腹腔输注耐受性树突状细胞诱导小鼠免疫耐受研究

Established the model by transfused tolerogenic dendritic cells in abdominal cavity to induce immunological tolerance in the mouse

目的通过腹腔输注体外培养的耐受性树突状细胞(tolerogenic dendritic cells,tDCs),在小鼠体内建立tDCs诱导特异性免疫耐受方法。方法采用雄性Balb/c小鼠为供体,雌性昆明小鼠为受体。取供体小鼠骨髓细胞,分离单个核细胞。在含GM-CSF、IL-4、VIP、DXM和LPS等因子的RPMI1640培养基中按不同的组合分为两组进行体外诱导培养:常规DC组(GM-CSF+IL-4)和tDC组(GM-CSF+VIP+DXM)。实验分4组:空白对照组(不经任何处理)、骨髓细胞组(输骨髓细胞)、常规DC组(输常规培养DC)和tDC组(输tDC)。腹腔输注细胞3d后移植供体脾片,移植后20d取移植脾片病理组织学检查,移植后1个月流式细胞术检测受体脾单个核细胞CD80、CD86、CD40、CD11c和CD4+CD25+的表达。结果小鼠体内tDCs输注实验:tDC组的移植脾片大体形态呈灰红色,与移植部位周围组织建立血液供应;病理学观察组织结构完整,未见明显变性坏死,与正常脾组织结构相似。而空白对照组、骨髓细胞组、常规DC组移植脾片大体呈灰白色、浅灰红色和灰褐色,与周围组织未建立血液供应;病理学观察组织结构受破坏,有的呈大片坏死。tDC组小鼠脾单个核细胞CD4+CD25+双表达细胞为(18.15±0.66)%,比正常小鼠的(6.5±0.55)%高近3倍,差异有显著性(P<0.05);CD40、CD11c、CD80和CD86的表达比正常小鼠低,差异有显著性(P<0.05)。结论在移植脾片小鼠模型,经腹腔注射tDCs能诱导受体对移植器官产生特异性免疫耐受。小鼠体内输注tDCs,其脾脏CD4+CD25+Treg细胞增高,CD11c、CD40、CD80和CD86表达降低。腹腔注射tDCs安全性较好,并能形成稳定的嵌合,且维持1个月以上。

[Objective] Transfuse tolerogenic dendritic cells （tDCs） cultured in vitro in abdominal cavity, in the mouse established the method of tDCs to induce specific immunological tolerance in vivo. [Methods] Male Balb/c mice are used as donor and female Kunming mice as receptor. Tolerogenic dendritic cells were generated from bone marrow precursors cultured with GM-CSF, IL-4, VIP, DXM and LPS. The cells were divied into two groups： common-DC group （GM-CSF＋IL-4）, tDC group （GM-CSF＋VIP＋DXM）. The animal transfusion experiment were divided into four groups： Blank group （not with any treatment）, bone marrow group （transfusion bone marrow）, common-DC group （transfusion common-DC）, tolerogenic-DC group （transfusion tolerogenic-DC）. Three days after transfusion transplant the pieces of donor＇s spleen to receptor. Twenty days after transplantation take the pieces of transplanted spleen into pathological section. A month after transplantation detect CD80, CD86, CD40, CD11c and CD4^＋CD25^＋ of spleen single caryoceils of receptor through FCM. [Results] The ceils possessed morphological character of typical DC. In the transfusion experiment of mouse, the tDC group present grayish red, and establish blood supply to surrounding structure; in the histopathologic examination it didn＇t see obviously denaturation and necrosis. But the Blank group, bone marrow group, common-DC group present grayish white, light grayish red, grayish brown, and didn＇t establish blood supply to surrounding structure; in the histopathologic examination it see obviously necrosis. The CD4^＋CD25^＋ cell of tDC group is （18.15±0.66）%,and the normal mouse is （6.5±0.55）% （P 〈0.05）. Compared to the common-DC group, the tDC group lowly express CDsc, CDs6, CD4o and CDnc （P 〈0.05）. [Conclusions] Transfused tDC to the mouse induce specific immunological tolerance in vivo. The CD,~CD~~ cell of tDC group is high,and lowly express CD80, CD86, CD40 and CD11c. The safety of transfused tDCs in abdominal cavity is high, tDCs could form stabilized engomphosis to the receptor, and persistant more than one month.