【作者】 魏民；

【导师】 薛耀明；

【作者基本信息】 南方医科大学 ， 内分泌与代谢病， 2008， 博士

【摘要】 【背景】糖尿病足部溃疡(diabetic foot ulcer,DFC)作为糖尿病的慢性并发症是导致截肢的主要原因之一。不仅给患者带来巨大的精神压力、严重地影响生活质量,同时也造成了沉重的社会经济负担。据统计足部溃疡占已诊断糖尿病患者的4%到10%,而且糖尿病足部溃疡患者3年后的再发生率高达50%。随着现代生活水平的提高,糖尿病患者逐年增多,深入研究糖尿病足部溃疡的发病机制,为临床提供切实可行的治疗方法已成为当今的一项重要课题。创面修复是一个复杂而有序的生物学过程,主要分为炎症期,肉芽增殖期,创面修复塑形期。在每个时期都有大量的细胞、细胞外基质、细胞因子和组织参与,任何影响创面修复过程参与者的因素都可能最终导致创面难愈合。目前,糖尿病患者皮肤组织发生的生物学和组织学改变在足部溃疡形成机制中扮演的角色越来越受到重视。以往的研究表明:(1)局部组织胰岛素水平低下导致的创面组织细胞代谢异常;(2)创面局部炎症反应失调;(3)晚期糖基化产物(advanced glycation end products,AGEs)引起的皮肤病理生理学改变;均与糖尿病足部溃疡创面难愈合相关。近年有研究显示,糖尿病皮肤溃疡难愈合的原因大部分为炎症反应的失调或者延迟。炎症反应是创面修复的第一步,可以分为急性期和慢性期过程。在急性炎症期主要是血管反应、炎症细胞的浸润。炎症细胞主要作用为吞噬局部细菌、异物,分泌炎症因子,趋化炎症细胞进一步浸润等等。在炎症慢性期,浸润的主要是巨噬细胞,淋巴细胞。其主要作用为分泌活性物质、抗原递呈、促进新血管生成和淋巴管的增殖等。如果炎症急性期或者慢性期迁延、失调,会导致创面修复的延迟和难愈合。在众多的炎症细胞中,巨噬细胞的功能和作用与皮肤愈合的关系已经得到肯定。炎症反应部位中巨噬细胞浸润数量和功能将直接影响创面愈合的进程。在炎症急性期,巨噬细胞的作用主要是吞噬和分泌。在炎症慢性期,巨噬细胞分泌的趋化因子,如:IL-1,促进其他炎症细胞浸润到炎症部位。在肉芽形成期,巨噬细胞分泌细胞因子,如:血管形成生长因子(Vascular endothelial growth factor,VEGF),参与血管的形成;在塑形期,巨噬细胞可以影响结缔组织、胶原的形成和降解。所以,巨噬细胞的数量和功能贯穿了整个创面修复过程。在糖尿病足部溃疡修复过程中,巨噬细胞是否存在数量和功能异常,我们还不得而知。以往的研究已经证实,血管形成是创面修复的一个重要方面。但是,在最近的研究却发现,在创面修复中淋巴管的形成要早于血管形成,并且血管形成生长因子受体3(Vascular endothelial growth factor receptor 3,VEGFR3)阳性淋巴管的形成是构成新生血管的必要准备。其实,在创面修复过程中,淋巴管不只是局部引流,减少创面组织水肿,还可以直接参与炎症反应过程。在炎症期淋巴管能够向炎症反应部位运输树突状细胞。所以,淋巴管的形成对于创面修复同样重要。国外学者研究发现活化的巨噬细胞和淋巴管内皮存在某种联系,炎症组织中激活的巨噬细胞表达淋巴管内皮标志物:淋巴管内皮透明质酸受体-1(Lymphatic Vessel Endothelial Receptor 1,LYVE-1),并且可能参与了淋巴管的形成。但是在人体创面修复过程中,激活的巨噬细胞是否能转化为淋巴管内皮细胞?这在以往的研究中未见报道。糖尿病患者足部溃疡皮肤中是否存在淋巴管形成障碍?其形成的原因是什么?此在以往的研究中也未见到报道。淋巴管内皮细胞主要来源于骨髓,并被称作淋巴管内皮祖细胞,其特异性标志为CD34~+/CD134~+/VEGFR3~+。在各种刺激因素,如:血管形成生长因子C(Vascular endothelial growth factor C,VEGFC)的作用下,淋巴管内皮祖细胞能够在外周组织形成淋巴管内皮。如果外周血中VEGFC作用不足或者骨髓祖细胞微环境受到破坏,将导致淋巴管内皮祖细胞数量减少。糖尿病患者和糖尿病足部溃疡患者体内的淋巴管内皮祖细胞的数量有何改变?这种改变和糖尿病足部溃疡难愈合是否有相关性?影响淋巴管内皮祖细胞改变的因素是什么?这在以往的研究中都没有明确的结论。在正常皮肤组织,巨噬细胞主要存在于表皮和真皮交界部位,其是皮肤防止感染侵害的第一道防线。如果皮肤破损,皮肤局部的巨噬细胞可以迅速浸润到创伤部位发挥作用。研究发现,在db/db小鼠的皮肤溃疡处的巨噬细胞数量减少,分泌VEGFC的功能降低,分析这可能是该小鼠创面难愈合的重要原因之一。那么,在离体状态下,高糖和/或AGEs是引起巨噬细胞分泌VEGFC功能的重要因素吗?这在以往的研究中尚未涉及。基于对以上已有研究的思考,本课题设计为三部分。分别探讨糖尿病足部非缺血性溃疡难愈合皮肤组织中巨噬细胞数量和功能的改变,以及淋巴管形成情况;糖尿病足部非缺血性溃疡患者外周血中淋巴管内皮祖细胞数量的改变,以及离体巨噬细胞在葡萄糖和/或AGEs刺激下VEGFC基础分泌量和吞噬功能的改变;为进一步探讨糖尿病足部非缺血性溃疡难愈合提供实验证据。第一部分糖尿病足部非缺血性溃疡皮肤组织巨噬细胞数量及功能损伤和淋巴管形成障碍的研究【目的】观察糖尿病足部非缺血性溃疡周边皮肤组织中巨噬细胞数量、分泌功能和淋巴管形成的情况,以及激活的巨噬细胞和淋巴管内皮的关系,并与相同溃疡形成时间的非糖尿病足部溃疡皮肤、正常皮肤组织比较。探讨巨噬细胞数量、功能和淋巴管形成在糖尿病足部非缺血性溃疡难愈合机制中的作用。【方法】1、以年龄、性别及溃疡形成时间相匹配的糖尿病足部非缺血性溃疡患者、非糖尿病足部溃疡患者以及其溃疡周边皮肤作为研究对象,以正常皮肤组织作为对照,分别命名为糖尿病足溃疡组(diabetic foot group,DF)、非糖尿病足溃疡组(non-diabetic foot group,NDF)和正常对照组(normal control group,NC)。2、采用HE常规染色观察三组皮肤组织学差异。3、免疫组织化学方法检测各组CD68、VEGFR3、LYVE-1、VEGFC蛋白表达。4、RT-PCR检测各组CD68mRNA、VEGFR3mRNA、LYVE-1mRNA、VEGFCmRNA表达。5、激光共聚焦显微镜检测各组CD68表达和LYVE-1表达的关系。【结果】1、NDF组、DF组和NC组比较,年龄无统计学差异(P＞0.05),NDF组和DF组比较,溃疡形成时间无统计学差异(P＞0.05)。2、组织学结果:DF组皮肤组织及NDF组皮肤组织中均有典型的组织学改变,与正常皮肤清晰的表皮复层结构、丰富且致密的胶原排列形成了鲜明的反差。