【作者】 任校峰；

【导师】 李世荣；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 硕士

【摘要】 目的糖尿病(Diabetes Mellitus,DM)是多病因引起的慢性代谢性疾病。皮肤病变如溃疡、坏疽、局部感染等是糖尿病患者极为常见的临床表现之一。创伤难愈并发症的出现与糖尿病患者皮肤组织的创伤愈合能力低下有直接关系。如何更快的促进创面修复,局部用药是一种重要的辅助治疗,而目前方法很多,但疗效往往不令人满意。本实验主要是利用NO在创面愈合中的重要作用,同时改变创面的微环境,为创面的愈合提供一种新的局部用药治疗方法:由促一氧化氮生成的L-精氨酸、硝普钠和超氧阴离子生成抑制剂夹竹桃麻素形成合剂,对糖尿病小鼠创面的进行联合应用,明确其方法在创面愈合中的作用。通过建立STZ诱导的I型糖尿病小鼠模型和创伤难愈模型,给予促NO合剂进行干预创面愈合过程,观察外源性药物在创伤愈合过程中对伤口愈合时间、肉芽组织生长及表皮细胞增殖的影响;应用免疫组化方法,揭示创面愈合时TGF-β1因子的表达规律和血管生成情况;通过光镜观察创面不同时间点的成纤维细胞数量及胶原纤维含量;研究NO合剂对创面愈合的影响,并探讨其机制,为临床上在治疗糖尿病难愈创面时提供理论依据。方法1.模型建立1.1建立STZ诱导的I型糖尿病小鼠模型小鼠标记称重后,连续4d腹腔注射STZ (50mg/ kg),小鼠禁食不禁水10h ,由尾静脉取血,连续8周跟踪检测小鼠的血糖浓度及体重变化。1.2建立STZ糖尿病小鼠创伤难愈模型空腹血糖浓度高于11.1 mmol/L,且连续出现多饮、多食、多尿者选为糖尿病模型小鼠。将小鼠以10%水合氯醛(30mg/kg)腹腔注射麻醉后,在背部中线两侧(间距1cm以上),以直径1cm打孔器打孔、止血,单笼喂养。跟踪检测其创口愈合速度直至完全愈合。2.NO合剂对小鼠创面愈合作用的观察实验分组:共设5个实验组,即糖尿病小鼠对照组、糖尿病小鼠L-精氨酸(L-Arg)治疗组(A组)、糖尿病小鼠夹竹桃麻素(APO)治疗组、糖尿病小鼠硝普钠(SNP)治疗组、糖尿病小鼠NO合剂治疗组,每组10只小鼠。给药方法:小鼠背部创面建立成功后,按照不同组别,分别以生理盐水、L-arg溶液(150g/L,去离子水配制)、APO溶液(1×10-4 mol/L,去离子水配制)、SNP溶液(0.1mmol/L,去离子水配制)及NO合剂(L-arg、APO、SNP溶液等比混合,临用前配制)在相应组小鼠创面用无菌注射器皮下注射各0.15ml,隔天一次。标本采集:在治疗后的第1、3、5、7、10天,应用数码照相机拍下创面情况,记录愈合的面积。于伤后第3、7、10天分别取创缘组织,10%福尔马林固定,石蜡包埋连续性切片后,常规HE、VG染色,观察创面成纤维细胞、血管新生及胶原沉积等情况,评价创面愈合效果。2.1计算创面愈合率使用透明方格纸描绘创口,用计算机图像分析软件计算面积,并按以下公式计算创面愈合率:创面愈合率=(创面初始面积-创面形成后第n天的面积)/创面初始面积×100%。2.2成纤维细胞密度测定400倍光镜下观察HE染色切片,分别在组织中央浅部、中央深部、两侧部各随机选取10个矩形视野(0.0052 mm2/视野),目测计数并计算切片内单位面积成纤维细胞数量。2.3.胶原纤维面密度测定400倍光镜下观察VG染色组织切片,分别在组织中央浅部、中央深部、两侧部各随机选取5个视野,利用HMIAS-2000高清晰度彩色医学图文分析系统计算红染的胶原纤维的面密度。3. NO合剂对小鼠创面血管生成和转化生长因子的研究3. 1血管密度的测定以抗血管VIII因子抗体对标本进行免疫组织化学染色,操作步骤按照试剂盒说明书进行。参照weidne的评判标准:呈现棕色的单个内皮细胞或内皮细胞簇均作为1个血管计数,肌层较厚及管腔面积大于8个红细胞直径的血管均不计数。400倍光镜下在每张切片的中央区、周边区各随机选取5个血管密集的视野,取其平均数作为该标本的平均血管密度(MVD)。3. 2转化生长因子测定以TGF-β1抗体对标本进行免疫组织化学染色,操作步骤按照试剂盒说明书进行。观察指标用光学显微镜(10×40倍)观察TGF-β1免疫阳性纤维的分布用Mplas-500多媒体彩色病理图文分析系统,通过显微摄像系统放大400倍,每张切片随机选取5个视野,在该视野中选定TGF-β1(+)标准,以图像分析系统自动测量出结果,单位μm2。采用SPSS 10.0软件包进行统计学分析,所有计量数据以( X±S)表示,进行t检验,p<0.05表示有统计学差异。结果1.连续8周跟踪检测小鼠的血糖浓度及体重变化;检测结果表明小鼠血糖浓度显著增高(p<0.05),而体重明显下降(p<0.05),且体征持续8周以上,与文献报道的STZ糖尿病小鼠模型一致;表明STZ在小鼠体内成功诱导I型糖尿病。2. STZ糖尿病小鼠创口的整个愈合过程比对照组明显滞后(p<0.05),说明手术使STZ糖尿病小鼠出现了创伤难愈现象,也说明糖尿病严重损伤了小鼠皮肤组织的创伤愈合能力。3.对照组于创伤后16-18天可以基本愈合;NO合剂治疗组肉芽组织生长好,愈合时间较对照组提前3-4天。其他单剂治疗组愈合情况良好,较对照组提前1-2天,创面形成后第3天起,各药物治疗组小鼠创面愈合率较对照组明显上升,以创面形成后7d内变化最为明显。4.各实验组创伤后3天可见细胞增生、胶原形成,7-10天成纤维细胞增生明显,胶原纤维排列整齐;实验组在创伤后3、7天成纤维细胞数密度(NA)均高于对照组水平,而在创伤后10天均低于对照组;其中,NO合剂治疗组较各药物单剂治疗组差异更为明显,具有统计学意义(P<0.05)。各组胶原纤维面密度(AA)均呈持续性增加的趋势,实验组在所有时间点AA均高于对照组(P<0.05),单剂各组比较差异不明显5. TGF-β1阳性细胞比率:NO合剂治疗组用药后3天细胞阳性染色明显,3-7天细胞中的TGF-β1在细胞中的阳性表达增强并达到高峰,创伤后7-14天细胞中的阳性表达逐渐减弱至相对维持稳定水平。实验组在3、7天时间点的阳性表达均高于对照组(P<0.01),对照组细胞中的阳性表达峰值明显滞后,且表达量少于其他组;而与其它单剂干预组组间比较差异不明显。6. NO合剂治疗组在创伤后3-7天内皮细胞增生活跃,较对照组有更多的毛细血管形成(P<0.01),与其他单剂干预组比较,血管生成的数量亦有差异(P<0.05)。对照组血管生成明显较少,且炎性反应较其他组严重。其中NO合剂治疗组与对照组的差异最为显著(P<0.01),L-Arg、APO、SNP治疗组组间差异不明显。结论在糖尿病小鼠创面的愈合过程中单独使用L-精氨酸或硝普钠或夹竹桃麻素可以促进创面的愈合,而促NO合剂则对创面的愈合具有更好的修复作用,可以明显缩短糖尿病小鼠创面的愈合时间,较L-Arg、APO、SNP单独使用效果更为显著。更多还原

