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背景:胰腺中存在的干细胞在合适的培养条件下可分化为具有内分泌功能的细胞,可以作为一种新的胰岛来源治疗糖尿病。目的:分析大鼠胰岛纯化程度与其中所携带的干细胞数量的关系。设计:以细胞为观察对象,对比实验。单位:绵阳市第三人民医院材料:实验于2005-03/2006-03在重庆医科大学附属一院中心实验室完成。选取30只清洁级健康雄性SD大鼠,鼠龄50 d。实验过程中对动物的处置符合动物伦理学标准。细胞角蛋白19和β-actin引物由上海英俊生物有限公司设计。Brdu试剂、小鼠抗大鼠Brdu单克隆抗体、兔抗鼠CK-19多克隆抗体、Brdu免疫组织化学试剂盒购于武汉博士德生物工程有限公司。Histostain~(TM)-DS免疫组织化学双染试剂盒购于北京中杉公司。方法:大鼠腹腔注射5-溴脱氧尿嘧啶核苷(Brdu)标记,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。离心后沉淀物进行如下处理:即,不纯化组:胰岛沉淀物不进行纯化;高密度介质纯化法组:胰岛沉淀物中加入体积分数为25%的Ficoll液纯化;两种不同密度梯度纯化法组:胰岛沉淀物依次加入25%及11%的Ficoll液纯化。主要观察指标:应用免疫组织化学法检测胰岛样本中Brdu及细胞角蛋白19阳性细胞的表达,应用反转录聚合酶链反应检测不同纯度胰岛中细胞角蛋白19 mRNA的表达。结果:①胰岛纯度:不纯化组最低,3组获得的胰岛细胞纯度比较,差异有显著性意义(F=89.42,P<0.05)。②胰岛样本中Brdu和细胞角蛋白19阳性细胞的表达:不纯化组的阳性表达高于高密度介质纯化法组和两种不同密度梯度纯化法组,差异有显著性意义(F=18.64,22.12,38.61,P<0.01)。③胰岛中细胞角蛋白19 mRNA的表达:不纯化组最强,两种不同密度梯度纯化法组最弱。结论:不同纯度的胰岛中有Brdu及细胞角蛋白19阳性细胞,在低纯度胰岛中含有较多的干细胞。
BACKGROUND: Stem cells present in the pancreas can differentiate into endocrine-competent cells under suitable culture conditions and can be used as a new source of islets to treat diabetes. OBJECTIVE: To analyze the relationship between the degree of islet purification and the number of stem cells carried in it. Design: Cells for the observation object, comparative experiment. Unit: Mianyang Third People's Hospital Materials: The experiment was performed at the Central Laboratory of the First Affiliated Hospital of Chongqing Medical University from March 2005 to March 2006. Thirty healthy male Sprague-Dawley (SD) rats were selected and aged 50 days. The handling of animals during the experiment is in line with animal ethics standards. Cytokeratin 19 and β-actin primers were designed by Shanghai Handsome Biological Co., Ltd. Brdu reagent, mouse anti-rat Brdu monoclonal antibody, rabbit anti-mouse CK-19 polyclonal antibody, Brdu immunohistochemical kit were purchased from Wuhan Boster Biological Engineering Co., Histostain ~ (TM) -DS immunohistochemical double staining kit was purchased from Beijing Zhongshan company. Methods: Rats were intraperitoneally injected with Brdu. All the rats were perfused with collagenase V through the pancreatic duct and the pancreas was excised. The pancreatic islets were excised and the islets were pelleted, washed, digested and centrifuged to obtain islet cell pellets. After centrifugation, the sediment was treated as follows: that is, the impure group: the islet sediment was not purified; the high-density medium purified method group: the islet sediment was added with the Ficoll volume fraction of 25%; two different density gradient purification methods Group: Islet sediment followed by 25% and 11% Ficoll fluid purification. MAIN OUTCOME MEASURES: The expression of Brdu and cytokeratin 19 positive cells were detected by immunohistochemistry. The expression of cytokeratin 19 mRNA in different purity islets was detected by reverse transcription polymerase chain reaction. Results: ① The purity of pancreatic islets was the lowest in the non-purified group. The purity of islet cells in the three groups was significantly different (F = 89.42, P <0.05). ② The expression of Brdu and cytokeratin 19 positive cells in islet samples: The positive expression of Brdu and cytokeratin 19 in implanted samples was higher than that in high-density media purification and two different density gradient purification methods (F = 18.64,22.12 , 38.61, P <0.01). ③ The expression of cytokeratin 19 mRNA in islets: the strongest in the impure group, and the weakest in the two different density gradient purification groups. Conclusion: There are Brdu and cytokeratin 19 positive cells in different purity islets and more stem cells in low purity islets.