿病足创面肉芽组织的表达及沉积情况,并在细胞水平观察肌成纤维细胞形成情况,研究NPWT对糖尿病足局部创面肉芽组织肌成纤维细胞形成的影响,探讨NPWT促进糖尿病足创面愈合的相关机制。研究方法:1.选取2014年1月到2016年6月符合纳入和排除标准的糖尿病足患者32例,将患者随机分为创面负压治疗组(NPWT组)和对照组(Control, Con组),每组各16例。NPWT组给予创面负压治疗,Con组给予常规换药治疗。2.分别测量两组患者治疗前及治疗后14d创面面积,比较NPWT和常规换药治疗对糖尿病足创面面积的影响。3.两组受试者分别于治疗前及治疗后14d取创面肉芽组织,行苏木精-伊红(hematoxylin-eosin,HE)染色、Masson 染色、免疫组织化学染色、Western blot、Real-time PCR及细胞培养。4.组织水平研究NPWT对糖尿病足创面肉芽组织中肌成纤维细胞生成的影响。应用HE染色、Masson染色观察两组新生肉芽组织形成情况。应用免疫组织化学染色法、Western blot法观察两组肉芽组织中肌成纤维细胞相关蛋白a-SMA、TGFβ1及EDA+FN的蛋白表达情况;应用Real-time PCR法观察两组肉芽组织中肌成纤维细胞相关蛋白a-SMA、TGFβ1及EDA+ FN的基因表达情况。5.细胞水平研究NPWT对糖尿病足创面肉芽组织中肌成纤维细胞形成的影响。取两组治疗前后肉芽组织,分离、培养成纤维细胞,免疫组织化学染色观察两组治疗前后a-SMA阳性成纤维细胞即肌成纤维细胞的数量,并应用Western blot和Real-time PCR法分析肌成纤维细胞a-SMA的蛋白和基因表达情况。研究结果:1. Con组治疗后,创面面积未见明显缩小(P0.05),NPWT组治疗后,创面面积明显缩小(P0.01)。2.组织水平研究结果显示:(1) HE染色和Masson染色结果显示,两组治疗后毛细血管数量及胶原纤维沉积较治疗前均增多(P0.01),但是与Con组相比,NPWT组治疗后毛细血管数量及胶原纤维沉积较治疗前增多更为显著(P0.01)。(2)免疫组织化学染色和Western blot结果显示,尽管两组治疗后a-SMA、TGFβ1和EDA+ FN的蛋白表达水平均高于治疗前(P0.01);但与Con组相比,NPWT组治疗后a-SMA、TGFβ1和EDA+FN蛋白表达水平较治疗前增加更为明显(P0.01)。(3) Real-time PCR结果显示,两组治疗后a-SMA、TGFβ 1和EDA+ FN mRNA表达量均高于治疗前(P0.01);但与Con组相比,NPWT组治疗后a-SMA、TGFβ1和EDA+ FN mRNA表达量较治疗前增加更为显著(P0.01)。3.细胞水平研究结果显示:(1)免疫组织化学染色结果显示,虽然两组治疗后肌成纤维细胞数量较治疗前均增多(P0.01),但是与Con组相比,NPWT组治疗后肌成纤维细胞数量较治疗前增多更为明显(P0.01)。(2) Western blot结果显示,尽管两组治疗后肌成纤维细胞a-SMA相对蛋白表达量均高于治疗前(P0.01),但与Con组相比,NPWT组治疗后肌成纤维细胞a-SMA相对蛋白表达量较治疗前增加更为显著(P0.01)。(3) Real-timePCR结果显示,两组治疗后肌成纤维细胞a-SMA相对mRNA表达量均高于治疗前(P0.01);但与Con组相比,NPWT组治疗后肌成纤维细胞a-SMA相对mRNA表达量较治疗前增加更为明显(P0.01)。研究结论:1.NPWT可明显促进糖尿病足创面肉芽组织的生成,明显缩小糖尿病足创面面积。2. NPWT可显著增加糖尿病足创面肉芽组织肌成纤维细胞相关蛋白a-SMA、TGFβ1和EDA+FN的蛋白和基因表达,可明显增加糖尿病足创面局部肉芽组织肌成纤维细胞的形成。3. NPWT可促进糖尿病足局部创面肉芽组织和肌成纤维细胞形成,这可能是NPWT促进糖尿病足创面愈合的机制之一。
[Abstract]:Background: diabetic foot is one of the serious chronic complications of diabetes. The wound of diabetic foot is different from the general surgical wound. Once it appears, it is often deferred. How to promote the healing of diabetic foot wound has become a difficult and hot issue in clinical. Wound negative pressure (negative pressure wound therapy, NPWT) can promote the wound surface. The application of NPWT to diabetic foot wound is increasing in recent years. Clinical randomized controlled trial and meta-analysis show that the healing of diabetic foot wound can be accelerated, but the specific mechanism for promoting the healing of diabetic foot wound is not very clear. In the process of wound healing, the cell is gradually transformed into myofibroblast, and the main difference between myofibroblast and fibroblast is that the former expresses a- smooth muscle actin (a-Smooth muscle actin, a-SMA), which makes myofibroblasts function as contractile function, and the myofibroblast is thinner than fibroblasts. Cell can play a role in secreting a lot of collagen fibers, so myofibroblast plays a more important role in the process of wound repair. Transforming growth factor beta 1 (Transforming growth factor beta 1, TGF beta 1) is the main cell growth factor to promote myofibroblast formation, EDA+ fibronectin (extradomainAfibronectin, EDA+FN). This study was the main extracellular matrix for the formation of myofibroblast. This study was conducted by NPWT and conventional dressing for diabetic foot wounds. The changes in wound area and the formation of new granulation tissue were observed. The expression and deposition of myofibroblast related protein a-SMA, TGF beta 1 and EDA+FN were observed in the granulation tissue of diabetic foot wounds. And the formation of myofibroblast at the cell level was observed and the effects of NPWT on the formation of myofibroblast in the granulation tissue of the diabetic foot were studied. The mechanism of NPWT to promote the healing of diabetic foot wound was discussed. 