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ProtEx~(TM)外源蛋白修饰技术在移植免疫研究中的作用

Application of ProtEx~(TM):an exogenous FasL protein modification technology in the study of immunomodulation

摘要:

目的 采用ProtEx~(TM)外源蛋白修饰技术将外源蛋白FasL修饰于靶细胞,观察其在移植免疫中的作用.方法 1.将嵌合蛋白FasL(SA-FasL)修饰于大鼠脾细胞,检测蛋白的表达效率及其对脾细胞表面免疫分子的影响;分别以近交系WF大鼠和ACI大鼠作为供者和受者,进行异位心脏移植,按围手术期注射供者细胞的不同将受者分为3组:(1)SA-FasL组(n=23):注射经SA-FasL修饰的供者脾细胞;(2)SA组(n=20):注经链霉亲和素蛋白(SA)修饰的供者脾细胞;(3)Control组(n=10):未注射任何细胞,作为空白对照.术后观察移植心的跳动情况,停止跳动为发生排斥反应,持续有力的跳动超过100d为移植心长期存活.2.以链脲佐菌素(STZ)静脉注射诱导C57BL/6小鼠产生糖尿病;将SA-FasL蛋白修饰于小鼠胰岛;以近交系BALB/c小鼠作为供者,糖尿病C57BL/6小鼠作为受者,进行胰岛移植,根据移植不同的供者胰岛将受者分为组:(1)islet-FasL组(n=21):移植经SA-FasL修饰的胰岛,(2)islet-SA组(n=21):移植经SA修饰的胰岛;(3)islet组(n=14):移植未经修饰的胰岛,作为空白对照.若受者尿糖阳性,并连续2d血糖≥250 mg/ml为移植排斥反应.尿糖阴性超过100d为移植物长期存活.结果 流式细胞术检测FasL蛋白在脾细胞的平均表达率为(94.49±4.27)%,免疫荧光染色显示FasL蛋白呈红色,表达于脾细胞表面;FasL蛋白的表达干扰脾细胞表面分子CD3、CD4、CD8、CD80以及主要组织相容性复合物I类抗原的表达.SA-FasL组70%的受者移植心获得长期存活,SA组仅为25%,两组间的差异有统计学意义(P<0.05).胰岛移植术后30d,islet-FasL组67%的受者移植胰岛功能良好,29%的受者移植胰岛获得长期存活,与其他两组(均为0)比较,差异均有统计学意义(P<0.05).结论 ProtEx~(TM)是简便、快速、安全和高效的外源蛋白修饰技术,过该技术将嵌合蛋白FasL直接表达于靶细胞,将对移植免疫应答发挥重要的调节作用.

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abstracts:

Objective By using ProtEx~(TM) technology to decorate target cells with an exogenous protein and observe its function for immunomodulation.Methods Donor splenocytes were decorated with chimeric SA-FasL protein.The expression of FasL and its impact on other molecules on the surface of splenocytes was detected.Heterotopic rat heart transplantation was performed from inbred WF rat to ACI rat.According to the different donor splenocytes perioperatively injected into recipients,the recipients were divided into 3 groups:SA-FasL group (n=23),SA group (n=20) and control group(n=10).Heart beating was examined by palpation on daily basis,the cessation was regarded as reiection,while surival over 100 days was considered as graft long-term survival.Diabetes models in C57BL/6 mice were induced by intravenous injection of STZ.Islets from BALB/C mice were decorated by SA-FasL protein,islets transplant was performed from BABL/c to C57BL/6 mice.Based on the different treatments of donor islets,recipients were divided into three groups:islet-FasL group (n=21),islet-SA group (n=21) and islet-untreated group(n=14).Rejection was identified while urine glucose was positive and blood glucose value was over 250 mg/ml for consecutive two days.Urine glucose negative over 100 days was regarded as graft long-term survival.Results The mean exDression rate of FasL in splenocytes was 94.49%±4.27% tested by flow cytometry and FasL showed red color 0n the surface of splenocytes by fluorescent stammg.FasL deeoration did not disturb the expression of CD3,CD4,D8,MHC I and CD80.The long-term survival rate o,f heart transplant showed significant difference between tWO groups(SA-FasL:70% vs SA:25%,P<0.05).67% FasL-decorated islets had normal function 30 days post transplant,and 29%FasL-decorated islets achieved long-term survival,which was significantly differed from the other groups(P<0.05).Conclusion ProtEx~(TM) is a simple,fast,safe and efficient approach for exogenous protein modmcation.Decoration of target cells with Chimeric FasL by this technique plays an umportant role in immunomodulatiorn.

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作者: 顾晓 [1] 杨进 [1] 赵鸿 [2]
分类号: R3
栏目名称: 实验研究
DOI: 10.3760/cma.j.issn.0254-1785.2010.03.004
发布时间: 2010-05-26
基金项目:
江苏省科学技术厅自然科学基金
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