【Abstract】 ObjectiveDiabetes Mellitus (DM) is a chronic metabolic disease. Wound healing impairments are the most common complications in DM. To promote wound repair, local administration would be optimal. However, even with thousands of methods, satisfactory effect can be hardly obtained. Our research was based on the NO’s important role in wound healing, by monitoring microenvironment, aiming to provide a new local drug therapy: Nitric oxide generator L - arginine, sodium nitrate, shevchenko and oxygen anion inhibitors oleander hemp meat. We establish a STZ induced type I diabetes and trauma refractory model in mice, and administrated the mixture to the wounds , wound healing time, granulation tissue growth and proliferation of epidermal cells were measured . Immunohistochemistry was applied to locate TGF-β1 expression and its relation with angiogenesis. Through the microscope observation at different time points we measure fibroblasts quantity and collagen fiber content. By researching on wound healing NO generating mixture, and discussing its mechanism, we are looking forward to providing more theoretical evidence for clinical treatment for diabetes refractory wounds.Methods1. Establish Model1.1 Establish STZ induction of type I diabetes model miceAfter weighing , and four consecutive days intraperitoneal injection STZ (50mg/ kg), mice banned food but not prohibiting to drink for 10h,was taken blood vein tail, and was tracked detection it’s glucose concentrations and variation of body weight eight weeks.1.2 Establish STZ diabetic mice trauma refractory modelWe choose the modle ,which fasting plasma glucose concentrations, and continuously appear above 11.1 mmol/L, food and drink more than urine .Mice with 10% water chloric aldehyde (30mg/kg) after intraperitoneal injection of anesthesia in line spacing both (back) in diameter, above the 1cm f.DaKongQi 1cm with diameter punch, hemostatic, single cage. Tracking the wound healing rate until healed completely.2. Generate NO of mice wound healing mixture of observationExperimental group: Total 5 group----diabetic mice, diabetic mice L - arginine (L - Arg in treatment group (group), diabetic mice oleander hemp meat (APO) treatment group, sodium nitrate diabetic mice doppler (SNPS) treatment group, diabetic mice NO mixture treatment groups, each group has 10 mice.Medication:After the wound is established in back, according to different groups success by physiological saline, L - 150g/L arg solution (compound, deionized water solution (1), the APO 10-4 mol/L, deionized water dispensing), mmol/SNPS solution (0.1 L, deionized water dispensing) and NO mixture (L - arg APO, SNPS solution, compares with preparation, mixing before in mice with corresponding wound sterile syringes subcutaneous injection, the next time the 0.15 ml.Specimen after treatment: 