1. the methods of research were studied in 32 cases of diabetic foot patients, which were included and excluded from January 2014 to June 2016. The patients were randomly divided into the negative pressure treatment group (NPWT group) and the control group (group Control, Con), 16.NPWT groups in each group were treated with negative pressure on the wound, and the group Con was given the routine change of.2. to measure the area of the wound surface before and after the treatment of the two groups respectively. The effect of NPWT and regular dressing on the area of diabetic foot wound was compared with the group of.3. two groups. The subjects were treated with the wound granulation tissue before and after the treatment, respectively, with hematoxylin eosin (hematoxylin-eosin, HE) staining, Masson staining, immunohistochemical staining, Western blot, Real-time PCR and cell culture at the.4. tissue level to study the effect of NPWT on myofibroblast formation in the granulation tissue of diabetic foot wounds. HE dye was applied. Color, Masson staining was used to observe the formation of new granulation tissue in the two groups. The expression of myofibroblast related protein a-SMA, TGF beta 1 and EDA+FN in the two groups of granulation tissue was observed by immunohistochemical staining, and the expression of TGF beta 1 and EDA+FN in the two groups of granulation tissues was observed. Real-time PCR method was used to observe the myofibroblast related protein a-SMA, TGF beta 1 in the two groups of meat buds. The effect of NPWT on the formation of myofibroblast in the granulation tissue of diabetic foot wounds. The effect of NPWT on the formation of myofibroblast in the granulation tissue of diabetic foot wound. Two groups of granulation tissue were taken before and after treatment, and the fibroblasts were isolated and cultured. The number of a-SMA positive fibroblasts, that is, the number of myofibroblast, was observed before and after the treatment of a-SMA positive fibroblasts in the two groups, and the number of myofibroblasts were observed before and after the treatment of a-SMA. The Western blot and Real-time PCR method were used to analyze the protein and gene expression of a-SMA in myofibroblast. The results were as follows: after 1. Con, the area of the wound was not significantly reduced (P0.05). After the treatment of group NPWT, the area of the wound was obviously reduced (P0.01).2. tissue level. (1) HE staining and Masson staining showed that two groups were treated. The amount of capillaries and collagen deposition increased after treatment (P0.01), but compared with group Con, the number of capillaries and collagen fibrils in group NPWT were more significant than before treatment (P0.01). (2) immunohistochemical staining and Western blot results showed that although the protein tables of a-SMA, TGF beta 1 and EDA+ FN after two groups were treated. The average amount of water was higher than that before the treatment (P0.01), but compared with the Con group, the expression level of a-SMA, TGF beta 1 and EDA+FN in the group NPWT was more obvious than that before the treatment (P0.01). (3) Real-time PCR results showed that the expression of a-SMA, TGF beta 1 and EDA+ before treatment in the two groups were higher than those before the treatment. The expression of beta 1 and EDA+ FN mRNA was more significant than that before treatment (P0.01).3. cell level studies showed: (1) immunohistochemical staining results showed that although the number of myofibroblasts increased in the two groups after treatment (P0.01), the number of myofibroblasts in the group NPWT was more than before the treatment, and the number of myofibroblasts in the group NPWT was more than before the treatment. (P0.01). (2) the results of (2) Western blot showed that the expression of a-SMA relative protein in myofibroblast in the two groups was higher than that before treatment (P0.01), but compared with the Con group, the a-SMA relative protein expression of myofibroblast in the NPWT group was more significant than that before the treatment (P0.01). (3) Real-timePCR results showed that the two groups were treated after the treatment. The relative mRNA expression of a-SMA in fibroblasts was higher than