NC组、NDF组和DF组表皮层的厚度分别是:0.970±0.052mm、1.761±0.983mm、1.790±0.102mm,真皮层厚度分别是2.787±0.109mm、1.471±0.063mml、1.434±0.062mm。NDF组和DF组溃疡周边皮肤与NC组比较表皮层增厚,真皮层变薄,差异有统计学意义(P＜0.001)。3、免疫组化结果:(1)CD68在NC组表皮组织中表达为阳性,其阳性细胞存在于表皮基底部。在NC组真皮组织可见CD68阳性细胞,NDF组和DF组真皮组织炎症部位有CD68阳性细胞浸润。NC组、NDF组和DF组表皮组织中CD68阳性细胞百分数分别为:77.200±2.588%;33.800±4.147%;19.400±1.140%,三组比较有统计学差异(P＜0.001)。NC组、NDF组和DF组真皮组织中CD68阳性细胞百分数分别为:9.400±1.140%;79.800±1.304%;57.600±2.074%,三组比较有统计学差异(P＜0.001)。(2)LYVE-1在NC组表皮组织表达阳性,阳性细胞集中在表皮基底部。在NC组真皮组织中,阳性细胞集中在真皮和表皮交界的部位。在DF组表皮组织基底层细胞中,LYVE-1表达为阴性,在真皮组织和表皮组织交界处未见阳性细胞,在真皮层中阳性细胞也减少。在NDF组表皮基底层细胞中,LYVE-1表达为弱阳性,在真皮组织和表皮组织交界处可见阳性细胞。在真皮中可见阳性细胞出现。NC组、NDF组和DF组表皮组织中LYVE-1阳性细胞百分数分别为:87.600±1.140%;16.600±1.673%;9.600±1.342%,三组比较有统计学差异(P＜0.001)。NC组、NDF组和DF组真皮组织中LYVE-1的积分光密度值为:92.000±0.707;187.000±3.033;76.600±1.673,以上三组比较有统计学差异(P＜0.001)。(3)VEGFR3在NC组表皮组织表达为阳性,阳性细胞主要集中在表皮组织基底部。在NC组真皮组织中,阳性细胞集中在真皮层和表皮层交界部。在DF组表皮组织基底层细胞为阴性,在真皮层中阳性细胞减少。在NDF组表皮组织基底层细胞中VEGFR3表达为弱阳性,在真皮组织中表达为阳性。NC组、NDF组和DF组表皮组织中VEGFR3阳性细胞百分数分别为:81.400±1.140%;16.600±1.140%;8.800±1.483%,三组比较有统计学差异(P＜0.001)。NC组、NDF组和DF组真皮组织中VEGFR3的积分光密度值为:82.800±1.643;179.400±2.608;71.800±3.347,以上三组比较有统计学差异(P＜0.001)。(4)VEGFC在NC组表皮组织表达为阳性,阳性信号集中在表皮组织基底层。在NC组真皮中,VEGFC有表达。在DF组表皮组织基底层细胞,VEGFC表达为阴性,在真皮层中阳性信号减少。在NDF组表皮组织基底层细胞表达呈弱阳性,真皮组织中可以见到阳性细胞。NC组、NDF组和DF组表皮组织中VEGFC阳性细胞百分数分别为:79.200±1.304%;22.600±1.140%;9.200±2.168%,三组比较有统计学差异(P＜0.001)。NC组、NDF组和DF组真皮组织中VEGFC的积分光密度值为:115.400±2.702;193.000±3.162;103.000±2.915,以上三组比较有统计学差异(P＜0.001)。4、RT-PCR结果:DF组和NC组、NDF组比较,CD68mRNA表达减少,差异有统计学意义(P＜0.001);NDF组和NC组比较,CD68mRNA表达增加,差异有统计学差异(P＜0.001)。NDF组和NC组比较,LYVE-1mRNA表达增加,差异有统计学意义(P＜0.001);DF组和NC组、NDF组比较,LYVE-1mRNA表达减少,差异有统计学意义(P＜0.001)。DF组与NC组、NDF组比较,VEGFR3mRNA表达减少,差异有统计学意义(P＜0.001);NDF组和NC组比较,VEGFR3mRNA表达增加,差异有统计学意义(P＜0.001)。DF组和NC组、NDF组比较,VEGFCmRNA表达减少,差异有统计学意义(P＜0.001);NDF组和NC组比较VEGFCmRNA表达增加,差异有统计学意义(P＜0.001)。5、激光共聚焦显微镜结果显示:NC组表皮组织基底层可见CD68阳性细胞,在表皮组织、表皮组织与真皮组织交界部位可以见到LYVE-1阳性信号,叠加后未见CD68和LYVE-1信号重叠。在NDF组中,表皮组织的CD68阳性信号消失,LYVE-1阳性信号也消失,叠加后发现,在真皮组织中部分管腔中可以见到CD68阳性的内皮信号,同一部位也出现LYVE-1阳性信号,叠加后发现,二者存在共阳性现象,并且以CD68阳性为主,提示可能二者存在关联。在DF组,表皮组织和真皮组织中的CD68、LYVE-1表达同NDF组,在真皮组织中,未见到在管腔内皮中的CD68阳性细胞、LYVE-1阳性细胞,但是在炎症部位可以见到大量的CD68阳性信号,这些细胞同时出现了LYVE-1阳性,但是没有形成管腔内皮,只是散在的细胞分布。【结论】1、糖尿病足部非缺血性溃疡和非糖尿病足溃疡皮肤组织均存在表皮组织增厚,真皮组织变薄、真皮组织胶原减少、排列紊乱等。2、相同溃疡形成时间的糖尿病足部非缺血性溃疡皮肤组织较非糖尿病足部溃疡皮肤组织巨噬细胞数量减少,分泌VEGFC的功能降低,淋巴管内皮形成减少。提示糖尿病足部非缺血性溃疡皮肤组织存在难愈合倾向。3、非糖尿病足部溃疡皮肤组织中激活的巨噬细胞能够转化为淋巴管内皮细胞。糖尿病足部溃疡皮肤中激活的巨噬细胞也存在LYVE-1阳性表达,未出现在淋巴管管腔内皮处,具体原因尚待进一步研究。第二部分糖尿病足部非缺血性溃疡患者外周血白细胞VEGFR3表达及VEGFR3~+细胞数量变化的研究【目的】探讨糖尿病足部非缺血性溃疡患者、糖尿病患者外周血白细胞中VEGFR3表达和VEGFR3~+细胞(淋巴管内皮祖细胞)数量的改变以及其可能的影响因素。【方法】1、分为糖尿病足部溃疡组(DF组)、糖尿病1组(DM1组,HbA1C＜7%)、糖尿病2组(DM2组,HbA1C≥7%)和正常对照组(NC组)。2、采集各组清晨静脉血5ml,抗凝分离得到白细胞作为研究对象。3、采用RT-PCR检测各组白细胞中VEGFR3mRNA的表达,并进行统计学分析。4、记录除NC组外的各组临床资料,主要包括:空腹血糖、甘油三酯、胆固醇、收缩压、舒张压、糖化血红蛋白、空腹C肽、餐后2hC肽等,并和VEGFR3mRNA表达进行相关性分析。