间充质干细胞包裹人胰岛减轻即刻经血液介导的炎症反应的体外研究

In vitro research of mesenchymal stem cell-coated human islets to alleviate instant blood-mediated inflammatory reaction

目的探讨间充质干细胞(MSC)包裹胰岛对胰岛移植即刻经血液介导的炎症反应(IBMIR)的影响。方法将经示踪剂标记的MSC与人胰岛放入超低吸附培养皿,间隔0.5 h吹打混匀2次,之后置37℃、5%CO_(2)条件下孵育培养24 h,实现MSC包裹胰岛,并检测MSC包裹胰岛的效果及胰岛的体外功能。体外构建血液循环管型模型,空白对照组加入0.2 mL胰岛培养液,胰岛组加入800胰岛当量未包裹的胰岛,MSC包裹组加入800胰岛当量MSC包裹的胰岛,37℃条件下循环60 min。在0、30、60 min 3个时间点各取0.5 mL血液进行血常规检测。循环60 min后将血液用70μm滤网过滤,收集血浆、血凝块及胰岛。血凝块及胰岛进行苏木素-伊红(HE)染色免疫组织化学(免疫组化)染色,观察组织形态学改变及CD11b阳性细胞在胰岛周围聚集情况。使用酶联免疫吸附试验检测血浆中凝血酶-抗凝血酶复合物(TAT)、组织因子(TF)、C3a、C5b-9、白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α、单核细胞趋化蛋白(MCP)-1、IL-8含量。结果MSC和胰岛共同孵育24 h后胰岛可以被MSC包裹,包裹程度约80%。胰岛组和MSC包裹组血凝块和胰岛周围均存在较多中性粒细胞和单核细胞,MSC包裹组较胰岛组CD11b阳性细胞少。与全血共孵育0、30、60 min后,MSC包裹组和胰岛组血小板、中性粒细胞和单核细胞数量均下降,且随着时间延长逐渐减少。与空白组比较,MSC包裹组血小板、单核细胞、中性粒细胞数量较少,TF含量较高;与胰岛组比较,MSC包裹组血小板、单核细胞、中性粒细胞数量较多,TAT、TF含量较低,差异均有统计学意义(均为P<0.05)。与空白组比较,MSC包裹组C3a、C5b-9、IL-6、TNF-α、IL-8表达水平较高;与胰岛组比较,MSC包裹组C3a、C5b-9、IL-1β、IL-6、TNF-α、IL-8、MCP-1表达水平较低,差异均有统计学意义(均为P<0.05)。结论MSC包裹胰岛可通过减少胰岛TF在血液中的暴露,在凝血反应阶段减轻IBMIR,从而减少移植早期胰岛移植物的损伤和丢失。

Objective To evaluate the effect of mesenchymal stem cell(MSC)coated-islets on instant blood-mediated inflammatory reaction(IBMIR)after islet transplantation.Methods MSC labeled with tracer and human islets were placed into an ultra-low adsorption cell culture dish,shaken and mixed twice at an interval of 0.5 h,and then incubated at 37℃and 5%CO_(2)for 24 h to obtain MSC-coated islets.The coating effect of MSC and in vitro function of the islets were assessed.A blood circulation tube-shaped model was established in vitro.In the blank control group,0.2 mL of islet culture solution was added.In the islet group,800 islet equivalent quantity(IEQ)of uncoated islets were supplemented.In the MSC-coated islets group,800 IEQ of MSC-coated islets were added,and circulated for 60 min at 37℃.A portion of 0.5 mL blood sample was taken for routine blood test at 0,30 and 60 min,respectively.After 60 min circulation,the blood sample was filtered with a 70μm filter to collect plasma,blood clots and islets.Blood clots and islets were subject to hematoxylin-eosin(HE)staining and immunohistochemical staining.Morphological changes and the aggregation of CD11b-positive cells surrounding the islets were observed.The contents of plasma thrombin-antithrombin complex(TAT),tissue factor(TF),C3a,C5b-9,interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,monocyte chemoattractant protein(MCP)-1 and IL-8 were determined by enzymelinked immune absorbent assay.Results After 24 h co-incubation,the islets were coated by MSC,with a coating degree of approximately 80%.In the islet and MSC-coated islet group,a large quantity of neutrophils and monocytes were observed surrounding the blood clots and islets,and the quantity of CD11b-positive cells in the MSC-coated islet group was less compared with that in the islet group.After co-incubation with the whole blood for 0,30 and 60 min,the quantity of platelets,neutrophils and monocytes was declined in the MSC-coated and islet groups,and gradually decreased over time.Compared with the blank control group,the quantity of platelets,monocytes and neutrophils was lower,whereas the TF content was higher in the MSCcoated islet group.Compared with the islet group,the quantity of platelets,monocytes and neutrophils was higher,whereas the TAT and TF contents were less in the MSC-coated islet group,the differences were statistically significant(all P<0.05).Compared with the blank control group,the expression levels of C3a,C5b-9,IL-6,TNF-αand IL-8 were up-regulated in the MSC-coated islet group.Compared with the islet group,the expression levels of C3a,C5b-9,IL-1β,IL-6,TNF-α,IL-8 and MCP-1 were down-regulated in the MSC-coated islet group,and the differences were statistically significant(all P<0.05).Conclusions MSC-coated islets may reduce the exposure of islet TF in the blood and prevent the incidence of IBMIR during the coagulation response stage,thereby mitigating the injury and loss of islet allograft in the early stage of islet transplantation.