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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。
Purpose Epidermal stem cells (ESCs) can actively participate in the repair of wounds and promote the re-epithelization of wounds. To establish a rat model of diabetes mellitus (DM) and observe the proliferation and differentiation-related proteins of keratin 19 (K19), β1-integrin, β-catenin (β- catenin and proliferating cell nuclear antigen (PCNA) in diabetic rats. Methods Twenty male SD rats weighing 250-300 g were randomly divided into DM group and normal control group, with 10 rats in each group. Rats in DM group were given a single intraperitoneal injection of streptozocin (STZ, 65 mg / kg) to prepare a DM rat model, and the normal control group was not treated. Four weeks after the administration, the pancreatic tissues of two groups were taken and the histological changes of islets were observed by HE staining. The whole skin of the back of the animals was taken from the two groups, and ESCs were isolated and cultured. The second generation of ESCs was taken for immunocytochemical staining to identify the positive expression of K19, β1-integrin, β-catenin and PCNA. The cell cycle was detected by flow cytometry. After the detection of two groups of cell clone formation rate. Results The success rate of modeling in DM rats was 90%. The number of pancreatic islet cells in DM group was significantly decreased, and the necrotic cells were necrotic. The islet cells in normal control group had clear structure without necrosis. The positive expressions of K19, β1-integrin, β-catenin and PCNA in DM group were 82.63 ± 14.77, 21.59 ± 4.71, 76.49 ± 6.58 and 90.77 ± 12.44, respectively, which were all lower than 151.24 ± 42.83 and 54.48 ± 17.43,116.39 ± 9.26,110.62 ± 20.67, the differences were statistically significant (P <0.01). The formation rate of ESCs in DM group was 6.43% ± 1.01%, which was significantly lower than that in normal control group (11.37% ± 1.62%, P <0.01). Flow cytometry analysis showed that 88.89% of cells were in G0 / G1 phase in DM group, the number of apoptotic cells was 3.98%; 91.50% of cells in normal control group were in G0 / G1 phase without apoptotic cells. Conclusion One-time intraperitoneal injection of 65mg / kgSTZ can effectively establish a DM rat model. The small number of ESCs in DM rats and the low proliferative and differentiation ability may be one of the most important mechanisms of refractory DM wound healing.