沉默CD86树突状细胞在异种胰岛混合淋巴细胞培养中的作用研究  

Effect of CD86 Gene Modified Recipient Dendritic Cell on Mix Cultured Donor-derived Islet with Recipient-derived Lymphocyte in vitro

作　　者：苏安平[1] 田伯乐[2] 张肇达[2] 李全生[3] 

机构地区：[1]四川大学华西医院甲状腺乳腺外科,成都610041 [2]四川大学华西医院胰腺外科,成都610041 [3]四川大学华西医院胆道外科,成都610041

出　　处：《四川大学学报（医学版）》2015年第4期501-507,共7页Journal of Sichuan University(Medical Sciences)

基　　金：四川省科技厅(No.2009SZ0156)资助

摘　　要：目的探讨沉默CD86的树突状细胞(DC)在异种胰岛混合淋巴细胞培养中的作用。方法分离培养BALB/c小鼠骨髓来源DC,流式细胞仪鉴定后,以CD86siRNA转染DC。分离纯化SD大鼠胰岛,吖啶橙(AO)/碘化丙啶(PI)双荧光染色方法检测胰岛细胞活性;提取SD大鼠脾脏淋巴细胞,与经siRNA沉默CD86的DC混合培养,使DC负载SD大鼠抗原。在400胰岛当量SD大鼠胰岛细胞+1×106个BALB/c小鼠淋巴细胞(对照组1)基础上,混合普通DC(负载SD大鼠抗原)(对照组2)或siRNA沉默CD86的DC(负载SD大鼠抗原)(实验组)进行体外培养,另以单纯SD大鼠胰岛细胞为空白组,培养3d后倒置显微镜下检测细胞形态学变化,测定上清液中反映淋巴细胞增殖的细胞因子白介素(IL)-2、IL-4、IL-10及干扰素(IFN)-γ水平,AO/PI染色观察胰岛活性,并行葡萄糖刺激胰岛素释放实验,计算刺激指数。结果培养所得的细胞具有典型的DC形态,流式细胞仪检测DC表面CD11c、CD80、CD86的表达阳性率分别为(86.26±9.73)%、(72.64±8.55)%、(77.18±10.23)%。CD86siRNA转染DC后CD86沉默。SD大鼠胰岛杂质较少,形态正常,细胞活性>95%。混合细胞培养3d后,与对照组1、2比较,实验组的IL-10水平升高(P<0.05),IL-4水平相当(P>0.05),IL-2、IFN-γ均降低(P<0.05),淋巴细胞增殖最少,胰岛功能最好,刺激指数最高。实验组4种细胞因子均高于空白组(P<0.05),刺激指数低于空白组(P<0.05)。结论沉默CD86的DC负载供体抗原后,与供体胰岛、受体淋巴细胞混合培养,可在一定程度上减轻免疫排斥反应,从而保护供体胰岛功能。Objective To investigate the effect of CD86 gene modified recipient dendritic cell（DC）on mix cultured donor-derived islet with recipient-derived lymphocyte in vitro.Methods DCs were separated from bone marrow of BALB/c mice and identified by flow cytometry.Chemically synthesized CD86 siRNA was transferred into DC.Donor islets were separated from the pancreas of SD rats.Acridine orange（AO）/Propidium iodide（PI）staining was conducted to assess the viability of islets.Lymphocytes were collected from the spleen of SD rats and then co-cultured with CD86 gene modified recipient DCs.CD86 gene modified recipient DC,donor-derived islet（400IEQ）and recipient-derived lymphocyte（1×10^6）were mix cultured in vitro.Four groups were set：blank group（islets of SD rat only）,control 1group（islets of SD rat with splenic lymphocyte of BALB/c mice）,control 2group（islets of SD rat,splenic lymphocyte of BALB/c mice with normal recipient DC）and experimental group（islets of SD rat,splenic lymphocyte of BALB/c mice with CD86 gene modified recipient DC）.After 3days culture,the cellular morphology of culture was observed with light inverted microscope.The levels of IL-2,IL-4,IL-10 and IFN-γin the culture supernatant were tested,and islets viability was assessed by AO/PI staining.GSIS was conducted and stimulation index（SI）was calculated.Results Typical DC morphology was found from the collectedcells.The positive rates of CD11 c,CD80and CD86 protein expression on DCs were 86.26%±9.73%,72.64%±8.55% and 77.18%±10.23%,respectively.The positive rate of CD86 protein expression on DCs after transfection was 23.64%±5.25%.The viability of islets was over 95%.After 3days culture,the level of IL-10 increased significantly and the levels of IL-2and INF-γdecreased significantly in experimental group（vs.control 1and control2 groups,P〈0.05）.The level of IL-4 was similar in control 1,control 2and experimental groups,but the proliferation rate of lymphocyte in the experimental group was the lowest one,the v

关 键 词：树突状细胞 异种胰岛移植 CD80/CD86 免疫耐受 糖尿病 

分 类 号：R657.5[医药卫生—外科学]