不同纯度的大鼠胰岛中干细胞标记物细胞角质蛋白19及胰十二指肠同源盒基因的表达

Expression of stem cell marker CK-19 and PDX-1 in different islets purity in rats

目的探讨不同纯度的大鼠胰岛中干细胞标记物细胞角质蛋白19(CK-19)及胰十二指肠同源盒基因1(PDX-1)的表达。方法将30只雄性SD大鼠随机平均分成3组,均采用胶原酶Ⅴ型通过胰管对胰腺进行灌注,切取胰腺,剪碎,吹打、消化、离心获得胰岛细胞沉淀物。A组:胰岛沉淀物不进行纯化;B组:胰岛沉淀物中加入25%的Ficoll液纯化;C组:胰岛沉淀物依次加入25%及11%的Ficoll液纯化。应用免疫组织化学、逆转录聚合酶链反应(RT-PCR),分析不同纯度胰岛中干细胞标记物CK-19及PDX-1蛋白和mRNA的表达。结果采用不同的纯化方法后,A、B、C3组获得胰岛的纯度不同,3组间比较,差异有统计学意义(P<0.05)。免疫组化和RT-PCR分析A、B、C3组中CK-19及PDX-1的蛋白和mRNA的表达,A组中CK-19及PDX-1蛋白和mRNA的表达明显高于B、C组,而C组表达最弱。结论不同纯度的胰岛中均有CK-19及PDX-1蛋白和mRNA的表达,表明在纯化胰岛中均有干细胞存在。

[Objective] To investigate expression of stem cell marker CK-19 and PDX-1 in the different islets purity in rats. [Methods] 30 male SD rats were randomly divided into 3 groups. To obtain sediment of islets, pan- creas was infused by pancreatic duct injection of collagenase and digested, then centrifuged. Group A： Sediment of islets was not purified; Group B： Sediment of islets was purified with Ficoll （25%） density gradient; Group C： Sediment of islets was purified with Ficoll （25%, 1 I%） density gradient. In the different islets purity, expression of pro- tein and mRNA of stem cell marker CK-19 and PDX-1 which were analyzed by immunohistochemlstry and RTPCR. [Results] Three different islets purity were produced by distinct purification methods. It was observed statistcally significance in each group （P 〈0.05）. Expression of protein and mRNA, it were predominantly expressed in group A, but poorly expressed in group C. [Conclusion] The protein and mRNA of CK-19 and PDX-1 are all expressed in different purity of islets, indicting that there are stem cells in the purified islets.