中国普外基础与临床杂志

中国普外基础与临床杂志

miRNA-155/PU.1信号通路阻滞对大鼠骨髓来源树突状细胞成熟及移植免疫的影响

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目的探讨miRNA-155/PU.1信号通路阻滞对骨髓来源树突状细胞(DCs)成熟及其对应用于大鼠小肠移植中的免疫功能的影响。 方法利用贴壁法培养诱导DCs,针对miRNA-155/PU.1信号通路中关键转录因子PU.1基因设计并合成3对PU.1 siRNA(PU.1沉默组、阴性对照组及对照组),用脂质体法转染细胞,并筛选一对高沉默效率PU.1 siRNA。流式细胞术分析PU.1沉默组、阴性对照组及对照组细胞表面CD80、CD86及主要组织相容性复合体(MHC)-Ⅱ的表达;ELISA法检测上清液中IL-10及IL-12p70的水平;混合淋巴细胞反应检测对同种异体T淋巴细胞的增殖作用;在大鼠小肠移植前7 d经受体尾静脉等量注射3组细胞,移植后观察各组受体存活情况及移植物病理情况。 结果①DCs表面分子CD80、CD86和MHC-Ⅱ表达率在PU.1沉默组分别为(27.0±5.6)%、(23.6±4.8)%及(36.8±6.8)%,阴性对照组分别为(74.0±9.4)%、(76.5±8.7)%及(87.8±11.3)%,PU.1沉默组均分别明显低于阴性对照组(P<0.05)。②PU.1沉默组IL-10水平较阴性对照组明显升高(P<0.05),IL-12p70则明显降低(P<0.05)。③PU.1沉默组DCs刺激同种异体T淋巴细胞增殖也较阴性对照组明显降低(P<0.05)。④将PU.1沉默组DCs术前注入受体行大鼠小肠移植后,受体平均存活时间为(14.3±3.3)d,明显长于阴性对照组的(7.8±1.5)d(P<0.05)和对照组的(8.0±2.5)d(P<0.05),且术后5 d时移植物病理显示移植肠组织轻度淋巴细胞浸润和绒毛水肿。 结论体内外实验表明,miRNA-155/PU.1信号通路阻滞的DCs成熟度明显受到抑制,并能稳定发挥诱导受体特异性免疫耐受的作用。

ObjectiveTo explore the influence of miRNA-155/PU.1 signaling pathway blockade on bone marrow-derived dendritic cells (DCs) maturation and immune function of rat small intestinal transplantation. MethodsThe DCs were induced by adherent culture.The critical transcription factor gene PU.1 was designed and PU.1 siRNA was synthe-sized.The DCs were transfected by liposome transfection and a pair of PU.1 siRNA was screened according to the high silencing efficiency.The expressions of DCs surface markers CD80, CD86, and MHC-Ⅱamong three groups (PU.1 silent group, negative control group, and control group) were analyzed by flow cytometry.The IL-10 and IL-12p70 secretion levels in the supernatant were tested by ELISA method.The allogeneic T lymphocyte proliferation was tested by mixed lymphocyte reaction.The transfected cells were intravenously injected into the recipient rat on day 7 before intestinal transplantation.The survival conditions as well as pathological changes were observed in each group recipients. Results①The surface molecules CD80, CD86, and MHC-Ⅱin the PU.1 silent group were (27.0±5.6)%, (23.6±4.8)%, and (36.8±6.8)%, respectively; versus (74.0±9.4)%, (76.5±8.7)%, and (87.8±11.3)% in the negative control group, respectively, which were significantly lower in former and showing an in creased trend (P < 0.05).②Compared with the negative control group, IL-10 secretion level was significantly increased (P < 0.05), IL-12p70 secretion level significantly decreased (P < 0.05) in the PU.1 silent group.③The proliferation of T lymphocytes in the PU.1 silent group was significantly lower than that in the negative control group (P < 0.05).④When the transfected DCs were injected into the intestinal transplantation rats on day 7 before operation, the survival time was (14.3±3.3) d, (7.8±1.5) d, and (8.0±2.5) d in the PU.1 silent group, negative control group, and control group, respectively, which in the PU.1 silent group were significantly longer than that in the other two groups (P < 0.05), and the graft pathology showed that there were mild intestinal tissue damage, lymphocyte infiltration or villus edema in the PU.1 silent group. ConclusionmiRNA-155/PU.1 signaling pathway blockade could reduce DCs maturation and induce receptor-specific immune tolerance, which are proved both in vivo and in vitro.

关键词: 树突状细胞; miRNA-155/PU.1; 免疫耐受; 小肠移植

Key words: Dendritic cell; miRNA-155/PU.1; Immune tolerance; Intestinal transplantation

引用本文: 徐兴伟, 丁博文, 朱传荣, 郑鹏, 冯涛, 嵇武. miRNA-155/PU.1信号通路阻滞对大鼠骨髓来源树突状细胞成熟及移植免疫的影响. 中国普外基础与临床杂志, 2014, 21(2): 168-172. doi: 10.7507/1007-9424.20140039 复制

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