1, 3, 5, 7, 10 days, the application of digital camera wound healing, under the circumstance of record. In the first 3 and 7 and 10 days were taken and margin, 10% formalin fixed, paraffin slice, wash. continuity, dyeing, observe VG wound fibroblasts, angiogenesis and collagen deposition etc, evaluation wound healing effect.2.1 calculate the wound healing rate Use transparent graph paper depicts wounds, using computer image analysis software area,and according to the following formula and wound healing wound healing rate: initial area ratio = (wound after the formation of the wound - wound formed n day area) / the initial area×100%2.2 Fibroblasts density measurement HE observed under microscope 400 times respectively in the organization, dyeing, the deep and shallow central randomly selected on both sides of the rectangular vision (10), 0.0052 was/vision and calculation counts in implementing slice of fibroblasts quantity per unit area.2.3. Collagen fiber surface density messurement 400 times observed under microscope VG dyeing biopsy,in the shallow、deep tissue and on both sides of the department of central randomly selected five vision, using HMIAS - 2000 high-resolution color medical graphic analysis system, calculation of collagen fiber dyed red surface density.3. NO generation of mice wound angiogenesis mixture and growth factors3.1 Vascular density measurement Use the antiangiogenesis factor VIII antibodies to immunohistochemical specimens,operating procedures in accordance with the kit instructions .According to weidne criteria: Presents the brown single endothelial cells or endothelial cell clusters are as a blood count, muscle thicker and lumen area more than 8 red blood vessels are not count in diameter.Under the microscope 400 times in each section of the central and peripheral zone randomly selected 5 vessels, the vision of dense as the average density of blood samples (MVD) average.3.2 Growth factor measurement With TGF - beta 1 antibodies to immunohisto- chemical specimens,operating procedures in accordance with the kit instructions .Observing indexes with optical microscope (10 by 40 times) TGF - beta 1 observe fiber distribution with positive immune Mplas-500 multi-media color pathological graphic analysis system, through microscopic imaging system amplification 400 times,each section randomly selected in this field, 5 in the field TGF selected beta 1 (+) - standard, image analysis system of automatic measurement results, a unit of mu m2.Using SPSS statistical analysis software, 10.0 all measurement data () says, t-test, p < 0.05 expressed statistically significant.Results1. Eight weeks tracking detection mouse glucose concentrations and variation of body weight;Test results show that mice glucose concentrations were significantly higher (p < 0.05), and weight decreased obviously (p < 0.05), and the signs for 