糖尿病皮肤病理生理改变及其机制的研究

目的研究糖尿病皮肤伤前潜在的病理生理改变及其形成的机制。方法将16只8周龄雄性SD大鼠随机分为糖尿病组(8只)和正常对照组(8只)。致病12周后,肝素抗凝血收集血浆,取背部全层皮肤组织标本。测量血浆中糖化蛋白(GSP)、丙二醛(MDA)和谷胱甘肽(GSH);观察皮肤组织学的改变,检测表皮厚度、表皮细胞周期、皮肤组织糖和AGEs含量;建立AGE-HSA干预的细胞培养体系,观测AGEs干预后表皮细胞周期和ECV304细胞的生长抑制。结果糖尿病大鼠毛色灰暗、枯黄、体质量减轻,皮肤明显变薄,表皮细胞数量减少,复层排列缺乏,表皮厚度(0.016±0.007)mm]明显薄于正常对照大鼠(0.037±0.007)mm],差异有统计学意义(P<0.01);真皮层部分胶原萎缩、肿胀,退化变性,并伴随程度不等的炎性细胞浸润;皮下脂肪进行性萎缩或消失。糖尿病大鼠血浆GSP和MDA含量升高,GSH下降,皮肤糖含量和胶原提取液的荧光值也显著升高(P均<0.01),真皮基质AGEs蛋白表达强烈且广泛,有的相互连接呈片状,正常大鼠真皮基质中亦出现AGEs蛋白的阳性表达,但均散在且淡染。糖尿病大鼠皮肤组织S期表皮细胞百分数(4.83±2.16)%与正常大鼠(5.01±2.47)%相比,差异无统计学意义(P>0.05),但进入G2/M期表皮细胞百分数(0.80±0.62)%比正常大鼠(2.86±1.10)%明显减少(P<0.05),而原代培养的表皮细胞经150μg/ml AGE-HSA干预48 h后,S期和G2/M期细胞比例均显著低于对照组(P<0.05)。ECV304细胞在12.5、25及50μg/ml AGE-HSA作用48 h后,细胞凋亡百分率与对照组比较仍无显著性差异,而在100或200μg/ml AGE-HSA作用6、12、24及48 h后,各时相点的细胞凋亡百分率明显高于对照组(P均<0.01),且呈时间和剂量依赖性。结论糖尿病皮肤组织局部高糖和AGEs等毒性物质蓄积所致的皮肤组织自身的细胞或基质功能不良,是糖尿病皮肤损害的重要机制之一。

Pathophysiological changes of dermal tissue in diabetic rats and their mechanisms

Objective To investigate pathophysiological changes of dermal tissue in diabetic rat and their mechanisms.Methods Sixteen 8-week-old male Sprague-Dawley(SD) rats were randomized into two groups: rats in DM group were intraperitoneally injected with streptozotocin(STZ) at a dose of 65 mg/kg;rats in NC group were injected with the same volume of citric acid buffer.All rats were sacrificed at week 12 after injection,blood and dermal tissue samples were collected.The histological changes in dermal tissue were observed,skin thickness and epidermal cell cycle were measured,the skin glucose concentration and advanced glycation end products(AGEs) were determined.Plasma glycosylated protein(GSP),malondialdehyde(MDA) and glutathione(GSH) levels were evaluated.The cultured epidermal cells and ECV304 cells were exposed to AGE-modified human serum albumin(AGE-HSA) and the effects were evaluated.Results The diabetic rats exhibited changes in skin tissue,including a decrease in thickness,disappearance of the multilayer epithelium structure,degeneration of collagen fibers,and an increase in infiltration of inflammatory cells,in addition to a significant increase in skin glucose and AGEs.Moreover,diabetic rats had increased plasma GSP and MDA,decreased plasma GSH levels.There were no differences in S phase percentage of epidermal cells between DM group(4.83±2.16)% and NC group(5.01±2.47)%(P>0.05).In contrast,there was a marked decrease in G2/M phase in DM group compared to control animals (0.80±0.62)% vs(2.86±1.10)%,P<0.05].After exposure to 150 μg/ml AGE-HSA for 48h,the percentage of epidermal cell in both phases was decreased(P<0.05).Exposure of ECV304 cells to 100 or 200μg/ml AGE-HSA for 6,12,24 or 48h led to a time-dependent and dose-dependent increase in apoptosis rate compared to the control group(P<0.01).Conclusion The high glucose in the skin tissue,coupled with the accumulation of toxic substances such as AGEs,results in the dysfunction of dermal cells and/or the matrix.This might be the mechanism of diabetes-induced early-stage endogenous skin damage.