5、采用细胞免疫荧光法检测各组VEGFR3蛋白表达。6、调整细胞浓度为2×10~6个/ml,采用细胞免疫荧光法检测各组白细胞中VEGFR3~+细胞的数量,并进行统计学分析。【结果】1、通过非参数检验分析DF组、DM1组、DM2组和NC组患者VEGFR3mRNA的表达,实验组和NC组比较VEGFR3mRNA表达均减少,差异有统计学意义(P＜0.001)。各组的结果为:DF组Mean Rank=8.50;DM2组Mean Rank=12.50;DM1组Mean Rank=25.50;NC组Mean Rank=35.50。2、外周血白细胞VEGFR3mRNA表达和临床指标的相关性分析结果:VEGFR3mRNA表达的相关因素分别为空腹血糖(Spearman’s r=-0.518 P＜0.01);收缩压(Spearman’s r=-0.55 1 P＜0.01);舒张压(Spearman’s r=-0.391 P＜0.05);糖化血红蛋白(Spearman’s r=-0.633 P＜0.01);与甘油三酯、胆固醇、空腹C肽,餐后2hC肽无显著相关。3、使用Mias-2000高清晰度计算机病理图文报告系统检测荧光强度,以每例标本表达量最强的5个高倍视野(×400)的积分光密度值作为该例测定值。DM2组和DF组和NC组比较,VEGFR3蛋白表达减少,差异有统计学意义(P＜0.001)。DM1组和NC组比较无统计学差异。4、细胞免疫荧光化学结果比较显示:DF组外周血白细胞中VEGFR3~+细胞数量最少,其次为DM2组和DM1组,与NC组比较数量减少,差异有统计学意义(P＜0.001)。【结论】1、糖尿病患者较正常人外周血白细胞中VEGFR3表达减少,并且随着糖化血红蛋白的升高,减少更为显著。当合并足部非缺血性溃疡时,无论患者糖化血红蛋白的高低,VEGFR3表达显著减少。2、糖尿病患者和糖尿病足部非缺血性溃疡患者外周血中VEGFR3mRNA表达减少和空腹血糖、糖化血红蛋白、收缩压以及舒张压呈显著负相关。3、糖尿病足部非缺血性溃疡患者外周血白细胞中VEGFR3~+细胞(淋巴管内皮祖细胞)数量减少,其可能为创面淋巴管内皮形成减少的原因之一。第三部分葡萄糖和/或AGE对U937细胞VEGFC基础分泌量和吞噬功能影响的研究【目的】探讨U937细胞在葡萄糖、AGE以及两者联合刺激下VEGFC基础分泌量、吞噬功能的改变。【方法】1、通过ELISA法检测不同浓度、不同刺激时间的葡萄糖、AGE条件下U937细胞VEGFC基础分泌量的改变,确定最适合的葡萄糖、AGE刺激时间和浓度,确定联合刺激组的组成。经预实验测定15mmol/L葡萄糖刺激24h为葡萄糖刺激组,25ug/mlAGE刺激24h为AGE刺激组,两者联合为联合刺激组。2、将相同培养环境中的U937细胞分为正常对照组(normal control,NC)、葡萄糖刺激组(glucose group,GG)、AGE刺激组(AGEs group,AG)以及联合刺激组(glucose and AGEs group,GAG)。3、采用台盼蓝染色法检测各组培养后的细胞存活数量。4、采用RT-PCR法检测各组VEGFCmRNA的表达。5、采用细胞免疫荧光法研究各组VEGFC蛋白的表达。6、采用墨汁吞噬实验检测各组U937细胞吞噬能力的改变。【结果】1、经过预实验确定15mmol/L葡萄糖刺激24h为葡萄糖刺激组,25ug/mlAGE刺激24h为AGE刺激组,两者联合为联合刺激组。2、台盼蓝染色实验结果:各组有活性细胞数量比较,差异没有统计学意义(P＞0.05)。3、RT-PCR法检测VEGFCmRNA,实验组和NC组比较VEGFCmRNA表达减少,差异有统计学意义(P＜0.001)。4、定性分析VEGFC蛋白表达,未加荧光抗体的阴性对照组为(-),NC组细胞内荧光很明亮,定义为高度阳性(+++),AG组荧光强度较阴性对照组强,较NC组弱,为(++),GG组细胞荧光强度较弱,和阴性对照组以及NC组比较,荧光强度为(+),GAG组呈不均匀的荧光,轮廓不清,荧光强度与阴性对照组基本相同为(-)。定量分析VEGFC蛋白表达,使用Mias-2000高清晰度计算机病理图文报告系统,以每例标本表达量最强的5个高倍视野(×400)的积分光密度值作为该例测定值。实验组和NC组比较,VEGFC蛋白表达减少,差异有统计学意义(P＜0.001)。两两比较的结果为:GG组和AG组比较,VEGFC蛋白表达减少,差异有统计学意义(P＜0.001);GAG组和GG组比较,VEGFC蛋白表达减少,差异有统计学意义(P＜0.001);GAG组和AG组比较,VEGFC蛋白表达减少,差异有统计学意义(P＜0.001)。5、墨汁吞噬实验结果:实验组和NC组比较吞噬功能降低,差异有统计学意义(P＜0.001)。【结论】1、15mmol/L葡萄糖刺激24h、25ug/mlAGE刺激24h以及两者联合刺激对于U937细胞的存活没有影响。2、15mmol/L葡萄糖、25ug/mlAGE、以及两者联合刺激24h均引起U937细胞VEGFCmRNA表达减少,以两者联合刺激减少最显著。3、15mmol/L葡萄糖、25ug/mlAGE、以及两者联合刺激均引起U937细胞VEGFC蛋白表达减少,以两者联合刺激减少最显著。4、15mmol/L葡萄糖、25ug/mlAGE、以及两者联合刺激24h均引起U937细胞吞噬功能降低,以两者联合刺激降低最显著。更多还原

【Abstract】 BACKGROUNDDiabetic foot ulcer is one of chronic complications of diabetes,and it is one of important reasons for amputation,which gave great stress to the patients,and brought economic burden to the society and the family of patients.According to reports,the lower limb skin ulcer patients accounted for 4%to 10%of patients with diabetes.And the risk of re-amputation of diabetic foot ulcer was 50%.With the improvement of our living standard,there will be more and