Expression of cytokeratin-19 as a stem cell marker in different-purity islet samples of rats

Chuang YANG; Jiming WANG; Chengyou DU; Yanjun HUANG; Dong XUE

ABSTRACT

BACKGROUND:

Pancreatic stem cells can be differentiated into endocrine functioning cells under a suitable cultivation condition, suggesting that pancreatic stem cells can be used as a new source of islet in treating diabetes mellitus.

OBJECTIVE:

To discuss the expression of stem cell in different purities of pancreatic islets.DESING A controlled observation based on cells.

SETTING:

Third People's Hospital of Mianyang City.MATERIALS The experiment was conducted in the Center Laboratory (Key Laboratory of Chongqing City), the First Affiliated Hospital of Chongqing Medical University from March 2005 to March 2006. Thirty healthy male SD rats, of clean grade, aged 50 days were used in this study. During the experiment, the disposal of animals corresponded to Animal Ethical Standard. Cytokeratin (CK)-19 and β-actin were designed by Shanghai Invitrogen Biological Co., Ltd.Brdu reagent, mouse anti-rat Brdu monoclonal antibody, rabbit anti-mouse (RAM) CK-19 polyclonal antibody and Brdu immunohistochemical kit were from Boster Co., Ltd. (Wuhan); HistostainTM -DS double immunohistochemical staining kit was purchased from Beijing Zhongshan Company.

METHODS:

Rats were intraperitoneally injected with Brdu for labeling, and subjected to perfusion with Ⅴ-type collagenase via pancreatic ducts, with pancreas being excised, ripped, beaten up, digested and centrifuged to obtain pancreatic islet sediments. After being precipitated, the sediments were divided into 3 groups. Group A The isolated sediments of islets were not purified; Group B The sediments were added slowly with 25% Ficoll-400 and Hanks solution for purification; Group C The sediments were slowly added with 25% Ficoll-400, 11% Ficoll-400 for purification in order.MAIN OUTCOME

MEASURES:

Expressions of Brdu and CK-19-positive cells in the islet samples were detected by immunohistochemical method, and mRNA expression of CK-19 in the different purities of islets was detected by reverse transcription-polymerase chain reaction.

RESULTS:

① The purity of islets in the group A was the lowest. The purity of islets obtained by different purification techniques showed significances among the three groups (F =89.42, P ＜ 0.05). ②Positive expressions of Brdu and CK-19 in the group A were significantly higher than those in the group B and group C (F =18.64, 22.12, 38.61. P ＜0.01). ③Expression of CK-19 mRNA in the group A was significantly higher than that in other groups, with group C showing the lowest.

CONCLUSION:

Expressions of Brdu and CK-19-positive cells exist in different purities of islets, suggesting the existence of more stem cells in the low purify of islets.