創(chuàng)面負壓治療對糖尿病足創(chuàng)面肉芽組織肌成纖維細胞影響的研究
發(fā)布時間：2018-08-07 12:59
【摘要】：研究背景:糖尿病足是糖尿病嚴重慢性并發(fā)癥之一,糖尿病足創(chuàng)面不同于一般的外科創(chuàng)面,一旦出現(xiàn),常常遷延不愈,如何促進糖尿病足創(chuàng)面愈合已成為臨床上的難點和熱點問題。創(chuàng)面負壓治療(negative pressure wound therapy, NPWT )可促進創(chuàng)面的愈合,近些年來,其在糖尿病足創(chuàng)面上的應用逐漸增多。臨床隨機對照試驗和薈萃分析顯示,NPWT可加速糖尿病足創(chuàng)面的愈合,但是其促進糖尿病足創(chuàng)面愈合的具體機制尚不十分明確,有待深入研究。成纖維細胞是肉芽組織的主要成份細胞,其在創(chuàng)面愈合過程中逐漸向肌成纖維細胞轉化,肌成纖維細胞與成纖維細胞的主要區(qū)別是前者表達a-平滑肌肌動蛋白(a-smooth muscle actin, a-SMA),這種特點使肌成纖維細胞在功能上具有收縮的作用,并且與成纖維細胞相比,肌成纖維細胞能具有分泌較大量膠原纖維的作用,因此,肌成纖維細胞在創(chuàng)面修復過程中起著更為重要的作用。轉化生長因子β1 (Transforming growth factor β1, TGFβ1)是促進肌成纖維細胞形成的主要細胞生長因子,EDA+纖連蛋白(extradomainAfibronectin,EDA+FN)是促進肌成纖維細胞形成的主要細胞外基質。本研究通過對糖尿病足創(chuàng)面進行NPWT和常規(guī)換藥治療,觀察創(chuàng)面面積的變化及新生肉芽組織生成情況,觀察肌成纖維細胞相關蛋白a-SMA、TGFβ1及EDA+FN在糖尿病足創(chuàng)面肉芽組織的表達及沉積情況,并在細胞水平觀察肌成纖維細胞形成情況,研究NPWT對糖尿病足局部創(chuàng)面肉芽組織肌成纖維細胞形成的影響,探討NPWT促進糖尿病足創(chuàng)面愈合的相關機制。研究方法:1.選取2014年1月到2016年6月符合納入和排除標準的糖尿病足患者32例,將患者隨機分為創(chuàng)面負壓治療組(NPWT組)和對照組(Control, Con組),每組各16例。NPWT組給予創(chuàng)面負壓治療,Con組給予常規(guī)換藥治療。2.分別測量兩組患者治療前及治療后14d創(chuàng)面面積,比較NPWT和常規(guī)換藥治療對糖尿病足創(chuàng)面面積的影響。3.兩組受試者分別于治療前及治療后14d取創(chuàng)面肉芽組織,行蘇木精-伊紅(hematoxylin-eosin,HE)染色、Masson 染色、免疫組織化學染色、Western blot、Real-time PCR及細胞培養(yǎng)。4.組織水平研究NPWT對糖尿病足創(chuàng)面肉芽組織中肌成纖維細胞生成的影響。應用HE染色、Masson染色觀察兩組新生肉芽組織形成情況。應用免疫組織化學染色法、Western blot法觀察兩組肉芽組織中肌成纖維細胞相關蛋白a-SMA、TGFβ1及EDA+FN的蛋白表達情況;應用Real-time PCR法觀察兩組肉芽組織中肌成纖維細胞相關蛋白a-SMA、TGFβ1及EDA+ FN的基因表達情況。5.細胞水平研究NPWT對糖尿病足創(chuàng)面肉芽組織中肌成纖維細胞形成的影響。取兩組治療前后肉芽組織,分離、培養(yǎng)成纖維細胞,免疫組織化學染色觀察兩組治療前后a-SMA陽性成纖維細胞即肌成纖維細胞的數(shù)量,并應用Western blot和Real-time PCR法分析肌成纖維細胞a-SMA的蛋白和基因表達情況。研究結果:1. Con組治療后,創(chuàng)面面積未見明顯縮小(P0.05),NPWT組治療后,創(chuàng)面面積明顯縮小(P0.01)。2.組織水平研究結果顯示:(1) HE染色和Masson染色結果顯示,兩組治療后毛細血管數(shù)量及膠原纖維沉積較治療前均增多(P0.01),但是與Con組相比,NPWT組治療后毛細血管數(shù)量及膠原纖維沉積較治療前增多更為顯著(P0.01)。(2)免疫組織化學染色和Western blot結果顯示,盡管兩組治療后a-SMA、TGFβ1和EDA+ FN的蛋白表達水平均高于治療前(P0.01);但與Con組相比,NPWT組治療后a-SMA、TGFβ1和EDA+FN蛋白表達水平較治療前增加更為明顯(P0.01)。(3) Real-time PCR結果顯示,兩組治療后a-SMA、TGFβ 1和EDA+ FN mRNA表達量均高于治療前(P0.01);但與Con組相比,NPWT組治療后a-SMA、TGFβ1和EDA+ FN mRNA表達量較治療前增加更為顯著(P0.01)。3.細胞水平研究結果顯示:(1)免疫組織化學染色結果顯示,雖然兩組治療后肌成纖維細胞數(shù)量較治療前均增多(P0.01),但是與Con組相比,NPWT組治療后肌成纖維細胞數(shù)量較治療前增多更為明顯(P0.01)。(2) Western blot結果顯示,盡管兩組治療后肌成纖維細胞a-SMA相對蛋白表達量均高于治療前(P0.01),但與Con組相比,NPWT組治療后肌成纖維細胞a-SMA相對蛋白表達量較治療前增加更為顯著(P0.01)。(3) Real-timePCR結果顯示,兩組治療后肌成纖維細胞a-SMA相對mRNA表達量均高于治療前(P0.01);但與Con組相比,NPWT組治療后肌成纖維細胞a-SMA相對mRNA表達量較治療前增加更為明顯(P0.01)。研究結論:1.NPWT可明顯促進糖尿病足創(chuàng)面肉芽組織的生成,明顯縮小糖尿病足創(chuàng)面面積。2. NPWT可顯著增加糖尿病足創(chuàng)面肉芽組織肌成纖維細胞相關蛋白a-SMA、TGFβ1和EDA+FN的蛋白和基因表達,可明顯增加糖尿病足創(chuàng)面局部肉芽組織肌成纖維細胞的形成。3. NPWT可促進糖尿病足局部創(chuàng)面肉芽組織和肌成纖維細胞形成,這可能是NPWT促進糖尿病足創(chuàng)面愈合的機制之一。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R587.2
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