more diabetes patients in our society.It is important for us to thorough research the mechanism of diabetic foot ulcer.The repair of injury is a complex but orderly biology process,including inflammation,granulation multiplies and moulding of injury and so on.There are many cells,extra cellular matrix,cytokine and tissue participating in every period of the repair of injury.There are many factors to have influence on the repair of injury and cause un-healing.It is important to research the change of the skin and foot ulcer of diabetes patients.Some studies showed that there were many factors which had relationship with injury repair of diabetic foot ulcer,including(1) cell metabolism disorder caused by low insulin level(2) disorder of inflammatory reaction;(3) influence of AGEs in skin of diabetes patients.Inflammatory reaction is the first step of injury repair;including acute stage and chronic phase.In acute stage of inflammatory,the main reactions are vascular reaction and infiltration of inflammatory cell,including neutrophilic granulocyte.The functions of these cells include phagocytizing bacterium,excreting cytokine and chemotaxis to other inflammatory cell.In chronic phase,the main cells in area are macrophage and lymphocyte.The functions of these cells are excreting cytokine, antigen presentation,promoting angiopoiesis and lymphangiogenesis.If damaged in acute stage or chronic phase,it would be delayed in repairing of injury.Recent study showed that delay and disorder of inflammatory reactions were the main reasons of impaired of diabetic foot ulcer.Macrophage is very important for injury repair in chronic phase of inflammatory.The number and function of macrophage have influence on process of injury repair.In chronic phase of inflammatory,macrophage can secret a lot of chemotatic factors,such as TNF-αand IL-1,which can attract inflammatory cells to injury area.In the stage of granulation,macrophage can secret factors,such as VEGFC,which participates in angiopoiesis and lymphangiogenesis.In the stage of moulding,macrophage can promote proliferation and cell division of fibroblasts.So those from above,the number and function of macrophage are correlated with all the stage of inflammatory.In the past studies,angiopoiesis was important for injury repair.But in recent studies,lymphangiogenesis was earlier than angiopoiesis,and lymphangiogenesis of VEGFR3 positives was the preparer for angiopoiesis.And in other studies,the author found that the function of lymphatic vessel was not only draining but also transporting dendritic cell to the injury area.So lymphangiogenesis was the same important for injury repair as angiopoiesis.So whether decrease of lymphatic vessel formation in diabetic foot ulcer or not? And what was the reason for it? All of