TGF-βRⅠ在糖尿病足创面肉芽组织中的表达

发布时间：2018-06-29 10:17

本文选题：转化生长因子β受体Ⅰ + 糖尿病足　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:糖尿病足创面经久不愈,修复机制复杂,目前尚不明确。转化生长因子β1(transforming growth factor-beta 1,TGF-β1)是多功能生物活性因子,在创面修复过程中发挥重要作用。已有研究证实,外源性TGF-β1可有效弥补糖尿病足创面中表达量减少的缺陷,加速创面的愈合。然而临床试验性应用TGF-β1的效果却不理想。探究其原因,可能与其受体异常表达相关。本文通过分析转化生长因子β受体Ⅰ(transforming growth factor-beta receptor typeⅠ,TGF-βRⅠ)在糖尿病足创面肉芽组织和非糖尿病性创面肉芽组织中的表达差异,探讨TGF-βRⅠ在糖尿病足创面修复过程中的作用,为糖尿病足创面修复机制和糖尿病足治疗提供新思路。方法:1糖尿病足组研究对象选自解放军白求恩国际和平医院内分泌科于2015年1月至2016年6月间收治并符合入排标准的糖尿病足患者10例;非糖尿病组(对照组)研究对象选自同时间段于本院烧伤科住院并符合入排标准的非糖尿病患者10例。分别收集两组患者的年龄、性别、空腹血糖(fast blood glucose,FBG)、糖化血红蛋白(glycosylated hemoglobin,HbA1C)、踝肱比(ankle-brachial index,ABI)、谷丙转氨酶、肌酐等一般资料,并进行统计学分析。2两组受试者均予以适当清创,清创时间为10-15天,清创完毕后取各组创面肉芽组织。每份肉芽组织分为三组,并以A、B、C标记。3 A组标本放置于4%多聚甲醛固定液中,4℃保存,待两组标本收齐后行石蜡包埋。4部分经石蜡包埋的A组标本行切片及HE染色,光镜下(400×)观察两组肉芽组织的形态及细胞组成;计数两组肉芽组织HE染色切片中毛细血管数并行统计学分析。5剩余石蜡包埋的A组标本行切片及免疫组化,光镜下(400×)观察两组肉芽组织切片中TGF-βRⅠ的表达情况。每份切片随机选取10个染色阳性区域进行拍照,并进行光密度分析,分析软件为数字医学图像分析系统(image-pro-plus6.0)。tgf-βrⅠ的表达量用平均光密度值(opticaldensity,od)表示,半定量分析tgf-βrⅠ的表达情况。6b组标本行蛋白印迹(westernblot)分析,quantityone软件分析两组tgf-βrⅠ的条带灰度值。每个条带中tgf-βrⅠ的表达量用tgf-βrⅠ与gapdh条带灰度比值表示,定量分析tgf-βrⅠ的蛋白表达情况。7c组标本应用real-timepcr检测tgf-βrⅠ的mrna表达量。对照组第一号样品为标准1,分别得到各组各样本目的基因的ct值,按照rq=2-△△ct,计算各组各样本目的基因的rq值,tgf-βrⅠ的mrna表达量用rq值表示,并对其行统计学分析。结果:1糖尿病足组与对照组年龄、性别、谷丙转氨酶、肌酐等一般资料无显著性差异(p0.05);与对照组相比,糖尿病足组fbg、hba1c明显升高,abi降低(p0.01)。2观察两组肉芽组织大体形态,可见糖尿病足组肉芽组织颜色晦暗,触感韧,触之不易出血;对照组肉芽组织颜色鲜红,颗粒状,触之柔软、湿润、出血多。光镜下观察两组肉芽组织he染色切片,糖尿病足组肉芽组织中毛细血管网稀疏,成纤维细胞少,炎性细胞大量浸润;对照组肉芽组织中毛细血管网密布,毛细血管网间可见大量增殖的成纤维细胞,炎性细胞浸润少。对两组肉芽组织he切片中毛细血管数进行统计学分析,结果显示:与对照组相比,糖尿病足组肉芽组织中毛细血管数量减少(p0.01)。3光镜下观察两组免疫组化染色切片,棕黄色或棕褐色颗粒即为染色阳性。可见糖尿病足组棕黄色染色颗粒少,着色浅;对照组棕黄色染色颗粒明显增多,且着色深;用od值表示tgf-βrⅠ的表达量,并行统计学分析,结果显示:与对照组相比,糖尿病足组tgf-βrⅠ的表达量降低(p0.01)。4westernblot分析结果显示:与对照组相比,糖尿病足组tgf-βrⅠ的蛋白表达降低(p0.01)。5real-timepcr分析结果显示:与对照组相比,糖尿病足组tgf-βRⅠ的mRNA表达降低(P0.01)。结论:1糖尿病足创面肉芽组织中毛细血管形成减少,肉芽组织老化严重。2糖尿病足创面肉芽组织中TGF-βRⅠ的蛋白表达量和基因表达量明显降低。3糖尿病足创面肉芽组织中TGF-βRⅠ的表达量降低,可能是导致糖尿病足创面肉芽组织形成不良、创面迁延不愈的原因之一。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2

【参考文献】