本文選題：轉(zhuǎn)化生長(zhǎng)因子β受體Ⅰ + 糖尿病足�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:糖尿病足創(chuàng)面經(jīng)久不愈,修復(fù)機(jī)制復(fù)雜,目前尚不明確。轉(zhuǎn)化生長(zhǎng)因子β1(transforming growth factor-beta 1,TGF-β1)是多功能生物活性因子,在創(chuàng)面修復(fù)過(guò)程中發(fā)揮重要作用。已有研究證實(shí),外源性TGF-β1可有效彌補(bǔ)糖尿病足創(chuàng)面中表達(dá)量減少的缺陷,加速創(chuàng)面的愈合。然而臨床試驗(yàn)性應(yīng)用TGF-β1的效果卻不理想。探究其原因,可能與其受體異常表達(dá)相關(guān)。本文通過(guò)分析轉(zhuǎn)化生長(zhǎng)因子β受體Ⅰ(transforming growth factor-beta receptor typeⅠ,TGF-βRⅠ)在糖尿病足創(chuàng)面肉芽組織和非糖尿病性創(chuàng)面肉芽組織中的表達(dá)差異,探討TGF-βRⅠ在糖尿病足創(chuàng)面修復(fù)過(guò)程中的作用,為糖尿病足創(chuàng)面修復(fù)機(jī)制和糖尿病足治療提供新思路。方法:1糖尿病足組研究對(duì)象選自解放軍白求恩國(guó)際和平醫(yī)院內(nèi)分泌科于2015年1月至2016年6月間收治并符合入排標(biāo)準(zhǔn)的糖尿病足患者10例;非糖尿病組(對(duì)照組)研究對(duì)象選自同時(shí)間段于本院燒傷科住院并符合入排標(biāo)準(zhǔn)的非糖尿病患者10例。分別收集兩組患者的年齡、性別、空腹血糖(fast blood glucose,FBG)、糖化血紅蛋白(glycosylated hemoglobin,HbA1C)、踝肱比(ankle-brachial index,ABI)、谷丙轉(zhuǎn)氨酶、肌酐等一般資料,并進(jìn)行統(tǒng)計(jì)學(xué)分析。2兩組受試者均予以適當(dāng)清創(chuàng),清創(chuàng)時(shí)間為10-15天,清創(chuàng)完畢后取各組創(chuàng)面肉芽組織。每份肉芽組織分為三組,并以A、B、C標(biāo)記。3 A組標(biāo)本放置于4%多聚甲醛固定液中,4℃保存,待兩組標(biāo)本收齊后行石蠟包埋。4部分經(jīng)石蠟包埋的A組標(biāo)本行切片及HE染色,光鏡下(400×)觀察兩組肉芽組織的形態(tài)及細(xì)胞組成;計(jì)數(shù)兩組肉芽組織HE染色切片中毛細(xì)血管數(shù)并行統(tǒng)計(jì)學(xué)分析。5剩余石蠟包埋的A組標(biāo)本行切片及免疫組化,光鏡下(400×)觀察兩組肉芽組織切片中TGF-βRⅠ的表達(dá)情況。每份切片隨機(jī)選取10個(gè)染色陽(yáng)性區(qū)域進(jìn)行拍照,并進(jìn)行光密度分析,分析軟件為數(shù)字醫(yī)學(xué)圖像分析系統(tǒng)(image-pro-plus6.0)。tgf-βrⅠ的表達(dá)量用平均光密度值(opticaldensity,od)表示,半定量分析tgf-βrⅠ的表達(dá)情況。6b組標(biāo)本行蛋白印跡(westernblot)分析,quantityone軟件分析兩組tgf-βrⅠ的條帶灰度值。每個(gè)條帶中tgf-βrⅠ的表達(dá)量用tgf-βrⅠ與gapdh條帶灰度比值表示,定量分析tgf-βrⅠ的蛋白表達(dá)情況。7c組標(biāo)本應(yīng)用real-timepcr檢測(cè)tgf-βrⅠ的mrna表達(dá)量。對(duì)照組第一號(hào)樣品為標(biāo)準(zhǔn)1,分別得到各組各樣本目的基因的ct值,按照rq=2-△△ct,計(jì)算各組各樣本目的基因的rq值,tgf-βrⅠ的mrna表達(dá)量用rq值表示,并對(duì)其行統(tǒng)計(jì)學(xué)分析。結(jié)果:1糖尿病足組與對(duì)照組年齡、性別、谷丙轉(zhuǎn)氨酶、肌酐等一般資料無(wú)顯著性差異(p0.05);與對(duì)照組相比,糖尿病足組fbg、hba1c明顯升高,abi降低(p0.01)。2觀察兩組肉芽組織大體形態(tài),可見(jiàn)糖尿病足組肉芽組織顏色晦暗,觸感韌,觸之不易出血;對(duì)照組肉芽組織顏色鮮紅,顆粒狀,觸之柔軟、濕潤(rùn)、出血多。光鏡下觀察兩組肉芽組織he染色切片,糖尿病足組肉芽組織中毛細(xì)血管網(wǎng)稀疏,成纖維細(xì)胞少,炎性細(xì)胞大量浸潤(rùn);對(duì)照組肉芽組織中毛細(xì)血管網(wǎng)密布,毛細(xì)血管網(wǎng)間可見(jiàn)大量增殖的成纖維細(xì)胞,炎性細(xì)胞浸潤(rùn)少。對(duì)兩組肉芽組織he切片中毛細(xì)血管數(shù)進(jìn)行統(tǒng)計(jì)學(xué)分析,結(jié)果顯示:與對(duì)照組相比,糖尿病足組肉芽組織中毛細(xì)血管數(shù)量減少(p0.01)。3光鏡下觀察兩組免疫組化染色切片,棕黃色或棕褐色顆粒即為染色陽(yáng)性�？梢�(jiàn)糖尿病足組棕黃色染色顆粒少,著色淺;對(duì)照組棕黃色染色顆粒明顯增多,且著色深;用od值表示tgf-βrⅠ的表達(dá)量,并行統(tǒng)計(jì)學(xué)分析,結(jié)果顯示:與對(duì)照組相比,糖尿病足組tgf-βrⅠ的表達(dá)量降低(p0.01)。4westernblot分析結(jié)果顯示:與對(duì)照組相比,糖尿病足組tgf-βrⅠ的蛋白表達(dá)降低(p0.01)。5real-timepcr分析結(jié)果顯示:與對(duì)照組相比,糖尿病足組tgf-βRⅠ的mRNA表達(dá)降低(P0.01)。結(jié)論:1糖尿病足創(chuàng)面肉芽組織中毛細(xì)血管形成減少,肉芽組織老化嚴(yán)重。2糖尿病足創(chuàng)面肉芽組織中TGF-βRⅠ的蛋白表達(dá)量和基因表達(dá)量明顯降低。3糖尿病足創(chuàng)面肉芽組織中TGF-βRⅠ的表達(dá)量降低,可能是導(dǎo)致糖尿病足創(chuàng)面肉芽組織形成不良、創(chuàng)面遷延不愈的原因之一。