these are not report in past study.There are lymphatic vessel ancestral cell in peripheral blood,and the mark is CD34~+/CD133~+/VEGFR3~+.The ancestral cell will form lymphatic vessel in injury area under VEGFC.It will be decreased of lymphatic vessel endothelial cell because of less VEGFC or reduced ancestral cell in bone marrow.But we did not know the change of VEGFR3~+ cells in peripheral blood of diabetes.And we did not know the relation between the number of VEGFR3~+ cells and diabetic foot ulcer.And we also did not know the influence of lymphatic vessel ancestral cell in diabetes patiens.All of this was not reported in past study.In normal skin,macrophages located in the basilar part between epidermis and dermis.The macrophages in basilar part are the first line of defense of our skin.If skin was damaged,the macrophages would transmigrate to the damaged area and produced a marked effect.In a past study,the number of macrophage in the skin of db/db mice was decreased.And the author thought it was the reason for un-healing. But there was not a study show what was the reason of macrophage damaging.Based on above,we divided our design into three parts.The aim was to explore the changes of number and function of macrophages and lymphatic vessel endothelial cells in diabetic foot ulcer skin,non-diabetic foot skin and normal skin tissue and the cause of those.We also wanted to explore the secretion of VEGFC in macrophages treated by glucose and AGEs. Chapter 1 Decreased macrophage number and activation and reduced lymphatic vessel formation in non-anemic diabetic foot ulcer skin tissueOBJECTIVETo investigate the number and function of macrophage and lymphangiogenesis in diabetic foot ulcer skin tissue,non-diabetic foot ulcer skin tissue and normal skin tissue.METHODS1.Diabetic foot ulcer skin tissue,non-diabetic foot ulcer skin tissue,matched with age,gender and the time of ulceration.,were experimental groups,and normal skin tissue was control group.2.Investigate the basic histological change of skin tissue in three groups.3.Detected protein of CD68,VEGFR3,LYVE-1,and VEGFC in skin tissue.4.Gene expression of CD68,VEGFR3,LYVE-1,VEGFC in skin tissue detected by RT-PCR.5.Detected protein of CD68 and LYVE-1 by confocal microscopy and investigate the relation between CD68 and LYVE-1.RESULT1.Age among DF group,NDF group and NC group were not significantly different (P＞0.05),and time of ulceration between DF group and NDF group was not significantly different(P＞0.05).2.Typical histological changes could be seen in skin tissue of DF group and NDF group,including thickening of epidermis,derangement of dermis and so on. Thickness of epidermis were increased in NDF group and DF group,1.761±0.983mm, 1.790±0.102mm than that of in NC 0.970±0.052mm(P＜0.001).Thickness