SOCS1基因调控未成熟DC及其预防小鼠移植物抗宿主病的功能研究  

Immature dendritic cell modified with SOCS1 gene and its role on prevention of graft-versus-host disease in mouse

作　　者：王苑[1] 张琦[1] 张艳霞[1] 张敬军[1] 夏荣[1] 

机构地区：[1]复旦大学附属华山医院输血科,上海200040

出　　处：《中国输血杂志》2015年第5期497-503,共7页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金面上项目(81170530);上海市自然科学基金面上项目(11ZR1405200)

摘　　要：目的构建慢病毒干扰的树突状细胞(DC)用于预防移植物抗宿主病(GVHD)。方法通过慢病毒转染小鼠骨髓来源DC,构建过表达细胞因子信号抑制因子1(SOCS1)基因的DC(DC-SOCS1)。用荧光实时定量PCR(RT-PCR)和Western blot方法检测慢病毒转染后的细胞SOCS1在基因和蛋白水平的表达量变化。通过脂多糖(LPS)刺激后,以流式细胞仪检测DC-SOCS1是否依然保持未成熟DC(i DC)的特性。将C57BL/6小鼠分为4组(n≥5),DC-GFP组:输注DC-GFP+异基因小鼠骨髓细胞和脾细胞;DC-SOCS1组:输注DC-SOCS1+异基因小鼠骨髓细胞和脾细胞;i DC组:输注i DC+异基因小鼠骨髓细胞和脾细胞;磷酸盐缓冲液(PBS)组:输注PBS+异基因小鼠骨髓细胞和脾细胞。输注的异基因骨髓细胞量为1×107/只,脾细胞量为2×107/只,DC-SOCS1和DC-GFP的输注量为2×106/只。通过眼静脉将指定细胞输入各组实验小鼠体内,观察小鼠体重、外周血和靶器官组织的病理学变化。以微量标本多指标流式蛋白定量技术(CBA)检测各组Th1细胞因子的分泌水平。结果 1)RT-PCR和Western blot均证实转染后DC-SOCS1细胞内SOCS1表达量明显提高,2)流式检测LPS刺激前后DC-GFP组、DC-SOCS1组和i DC组胞表面共刺激分子表达量,CD80:LPS刺激前为25.34%、23.74%、29.16%,LPS刺激后为67.92%、52.17%、82.42%;CD86:LPS刺激前为46.61%、44.77%、46.18%,LPS刺激后为:80.47%、57.11%、74.10%;MHC-Ⅱ:LPS刺激前27.61%、23.67%、22.86%,LPS刺激后为:90.33%、48.57%、94.58%。3)移植后21 d各组小鼠血细胞恢复情况,WBC(×109/L):2.18±0.32、3.37±0.37、1.12±0.05、1.48±0.20;Plt:330.83±53.04、563.25±71.76、153.27±16.97、101.00±6.01;Hb(g/L):103.83±8.24、128.00±6.19、88.14±7.45、99.75±8.16(P<0.05)。4)与DCSOCS1组小鼠相比,其他各组均有不同程度靶组织损伤表现:小肠绒毛断裂坏死,粘膜下层及肌层水肿;肺组织水肿,肺泡腔破坏以及炎性渗出;肝汇管区结构破坏,炎性细胞浸润,肝组织结�Objective To conceptualize the role of lentivirus vector with SOCS1 gene interference in dendritic cells（ DCs） on the prevention of graft-versus-host disease（ GVHD）. Methods DCs were obtained from mouse bone marrow and transfected with lentivirus vectors which upregulated the expression of SOCS1 gene. After transfection,RT-PCR and Western blot were used to test the changes of SOCS1 expression on gene and protein levels. Flow cytometry was used to detect if DCSOCS1 can maintain the characteristics of immature dendritic cell after stimulation with LPS. C57 BL /6 mice were divided into 4 groups： DC-SOCS1 group contained DC-SOCS1 ＋ allogeneic bone marrow cells and spleen cells,DC-GFP group contained DC-GFP ＋ allogeneic bone marrow cells and spleen cells,i DC group contained i DC ＋ allogeneic bone marrow cells and spleen cells,and PBS group contained PBS ＋ allogeneic bone marrow cells and spleen cells. The number of cells in each group were as followed： 1 × 10^7 bone marrow cells per mouse,2 × 10^7 spleen cells per mouse,and 2 × 10^6DC-SOCS1 and DC-GFP per mouse. Cells were injected into mice through ophthalmic vein. Their body weights,peripheral blood changes and pathological changes in target organs were then observed. The levels of Th1 cytokines were tested by cytometric bead array（ CBA）. Results（ 1） RT-PCR and Western blot confirmed that the SOCS1 levels of gene and protein significantly improved after transfection.（ 2） The expression of cell surface costimulatory molecules in DC-GFP,DC-SOCS1,and i DC groups was detected by flow cytometry before and after LPS stimulation. Before stimulation,CD80： 25. 34%,23. 74%,29. 16%; after stimulation,67. 92%,52. 17%,82. 42%. CD86： before： 46. 61%,44. 77%,46. 18%,after： 80. 47%,57. 11%,74. 10%. MHC-Ⅱ： before： 27. 61%,23. 67%,22. 86%,after： 90. 33%,48. 57%,94. 58%.（ 3） 21 days after transplantation,the recovery of mice blood cells were as followed： WBC（ × 10^9/ L） ： 2. 18 ± 0. 32,3. 37 ± 0. 37,1. 12 ±0. 05,1. 48

关 键 词：树突状细胞 未成熟DC 移植物抗宿主病 异基因移植骨髓移植 细胞因子信号抑制因子1 慢病毒载体 

分 类 号：R456[医药卫生—治疗学] R457.2[医药卫生—临床医学]