[Abstract]:Objective: the wound of diabetic foot has long been unhealed and the mechanism of repair is complex. It is not clear at present. Transforming growth factor beta 1 (transforming growth factor-beta 1, TGF- beta 1) is a multifunctional bioactive factor and plays an important role in the process of wound repair. It has been proved that exogenous TGF- beta 1 can effectively compensate for the reduction in the expression of diabetic foot wound. However, the effect of TGF- beta 1 in clinical trials is not ideal. To explore the cause, it may be related to the abnormal expression of its receptor. In this paper, we analyzed the growth factor beta receptor I (transforming growth factor-beta receptor type I, TGF- beta R I) in the granulation tissue and non diabetes of diabetic foot wounds. The effect of TGF- beta R I on the repair of diabetic foot wound, and to provide new ideas for the repair mechanism of diabetic foot wound and the treatment of diabetic foot. Methods: 1 the subjects of diabetic foot group were selected from the Department of Endocrinology, Bethune international Heping Hospital, from January 2015 to June 2016. 10 cases of diabetic foot patients were treated in accordance with the standard of admission. The subjects of non diabetic group (control group) were selected from 10 non diabetic patients who were hospitalized in the Department of burn in the same time section and were in line with the standard of discharge. The age, sex, fast blood glucose, FBG, glycated hemoglobin (glycosylated hemoglobin) were collected respectively. HbA1C), the general data of ankle brachial ratio (ankle-brachial index, ABI), glutamic pyruvic aminotransferase, creatinine and other general data, and statistical analysis of.2 two subjects were properly debrided and debridement time was 10-15 days. After debridement was completed, the granulation tissue of each group was divided into three groups, and A, B, C labeled.3 A groups were placed in 4% polyformaldehyde. In the solution, 4 centigrade was preserved. After two groups of specimens were collected, the paraffin embedded.4 part of the A group was sliced and stained with HE. The morphology and cell composition of two groups of granulation tissue were observed under light microscope (400 *), and the number of capillaries in the two groups of granulation tissue HE staining sections was counted and the A group specimens of.5 remaining paraffin embedded in the.5 group were cut down. The expression of TGF- beta R I in two groups of granulation tissue sections was observed under the light microscope (400 x). 10 stained positive regions were selected for each slice, and the light density analysis was carried out. The analysis software was used to use the mean optical density (opticaldens) for the expression of.Tgf- beta R I in the digital medical image analysis system (image-pro-plus6.0). Ity, OD) expressed, semi quantitative analysis of the expression of tgf- beta R I,.6b group mark of Western blot (Westernblot) analysis, quantityone software analysis of the band gray value of the two groups of tgf- beta r i. The expression of tgf- beta R I in each band was expressed by the ratio of tgf- beta and gray level I with the gray level. Use real-timepcr