that before the treatment (P0.01), but compared with the Con group, the relative mRNA expression of a-SMA in myofibroblast in NPWT group was more obvious than that before the treatment (P0.01). Conclusion: 1.NPWT can obviously promote the formation of granuloma in the wound of diabetic foot, and obviously reduce the.2. NPWT of the area of diabetic foot wound. The protein and gene expression of myofibroblast related protein a-SMA, TGF beta 1 and EDA+FN can obviously increase the formation of.3. NPWT in the local granulomatous myofibroblast of diabetic foot wound, which can promote the formation of granulomatous and myofibroblast in the local wound of diabetic foot, which may be NPWT promoting. One of the mechanisms of wound healing in diabetic foot.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2
[Abstract]:Background: diabetic foot is one of the serious chronic complications of diabetes. The wound of diabetic foot is different from the general surgical wound. Once it appears, it is often deferred. How to promote the healing of diabetic foot wound has become a difficult and hot issue in clinical. Wound negative pressure (negative pressure wound therapy, NPWT) can promote the wound surface. The application of NPWT to diabetic foot wound is increasing in recent years. Clinical randomized controlled trial and meta-analysis show that the healing of diabetic foot wound can be accelerated, but the specific mechanism for promoting the healing of diabetic foot wound is not very clear. In the process of wound healing, the cell is gradually transformed into myofibroblast, and the main difference between myofibroblast and fibroblast is that the former expresses a- smooth muscle actin (a-Smooth muscle actin, a-SMA), which makes myofibroblasts function as contractile function, and the myofibroblast is thinner than fibroblasts. Cell can play a role in secreting a lot of collagen fibers, so myofibroblast plays a more important role in the process of wound repair. Transforming growth factor beta 1 (Transforming growth factor beta 1, TGF beta 1) is the main cell growth factor to promote myofibroblast formation, EDA+ fibronectin (extradomainAfibronectin, EDA+FN). This study was the main extracellular matrix for the formation of myofibroblast. This study was conducted by NPWT and conventional dressing for diabetic foot wounds. The changes in wound area and the formation of new granulation tissue were observed. The expression and deposition of myofibroblast related protein a-SMA, TGF beta 1 and EDA+FN were observed in the granulation tissue of diabetic foot wounds. And the formation of myofibroblast at the cell level was observed and the effects of NPWT on the formation of myofibroblast in the granulation tissue of the diabetic foot were studied. The mechanism of NPWT to promote the healing of diabetic foot wound was discussed. 1. the methods of research were studied in 32 cases of diabetic foot patients, which were included and excluded from January 2014 to June 2016. The patients were randomly divided into the negative pressure treatment group (NPWT group) and the control group (group Control, Con), 16.NPWT groups in each group were treated with negative pressure on the wound, and the group Con was given the routine change of.2. to measure the area of the wound surface before and after the treatment of the two groups respectively. The effect of NPWT and regular dressing on the area of diabetic foot wound was compared with the group of.3. two groups. The subjects were treated with the wound granulation tissue before and after the treatment, respectively, with hematoxylin eosin (hematoxylin-eosin, HE) staining, Masson staining, immunohistochemical staining, Western blot, Real-time PCR and cell culture at the.4. tissue level to study the effect of NPWT on myofibroblast formation in the granulation tissue of diabetic foot wounds. HE dye was applied. Color, Masson staining was used to observe the formation of new granulation tissue in the