of dermis were decreased in NDF group and DF group,1.471±0.063mm、1.434±0.062mm than that of in group NC 2.787±0.109mm(P＜0.001).3.Protein of CD68,VEGFR3,LYVE-1 and VEGFC located in basilar part of NC group but disappeared in skin tissue of DF group and NDF group.In inflammatory area of dermis,expression of CD68 in NDF group was more than that of in DF group. The VEGFR3 positive cells,LYVE-1 positive cells were located in the borderline between dermis and epidermis in NDF group,but less in DF group.VEGFC was located around collagen in dermis in NDF group,but less in DF group.Protein of CD68 in epidermis in group NC,NDF and DF were 77.200±2.588%; 33.800±4.147%;19.400±1.140%.Protein of CD68 in dermis in group NC,NDF and DF were 9.400±1.140%;79.800±1.304%;57.600±2.074%.Protein of LYVE-1 in epidermis in group NC,NDF and DF were 87.600±1.140%;16.600±1.673%; 9.600±1.342%;Protein of LYVE-1 in dermis in group NC,NDF and DF were 92.000±0.707;187.000±3.033:76.600±1.673.Protein of VEGFR3 in epidermis in group NC,NDF and DF were 81.400±1.140%;16.600±1.140%;8.800±1.483%; Protein of VEGFR3 in dermis in group NC,NDF and DF were 82.800±1.643; 179.400±2.608;71.800±3.347.Protein of VEGFC in epidermis in group NC,NDF and DF were 79.200±1.304%;22.600±1.140%;9.200±2.168%;Protein of VEGFC in dermis in group NC,NDF and DF were 115.400±2.702;193.000±3.162; 103.000±2.915.4.Gene expression of CD68 in group DF was significantly less than group NC (P＜0.001).Gene expression of CD68 in group NDF was significantly higher than group NC(P＜0.001).Gene expression of CD68 in group DF was significantly less than group NDF(P＜0.001).Gene expression of VEGFR3 in group DF was significantly less than group NC(P＜0.001).Gene expression of VEGFR3 in group NDF was significantly higher than group NC(P＜0.001).Gene expression of VEGFR3 in group DF was significantly less than group NDF(P＜0.001).Gene expression of LYVE-1 in group DF were significantly less than group NC(P＜0.001). Gene expression of LYVE-1 in group NDF was significantly higher than group NC (P＜0.001).Gene expression of LYVE-1 in group DF was significantly less than group NDF(P＜0.001).Gene expression of VEGFC in group DF was significantly less than group NC(P＜0.001).Gene expression of VEGFC in group NDF was significantly higher than group NC(P＜0.001).Gene expression of VEGFC in group DF was significantly less than group NDF(P＜0.001).5.CD68 positive cells were in basilar part of epidermis,and LYVE-1 positive cells were in borderline between epidermis and dermis.CD68 positive cells and LYVE-1 positive cells were not overlapping after built up by confocal microscopy.CD68 positive cells were disappeared in epidermis in group NDE neither been of LYVE-1. But CD68 positive cells and LYVE-1 positive cells could be conduplicate in lympgatic