to detect the mRNA expression of tgf- beta r i. The first sample of the control group was a standard 1, and the CT value of the target genes of each group was obtained. The RQ value of the target genes in each group was calculated according to rq=2- delta CT, and the mRNA expression of tgf- beta R I was expressed with RQ values, and the results were statistically analyzed. Results 1 diabetic foot group and control There was no significant difference in age, sex, glutamic pyruvic transaminase and creatinine (P0.05). Compared with the control group, the FBG and HbA1c in the diabetic foot group increased significantly, and the ABI decreased (P0.01).2 to observe the gross morphology of the two groups of granulation tissue. The color of the granulation tissue in the diabetic foot group was dark, tactile toughened, and the control group was bright red in color. Two groups of granulated tissue he stained slices were observed under light microscope. The capillary network in the granulation tissue of the diabetic foot group was sparse, the fibroblasts were few, the inflammatory cells were infiltrated, the capillary network in the granuloma of the control group was densely distributed, and the proliferating fibroblasts and inflammatory cells were seen between the capillary network and the inflammatory cells. The number of capillary vessels in the he section of two groups of granulation tissue was statistically analyzed. The results showed that compared with the control group, the number of capillaries in the granulation tissue of the diabetic foot group decreased (P0.01) under the.3 light microscope, and the two groups of immunohistochemical staining sections were observed. The brown yellow or brown brown granules were stained positive. The color particles were less and the coloring was shallow, and the brown yellow staining particles in the control group were significantly increased and the coloring was deep. The expression of tgf- beta R I was expressed with OD value, and the results showed that the expression of tgf- beta R I in the diabetic foot group decreased (P0.01).4westernblot analysis results showed that the diabetic foot group was tgf- beta R I compared with the control group. The results of protein expression reduction (P0.01).5real-timepcr analysis showed that the mRNA expression of tgf- beta R I in the diabetic foot group was lower than that of the control group (P0.01). Conclusion: 1 the decrease of capillary formation in the granulation tissue of diabetic foot wound tissue and the protein expression and gene expression of TGF- beta R I in the granulation tissue of.2 diabetic foot on the granulation tissue The decrease of the expression of TGF- beta R I in the granulation tissue of.3 diabetic foot was significantly reduced, which may be one of the reasons for the bad formation of granulation tissue in the wound of diabetic foot and the non healing of the wound.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2

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