上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (5): 567-.doi: 10.3969/j.issn.1674-8115.2012.05.007

• 论著（基础研究） • 上一篇    下一篇

三种消化酶分离胰岛对移植胰岛存活的影响

刘定志, 杜成友, 罗诗樵, 肖 衡, 潘 龙   

  1. 重庆医科大学附属第一医院肝胆外科, 重庆 400016
  • 出版日期:2012-05-28 发布日期:2012-06-01
  • 通讯作者: 杜成友, 电子信箱: duchengyou@126.com。
  • 作者简介:刘定志（1983—), 男, 硕士生；电子信箱: liudingzhi123_0@163.com。
  • 基金资助:

    国家自然科学基金（30972945）；重庆市科委基金（CSTC2007BB5263）；渝卫科教基金（06-2-016）

Influence of three digestive enzymes for islet isolation on survival of islet grafts

LIU Ding-zhi, DU Cheng-you, LUO Shi-qiao, XIAO Heng, PAN Long   

  1. Department of Hepatobiliary Surgery, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
  • Online:2012-05-28 Published:2012-06-01
  • Supported by:

    National Natural Science Foundation of China, 30972945；Chongqing Science and Technology Committee Foundation, CSTC2007BB5263；Chongqing Health Science and Education Foundation, 06-2-016

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摘要：

目的 比较三种消化酶分离胰岛的效率和质量及对移植胰岛存活的影响，为临床胰岛移植消化酶的选取提供参考。方法 相同条件下采用Liberase TL （A1组）、Collagenase P （B1组）和CollagenaseⅤ（C1组）（n=35）消化酶消化小鼠胰腺，Ficoll-400密度梯度纯化获得胰岛；DTZ染色观察胰岛细胞形态，并计算IEQ(直径>150 μm的胰岛细胞团定义为1个IEQ)和纯度；锥虫蓝染色检测胰岛细胞成活率。C57BL/6糖尿病（DM）小鼠作为受体分为A2、B2和C2三组（n=7），采用同基因胰岛肾被膜下移植，监测术后血糖浓度变化；于移植成功后15 d切取移植物，HE染色观察组织学变化。结果 3种消化酶分离C57BL/6小鼠胰腺可获得的IEQ、胰岛纯度和胰岛细胞成活率均为A1组＞B1组＞C1组，三组间两两比较差异均有统计学意义（P<0.05）。每只受体小鼠移植（900±30）个IEQ胰岛；移植前各组小鼠血糖浓度比较差异均无统计学意义（P>0.05）；植术后第2天血糖浓度达最低值，第3天有一个短暂的回升，随后达到一个相对较平稳的状态，至第13天血糖浓度依次为A2组＜B2组＜C2组，组间比较差异均有统计学意义（P<0.05）。HE染色显示A2组胰岛形态完整，胞质、胞核区分明显，存活状态良好，炎症浸润不明显；B2组呈圆形或椭圆形，胞质淡染，多核，炎症细胞浸润较A2组多；C2组炎症细胞浸润明显，伴胰岛破坏，形态欠规则。结论 与Collagenase P和CollagenaseⅤ比较，Liberase TL消化小鼠胰腺可提高胰岛细胞团的收获量、活性和功能。

关键词: 胰岛移植, 同基因, 释放酶, 胶原酶, 小鼠

Abstract:

Objective To compare the efficiency and quality in islet isolation and survival of islet grafts among three digestive enzymes for islet isolation so as to provide reference for enzyme selection in clinical islet transplantation. Methods Under the same conditions, digestive enzymes of Liberase TL (A1 group), Collagenase P (B1 group) and Collagenase Ⅴ(C1 group)   were used for pancreas digestion in mice, with 35 mice in each group. Ficoll-400 density gradient method was adopted for islet purification. The morphology of islet cells was observed by DTZ staining, and IEQ (islet cell cluster with diameter>150 μm) and purification of islet cells were calculated. The survival of islet cells was determined by trypan blue staining. C57BL/6 diabetic mice were served as receptors, and were randomly divided into A2 group, B2 group and C2 group, with 7 mice in each group. Syngenetic islet transplantation under renal capsule was performed, and changes of blood glucose were monitored after operation. The graft samples were obtained 15 d after transplantation, and histological changes were observed with HE staining. Results The quantity of IEQ, purity of pancreas and survival of islet cells in A1 group were the highest, those in C1 group were the lowest, and there were significant differences among groups (P<0.05). Each receptor mouse obtained (900±30) IEQ islets, and there was no significant difference in blood glucose among groups before transplantation (P>0.05). The blood glucose was the lowest 2 d after transplantation, increased on the third day, then maintained at a relatively stable state, and on the thirteenth day, the blood glucose in A2 group was lower than that in B2 group, while the blood glucose in C2 group was higher than that in B2 group, with significant differences among groups (P<0.05). HE staining revealed that the appearance of islet in A2 group was intact, the cytoplasm and nucleus were clearly discriminated, the survival status was favorable, and the inflammatory infiltration was not significant. The appearance of islet in B2 group was round or oval, the cells were multinuclear, with understained cytoplasm, and the inflammatory cell infiltration was more significant than that in A2 group. The inflammatory cell infiltration was significant in C2 group, with damaged islet and irregular morphology. Conclusion Compared with Collagenase P and Collagenase Ⅴ, Liberase TL can improve the quantity, activity and function of islet cell clusters in pancreas digestion in mice.

Key words: islet transplantation, syngeneic, Liberase, Collagenase, mouse