vessel endothelial cell by confocal microscopy,and the main signal was CD68.CD68 positive cells and LYVE-1 positive cells were disappeared in epidermis in group DF.In inflammation area of dermis,we found CD68 positive cells and LYVE-1 positive cells,but we could not find stacked signal in lymphatic vessel endothelial cell.CONCLUTIONS1.It was more thickening of epidermis in diabetic foot than in non-diabetic foot.And it was more thinning of dermis in diabetic foot than in non-diabetic foot.And it was absent and disorder of collagen in dermis in DF and NDF.It maybe had relations with débridement and chronic inflammation. 2.It was decreased of the number,function of macrophages in the skin tissue of DF. And it was decreased of the lymphatic vessel formation in the skin tissue of DF.But it was increased of the number,function of macrophages in the skin tissue of NDF.And it was increased of the lymphatic vessel formation in the skin tissue of NDF We supposed that it was related with impaired wound healing.3.The macrophages were appeared in the lymphatic vessel in NDF.And it was negative in DF.We supposed that the macrophages in NDF skin tissue could change into lymphatic vessel endothelial cell.,but couldn’t in DF.Chapter 2 Expression of VEGFR3 and number of VEGFR3 positive cells in peripheral blood leukocyte of non-ischemia diabetic foot ulcer patientsOBJECTIVETo investigate gene expression of VEGF3 and number of VEGFR3~+ cells in peripheral blood leukocyte of diabetes patients and non-ischemia diabetic foot ulcer patients.METHODS1.Leukocytes were from four groups,group DF,group DM1(HbA1C＜7%),group DM2(HbA1C≥7%) and group NC.2.Leukocytes were from anticoagulated blood 5ml. 3.Gene and protein expression of VEGFR3 of leukocyte in every group was detected by RT-PCR and by immunofluorescence.4.Analysis relation between gene expression of VEGFR3 and clinical data,such as FBG,TG,CH,SBP,DBP,HbA1C,CP and so on.5.Number of VEGFR3 positive leukocyte was detected by immunofluorescence.RESULTS1.Gene expression of VEGFR3 in group DF,in group DM1 and in group DM2 were significantly less than that of in group NC(P＜0.001).Gene expression of VEGFR3 in every group were group DF(Mean Rank=8.50),group DM2(Mean Rank=12.50), and group DM1(Mean Rank=25.50).2.Gene expression of VEGFR3 had relation with FBG(Spearman’s r=-0.518 P＜0.01);with SBP(Spearman’s r=-0.551 P＜0.01);with DBP(Spearman’s r=-0.391 P＜0.05) and with HbA1C(Spearman’s r=-0.633 P＜0.01).3.Protein of VEGFR3 of group DM1 and group DF were significantly less than that of in group NC(P＜0.001).And Protein of VEGFR3 of group DM2 was significantly less than that of in group NC(P＜0.001).4.Number of VEGFR3 positive leukocyte in group DF,in group DM1 were significantly less than that of in group NC(P＜0.001).CONLUSION1.Gene expression of VEGFR3 of diabetes patients was less than that in normal control group,and the higher of HbA1C,the less of gene expression of VEGFR3. Gene expression of VEGFR3 had no relation with HbA1C in non-ischemia diabetic foot ulcer patients. 