CAJ | 학술논문

目的观察应用西罗莫司联合可溶性肿瘤坏死因子受体I(sTNFRI)-IgGFc基因转染对大鼠骨髓源性树突状细胞(DC)形态、表型、功能和分化的影响及抑制DC成熟的作用.方法获取和培养Wistar大鼠骨髓源性DC.实验分5组,未成熟DC组(imDC组),成熟DC组(mDC组),西罗莫司组(经西罗莫司处理的DC),sTNFRI基因转染组(转染sTNFRI-IgGFc基因的DC)与联合处理组(经西罗莫司处理和sTNFRI基因转染的DC).采用流式细胞技术检测各组DC表面分子MHCⅡ、CD80、CD86的表达情况,进行单向式混合淋巴细胞反应后进行MTT实验检测各组DC活化T淋巴细胞能力,采用酶联免疫吸附试验试剂盒检测各组DC培养上清液中白细胞介素12(IL-12)、γ干扰素(IFN-γ)及sTNFRI-IgGFc蛋白的表达状况.结果培养第10天,rnDC组DC表面MHCⅡ、CD80、CD86的表达量显著高于其他4组(P＜0.05);联合处理组较imDC组CD86的表达显著降低(P＜00.05).mDC组DC刺激T淋巴细胞增殖能力显著高于其他4组(P＜0.05),联合处理组显著低于imDC组、mDC组和西罗莫司组(P＜0.05).mDC组培养液中IL-12和INF-γ的含量显著高于其他4组(P＜0.05),而联合处理组较其他4组培养液中IL-12和INF-γ的水平显著降低(P＜0.05).结论西罗莫司和sTNFRI-IgGFc基因修饰均能抑制DC的成熟,二者联合应用具有协同效应,较单独使用具有更强地抑制DC成熟的作用.
목적 관찰응용서라막사연합가용성종류배사인자수체I(sTNFRI)-IgGFc기인전염대대서골수원성수돌상세포(DC)형태、표형、공능화분화적영향급억제DC성숙적작용.방법 획취화배양Wistar대서골수원성DC.실험분5조,미성숙DC조(imDC조),성숙DC조(mDC조),서라막사조(경서라막사처리적DC),sTNFRI기인전염조(전염sTNFRI-IgGFc기인적DC)여연합처리조(경서라막사처리화sTNFRI기인전염적DC).채용류식세포기술검측각조DC표면분자MHCⅡ、CD80、CD86적표체정황,진행단향식혼합림파세포반응후진행MTT실험검측각조DC활화T림파세포능력,채용매련면역흡부시험시제합검측각조DC배양상청액중백세포개소12(IL-12)、γ간우소(IFN-γ)급sTNFRI-IgGFc단백적표체상황.결과 배양제10천,rnDC조DC표면MHCⅡ、CD80、CD86적표체량현저고우기타4조(P＜0.05);연합처리조교imDC조CD86적표체현저강저(P＜00.05).mDC조DC자격T림파세포증식능력현저고우기타4조(P＜0.05),연합처리조현저저우imDC조、mDC조화서라막사조(P＜0.05).mDC조배양액중IL-12화INF-γ적함량현저고우기타4조(P＜0.05),이연합처리조교기타4조배양액중IL-12화INF-γ적수평현저강저(P＜0.05).결론 서라막사화sTNFRI-IgGFc기인수식균능억제DC적성숙,이자연합응용구유협동효응,교단독사용구유경강지억제DC성숙적작용.
Objective To explore the morphology,cell phenotype and cell function in dendritic cells (DCs) derived from bone marrow after treatment with Sirolimus or Sirolimus combined with sTNFRI-IgGFc gene segment transfection.Method DCs were divided into 5 groups (imDCs,mDCs,Rapa-DCs,sTNFRI-DCs and Rapa-sTNFRI-DCs) according to different interventions.The expresson of MHC-Ⅱ,CD80 and CD86 was detected by flow cytometry.T cell proliferation of the mixed lymphocyte reaction was evaluated by MTT method.The levels of IL-12,IFN-γ and sTNFRI-IgGFc were determined by ELISA.Result On the day 10,the flow cytometry showed that the expression levels of MHC-Ⅱ,CD80 and CD86 on the cell surface in Sirolimus group were significantly higher than the other groups (P＜0.05).The expression level of CD86 in Rapa-sTNFRI group was significantly lower than in imDC group (P＜0.05).MTT results demonstrated that T cell proliferation ability in Sirolimus group,sTNFRI group and Rapa-sTNFRI group were reduced as compared with mDC group (P＜0.05).The ELISA results revealed that the levels of IL-12 and INF-γ in rnDC group were significantly higher than other groups (P＜0.05).The levels of IL-12 and INF-γ in Rapa-sTNFRI group were significantly lower than other groups (P＜0.05).Conclusion Sirolimus combined with modified sTNFRI-IgGFc gene could synergistically inhibit maturation of DCs more effectively than Sirolimus or modified sTNFRI-IgGFc gene used alone.