two groups. The expression of myofibroblast related protein a-SMA, TGF beta 1 and EDA+FN in the two groups of granulation tissue was observed by immunohistochemical staining, and the expression of TGF beta 1 and EDA+FN in the two groups of granulation tissues was observed. Real-time PCR method was used to observe the myofibroblast related protein a-SMA, TGF beta 1 in the two groups of meat buds. The effect of NPWT on the formation of myofibroblast in the granulation tissue of diabetic foot wounds. The effect of NPWT on the formation of myofibroblast in the granulation tissue of diabetic foot wound. Two groups of granulation tissue were taken before and after treatment, and the fibroblasts were isolated and cultured. The number of a-SMA positive fibroblasts, that is, the number of myofibroblast, was observed before and after the treatment of a-SMA positive fibroblasts in the two groups, and the number of myofibroblasts were observed before and after the treatment of a-SMA. The Western blot and Real-time PCR method were used to analyze the protein and gene expression of a-SMA in myofibroblast. The results were as follows: after 1. Con, the area of the wound was not significantly reduced (P0.05). After the treatment of group NPWT, the area of the wound was obviously reduced (P0.01).2. tissue level. (1) HE staining and Masson staining showed that two groups were treated. The amount of capillaries and collagen deposition increased after treatment (P0.01), but compared with group Con, the number of capillaries and collagen fibrils in group NPWT were more significant than before treatment (P0.01). (2) immunohistochemical staining and Western blot results showed that although the protein tables of a-SMA, TGF beta 1 and EDA+ FN after two groups were treated. The average amount of water was higher than that before the treatment (P0.01), but compared with the Con group, the expression level of a-SMA, TGF beta 1 and EDA+FN in the group NPWT was more obvious than that before the treatment (P0.01). (3) Real-time PCR results showed that the expression of a-SMA, TGF beta 1 and EDA+ before treatment in the two groups were higher than those before the treatment. The expression of beta 1 and EDA+ FN mRNA was more significant than that before treatment (P0.01).3. cell level studies showed: (1) immunohistochemical staining results showed that although the number of myofibroblasts increased in the two groups after treatment (P0.01), the number of myofibroblasts in the group NPWT was more than before the treatment, and the number of myofibroblasts in the group NPWT was more than before the treatment. (P0.01). (2) the results of (2) Western blot showed that the expression of a-SMA relative protein in myofibroblast in the two groups was higher than that before treatment (P0.01), but compared with the Con group, the a-SMA relative protein expression of myofibroblast in the NPWT group was more significant than that before the treatment (P0.01). (3) Real-timePCR results showed that the two groups were treated after the treatment. The relative mRNA expression of a-SMA in fibroblasts was higher than that before the treatment (P0.01), but compared with the Con group, the relative mRNA expression of a-SMA in myofibroblast in NPWT group was more obvious than that before the treatment (P0.01). Conclusion: 1.NPWT can obviously promote the formation of granuloma in the wound of diabetic foot, and obviously reduce the.2. NPWT of the area of diabetic foot wound. The protein and gene expression of myofibroblast related protein a-SMA, TGF beta 1 and EDA+FN can obviously increase the formation of.3. NPWT in the local granulomatous myofibroblast of diabetic foot wound, which can promote the formation of granulomatous and myofibroblast in the local wound of diabetic foot, which may be NPWT promoting. One of the mechanisms of wound healing in diabetic foot.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2
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