单纯酶和复合酶消化液对成猪胰岛细胞分离的作用及其机制

Effects and mechanism of single enzyme and compound enzyme digestive juice on pancreatic islet cell isolation of adult pigs

背景:近来,从大动物获得大量存活的胰岛细胞技术已经日趋成熟,并且已经应用到了临床。然而大量研究表明,同小动物相比,猪胰岛细胞的收获量,还是相对过低,究其原因,是否与猪胰腺外分泌组织生化成分有关?此研究旨在为糖尿病患者提供可靠的胰岛细胞来源。目的:探讨单纯酶和复合酶消化液对成猪胰岛细胞分离的作用及其机制,为糖尿病患者的二级康复措施介入提供理论依据。设计:以动物为研究对象,随机对照实验研究。单位:一所大学医院的普外科和老年病科。材料:实验于2003-06/2004-05在哈尔滨医科大学附属第二医院完成。哈尔滨市哈达屠宰厂提供成年杂种活猪20头,猪龄12～24个月,体质量100kg左右。干预:屠宰放血后在相对无菌情况下,仔细取出胰腺脾部放在冷RP-MI1640液内,迅速送到实验室。快速去除胰周组织,脂肪、血管等。用1∶5000洗必泰浸泡3min,然后用冷RPMI1640液冲洗,4℃条件下在平皿中用眼科剪剪成1mm×1mm×1mm左右的碎片,均分成两份,分别放入含有单纯酶溶液和和复合酶溶液的锥形瓶中,分级消化获得胰岛细胞。主要观察指标:经双硫腙染色计数胰岛细胞,并做椎虫蓝染色测定胰岛细胞活性,胰岛素释放试验观察胰岛细胞的功能;电子显微镜对胰岛细胞团检查,检验其结构的完整性。结果:复合酶消化制备物在平均产量上?

BACKGROUND:Recently,the technique of isolation procedure of viable islets cell from large animals has been mature and used in clinical islet cell transplantation.Some study results indicate that low yields of pig islet cell isolations is probably not due to the presence of higher or more aggressive enzyme activities during the porcine isolation procedure.Therefore,the causative factors resulting in the inconsistent results should be sought for the intrinsic properties of the pancreatic islet and pancreas.The purpose of the study is to get more pancreatic islet cells for diabetic patients.OBJECTIVE:To investigate the effect of single enzymatic solution (SES) and multi enzymatic solution(MES) on adult porcine pancreatic islet cell isolation,and to provide the theory base for diabetic patients rehabilitation. DESIGN:A completely randomized and controlled experiment based on animals.SETTING:General surgery and gerontology department in a university hospital.MATERIALS:The experiment was conducted in the Second Affiliated Hospital of Harbin Medical University from June 2003 to May 2004.Twenty consecutive random bred adult pigs,12- 24 months old,weighing average 100 kg,were selected for experiment from Hada Slaughterhouse of Harbin.INTERVENTIONS:After slaughtering,the spleentic lobe of the pancreas was excised using sterile surgical gloves and surgical instruments.Each was immediately transported in 500 mL of sterile RPMI1640 solution at 4 ° C to the laboratory for processing.After removal of fat peritoneum and superficial blood vessel,each pancreas was first immersed in 1∶ 5 000 Liquor chlorhexidine for 3 minutes, then washed three times with cold RPMI1640,next mechanically minced into 1 mm × 1 mm × 1 mm fragments,weighed,and equally divided into two groups.Adult porcine islets were isolated with two different collagenase solutions(SES and MES). MAIN OUTCOME MEASURES:Islet count was performed with DTZ staining.The viability was assessed by trypan blue staining. Porcine islet insulin secretory function was assessed by insulin content of cultured porcine islets and by insulin release.Islet morphological integrity was finally established by electron microscope examination.RESULTS:Overall islet cell content were statistically different between the two methods[(1 782± 427) IE/g vs (1 293± 451) IE/g,P< 0.05]. No significant difference was found in viability, function or morphology of islet between MES and SES(P >0.05).CONCLUSION:An average of islet isolated with MES method represents a uniquely massive yield in comparison with that by SES method.A good viability was also confirmed by static incubation and culture.The transplantation of those pancreatic islet cells can help to control the syndromes of diabetes.