8 weeks, and reports in the literature of the above model STZ diabetic mice, STZ that success in mice induced type I diabetes.2. The whole STZ diabetic mice wound healing process is slower than the control group (p < 0.05), and explain the operation made STZ diabetic mice appeared trauma and diabetes phenomenon of refractory severely damaged skin tissue in the wound healing abilities.3. Controls in 16-18 days after trauma can heal, NO mixture of granulation tissue growth in treatment group than controls, healing time of 3-4 days in advance. Other single-agent intervention group, a group healed ahead 1-2 days, three days after the formation of wounds, each drug treatment group than controls wound healing rate in mice with wounds, after forming most obvious changes within the 7d.4. Each group, three days after traumatic visible cell hyperplasia, collagen formation, 7-10 tiancheng fiber cell hyperplasia of collagen fibers, aligned, In march, after traumatic group, 10 tiancheng fiber cell density (making) were higher than those in the control group (P < 0.05), and in 14 days after trauma were lower than that of the control group (P < 0.05).But NO treatment group than other mixture single-agent therapy is more obvious (P < 0.01). Each collagen fiber surface density (AA) are continuous increasing trend, at all time points AA are higher than the control group (P < 0.01), single-agent intervention group, with no obvious difference in the APO slightly higher than the other two treatment groups.5. TGF - beta cell 1 positive rate:NO mixture treatment group after 3 days, positive dyeing cells,3-7 days of TGF - beta cells in the positive and express enhancement cell peak, traumatic 7-14 days of cells express weakened gradually to the positive relatively stable level;On July 3, group, 10 days of positive point above expression in control group (P < 0.01), the positive control cells express clearly, and express peak lag is less than other groups,as with any other single agent intervention group no obvious difference (P > 0.05).6. NO mixture in treatment group 3,5,7 days after traumatic endothelial cells, hyperplasia of active control more capillary formation (P < 0.01), and other single-agent intervention group, the number of angiogenesis is also has difference (P < 0.05). Control and angiogenesis significantly less serious than any other group of the inflammatory response. NO one in treatment group and control group of mixture is the most significant difference (P < 0.01), L - Arg APO, between treatment group and SNPS difference.ConclusionsIn diabetic mice wound healing process ,single use L - arginine, or Apocynin, or sodium nitroferricyanide can promote wound healing, while promoting NO generation of wound healing mixture with better effect,can significantly reduce diabetic mice, the wound healing time L - Arg APO SNPS, alone, especially.更多还原