2.There were positive correlations among gene expression of VEGF3 and FBG, HbA1C,SBP and DBP.3.Number of VEGFR3 positive cells in group DF,DM2 and DM1 was significantly less than that of in group NC,and it was a possible reason for impaired diabetic foot ulcer.Chapter 3 Basal secretion of VEGFC and phagocytosis ability in U937 cell line influenced by high Glucose and/or AGEOBJECTIVETo explore basal secretion of VEGFC and phagocytosis ability in U937 cell line influenced by high Glucose and/or AGE.METHORDS1.Detected the basal secretion of VEGFC influenced by Glucose and/or AGEs in different time and/or in different concentration.2.We divided U937 cell into four groups,glucose group(15mmol/L G,24H), AGEs group(25ug/mlAGE,24H),combination group(15mmol/L G,24H and 25ug/mlAGE,24H) and NC group.3.Cytoactive was detected by trypan blue staining.4.Gene expression of VEGFC was detected by RT-PCR.5.Protein of VEGFC was detected by immunofluorescence.6.Ability of phagocytosis was detected by Indian ink phagocytosis test. RESULTS1.There were four groups through preliminary experiment,include G group (15mmol/L G,24H),AGE group(25ug/mlAGE,24H),combination group(15mmol/L G,24H and 25ug/mlAGE,24H) and NC group.2.Cytoactive was the same in every group.There was no significant difference in every group.3.Gene expression of VEGFC in all group was significantly less than that of in group NC(P＜0.001).Gene expression of VEGFC in group combination and group AGE were significantly less than that of in group NC(P＜0.001).4.Result of immunofluorescence was that we selected 5 high power field randomly and calculated gray scale.Experimental group was decreased of protein VEGFC than group NC.Group G was decreased of protein VEGFC than group AG. Group GAG was decreased of protein VEGFC than group GG.Group GAG was decreased of protein VEGFC than group AG.5.Ability of phagocytosis in group combination was significantly lower than that of in group NC(P＜0.001).Ability of phagocytosis in group G was significantly lower than that of in group NC(P＜0.01).Ability of phagocytosis in group AGE was significantly lower than that of in group NC(P＜0.05).CONCLUSION1.High glucose,AGE and combination of glucose and AGE had no influence on the activity of U937 cell line.2.Gene expression of VEGFC in group combination was the least,the second was group G,and the third is group AGE.3.Protein of VEGFC was the same as gene expression of VEGFC. 4.Ability of phagocytosis in group combination was the least,the second was group G and the third was group AGE.更多还原