胶原酶浓度对大鼠胰岛分离纯化产量的影响

目的观察胶原酶浓度对大鼠胰岛分离纯化产量的影响,筛选出获取高产量胰岛的最佳消化液的胶原酶浓度。方法 75只雄性SD大鼠按胶原酶浓度分为5组：Ⅰ组（0.5 mg/ml）、Ⅱ组（0.75 mg/ml）、Ⅲ组（1.0 mg/ml）Ⅳ组（1.2 mg/ml）和Ⅴ组（1.5 mg/ml）,每组15只。通过胆灌注胶原酶V来消化大鼠胰腺,分离胰岛,采用不连续密度梯度Ficoll离心法纯化胰岛,根据胰岛直径计数所获胰岛的数量及胰岛当量,并利用葡萄糖刺激胰岛素释放试验评估胰岛功能。结果分离的大鼠胰岛具有较好的质量,胰岛纯度为95%左右,活性率为90%左右,体外培养生长良好。随着胶原酶的浓度增加,胰岛数量和胰岛当量明显增加（P〈0.05）,并且最大直径的胰岛数量也在增加（P〈0.05）,在胶原酶浓度1.0 mg/ml时获得的胰岛产量最高,其次为1.2 mg/ml,在其他浓度时,胰岛产量都较低（P〈0.05）。在低糖和高糖刺激下胰岛素的释放分别为（1.346±0.128）ng/ml和（2.565±0.208）ng/ml,差异具有显著性意义（P〈0.05）,刺激指数为（1.908±0.152）,提示胰岛细胞功能良好,电镜照片显示胰岛生长状况良好。结论进行大鼠胰岛分离纯化时最适宜的胶原酶浓度为1.0~1.2 mg/ml,可获得稳定高产量的胰岛,过低和过高的浓度都是不适宜的。

The Effect of Collagenase Concentration on Purification Yield Of Rat Islets

Objective To investigate the effect of collagenase concentration on the yield of isolation and purification of pancreatic islets in rats.Methods The male SD rats were randomly divided into five groups according to the collagenase concentration,groupⅠ（0.5 mg/ml）,group Ⅱ（0.75 mg/ml）,group Ⅲ（1.0 mg/ml）,groupⅣ（1.2 mg/ml） and groupⅤ（1.5 mg/ml）,（n = 15,each group）.Collagenase V was injected into the common bile duct of the rat for pancreas digestion.The pancreatic islets were isolated and purified by using discontinuous Ficoll density gradient centrifugation.The pancreatic islets were identified by dithizone staining,and the viability was assessed by acridine orange and propidium iodide staining.The yield of pancreatic islets was calculated by islet equivalent according to islets diameter.Electron microscopy examination was performed to investigate rat islet morphological character and islet function was measured by glucose-evoked insulin release.Results The isolated pancreatic islets showed excellent quality and grew well in vitro.The purity and viability was about 95% and 90% respectively.The yield of pancreatic islets and pancreatic islet equivalent increased significantly when the collagenase concentration increased（P 0.05）.Highest yield of islets and islet equivalent were found in the groups with the concentration of 1.0 mg/ml and 1.2 mg/ml（P 0.05）.Electron microscopy studies showed the islets without morphological change.The release of insulin stimulated by low and high concentration of glucose were（1.346 ± 0.128） ng/ml and（2.565 ± 0.208） ng/ml respectively.The results had significant difference between those groups（P 0.05） and the stimulation index was（1.908 ± 0.152）,suggesting the islet cells function was well.Conclusions To obtain high yield islets in rat,the best concentration of collagenase applied in digestion solution is 1.0 ~ 1.2 mg/ml.