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目的探讨大鼠的不同纯度胰岛中干细胞标志物细胞角质蛋白19(CK-19)和胰十二指肠同源盒基因1(PDX-1)mRNA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体积分数为25%的Ficoll-400液纯化;C组胰岛沉淀物依次加入25%及11%的Ficoll-400纯化。分别将上述3组纯化后的胰岛抽提RNA行逆转录聚合酶链反应(RT-PCR),分析胰岛干细胞标志物CK-19及PDX-1mRNA的表达。结果采用不同的纯化方法后,A、B、C三组获得胰岛的纯度分别为(43.6±6.29)%、(65.3±4.40)%和(77.6±6.36)%,三组间比较,差异有统计学意义(P<0.05)。A组胰岛中CK-19和PDX-1mRNA的表达明显高于B组和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-1mRNA的表达,表明在纯化胰岛中均有干细胞存在。
Objective To investigate the expression of stem cell marker cytokeratin 19 (CK-19) and pancreatic duodenal homeobox 1 (PDX-1) mRNA in rat pancreatic islets with different purity. Methods Thirty male Sprague-Dawley rats were randomly divided into three groups randomly. All the rats were perfused with pancreatic duct by type Ⅴ collagenase and the pancreas was excised. The pancreatic islets were cut, sheared, blow, digested and centrifuged to obtain islet cell pellets. A group of islet sediment was not purified; group B islet volume fraction of 25% added Ficoll-400 liquid purification; C group pancreatic islet followed by 25% and 11% Ficoll-400 purification. The above 3 groups of purified islet RNA were respectively analyzed by reverse transcription-polymerase chain reaction (RT-PCR) to analyze the expression of islet stem cell markers CK-19 and PDX-1mRNA. Results The purities of islets obtained in groups A, B and C were (43.6 ± 6.29)%, (65.3 ± 4.40)% and (77.6 ± 6.36)%, respectively, with different purification methods. The differences among the three groups were statistically significant Significance (P <0.05). The expression of CK-19 and PDX-1 mRNA in group A was significantly higher than that in group B and C, while the expression in group C was the weakest. Conclusion The expression of CK-19 and PDX-1 mRNA in islets of different purity shows that all the stem cells exist in the purified islets.