糖尿病大鼠表皮干细胞及其增殖分化相关蛋白的研究  

STUDY ON RELATED PROTEINS OF PROLIFERATION AND DIFFERENTIATION OF EPIDERMAL STEM CELLS IN DIABETIC RATS

作　　者：钟清玲[1] 刘德伍[2] 刘繁荣[3] 彭燕[2] 张向荣[2] 蓝蔚[2] 

机构地区：[1]南昌大学医学院基础护理教研室,南昌330006 [2]南昌大学第一附属医院烧伤研究所 [3]南昌大学医学院病理教研室,南昌330006

出　　处：《中国修复重建外科杂志》2010年第2期133-137,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30560058);江西省科技厅重大科技招标项目(200604)~~

摘　　要：目的表皮干细胞(epidermal stem cells,ESCs)能主动参与创面修复,促进创面再上皮化。建立糖尿病(diabetes mellitus,DM)大鼠模型,观察DM大鼠皮肤中ESCs增殖分化相关蛋白——角蛋白19(keratin19,K19)、β1整合素(β1-integrin)、β-连环素(β-catenin)及增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达,探讨DM大鼠皮肤创面难愈合的潜在机制。方法20只雄性SD大鼠,体重250～300g,随机分为DM组和正常对照组,每组10只。DM组大鼠一次性腹腔注射链脲佐菌素(streptozocin,STZ,65mg/kg)制备DM大鼠模型,正常对照组不作处理。给药后4周取两组大鼠胰腺组织,HE染色观察胰岛组织学变化。取两组动物背部全层皮肤,分离培养ESCs,取第2代ESCs行免疫细胞化学染色鉴定K19、β1-integrin、β-catenin和PCNA阳性表达,流式细胞仪检测细胞周期,细胞接种1周后检测两组细胞克隆形成率。结果DM大鼠造模成功率为90%。DM组胰腺HE染色可见胰岛细胞数明显减少,出现变性坏死;正常对照组胰岛细胞结构清楚,无变性坏死。DM组大鼠ESCs的K19、β1-integrin、β-catenin及PCNA阳性表达分别为82.63±14.77、21.59±4.71、76.49±6.58、90.77±12.44,均低于正常对照组的151.24±42.83、54.48±17.43、116.39±9.26、110.62±20.67,差异均有统计学意义(P<0.01)。DM组ESCs克隆形成率为6.43%±1.01%,明显低于正常对照组的11.37%±1.62%,差异有统计学意义(P<0.01)。流式细胞仪分析显示DM组88.89%细胞处于G0/G1期,凋亡细胞数为3.98%;正常对照组91.50%细胞处于G0/G1期,无凋亡细胞。结论通过腹腔一次性注射65mg/kgSTZ可有效建立DM大鼠模型。DM大鼠ESCs数量少、增殖分化能力低可能是DM创面难愈合的重要机制之一。Objective Epidermal stem cells（ESCs）can actively participate in wound healing and enhance reepithelialization.To establish ideal diabetes mellitus（DM）rat models and to investigate the expression of keratin 19（K19）,β1-integrin,β-catenin,and proliferating cell nuclear antigen（PCNA）in ESCs of DM rat model,then to study the potential mechanism of difficult recovering wounds of diabetic skin.Methods Twenty male SD rats（weighing 250-300 g）were divided into DM group and normal control group randomly（n=10）.The DM rat model was made by intraperitoneal injected 65 mg/kg streptozocin（STZ）,the normal control group was not treated.At 4 weeks after injection,pancreatic tissue was harvested for HE staining in two groups.The ESCs isolated from full-thickness skins of the back of two group rats were culutured and identified.The 2nd passage of ESCs were obtained for immunocytochemical staining of K19,β1-integrin,β-catenin,and PCNA.Meanwhile,the cell cycle were measured by flow cytometry.The cell colony formation rates were detected after 1 week.Results The achievement ratio of DM rat model was 90%with good stability.HE staining showed that the number of islet cells significantly decreased with degeneration and necrosis in DM group;the structure of islet cell was clear without degeneration and necrosis in normal control group.The integral absorbance values of positive expression for K19,β1-integrin,β-catenin,and PCNA in ESCs of DM group（82.63±14.77,21.59±4.71,6.49±6.58,and 90.77±12.44,respectively）were significantly lower than those in the normal control group（151.24±42.83,54.48±17.43,116.39±9.26,and 110.62±20.67,respectively）（P0.01）.The clone forming efficiency of ESCs in DM group（6.43%±1.01%）was significantly lower than that in the normal control group（11.37%±1.62%）（P0.01）.Flow cytometry indicated that 88.89%of cultured ESCs in the DM group were in resting state/pre-DNA-synthetic gap（G0/G1）,and the apoptosis rate was 3.98%;91.50%in the normal control group an

关 键 词：表皮干细胞 糖尿病大鼠模型 角蛋白19 Β1整合素 Β-连环素 增殖细胞核抗原大鼠 

分 类 号：R587.2[医药卫生—内分泌] R332[医药卫生—内科学]