CD86及NF-κB在IL-10修饰的DC诱导大鼠角膜移植免疫耐受中的作用

发布时间：2018-03-14 17:09

本文选题：白细胞介素-10　切入点：树突状细胞　出处：《辽宁医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的研究CD86及NF-κB在IL-10-DC诱导大鼠同种异体角膜移植免疫耐受中的作用。方法 1. DC培养与鉴定:参照经典Hermans方案直接分离诱生法进行细胞培养,分三组,分别为6-DC组、12-DC组和IL-10-DC组。在经典分离诱导方案(GM-CSF+IL-4)的基础上培养至第6天为6-DC;第二组细胞继续培养至12天,即为12-DC;第三组在第6天加入IL-10继续培养至12天,为IL-10-DC。光镜观察DC形态,用流式细胞仪(FCM)检测所得DC表型。 2.用培养的供体来源的6-DC及IL-10-DC预处理受体,建立Wistar-SD大鼠穿透性角膜移植模型。供体Wistar大鼠21只;受体SD大鼠42只,受体鼠随机分为3组,每组均为14只,角膜移植前3d,每只受体鼠经尾静脉注射不同物质。A组:注射PBS;B组:注射6-DC,细胞数为2×10~6个/只;C组:注射IL-10-DC,细胞数为2×10~6个/只。以混浊、水肿和新生血管3项指标作为临床评估标准,观察术后各组受体角膜植片的存活时间。术后第14天各组随机抽取4只鼠取术眼角膜行组织病理学检查及免疫组化法检测CD86及NF-κb的表达。结果 1.DC的生长情况、形态和表型:新分离的细胞呈圆形,胞体小。培养至第2天,细胞开始集落生长;第4天集落最大,随着培养时间的延长,细胞体积增加呈现典型的树突状细胞形态。流式细胞仪检测DC的表型:IL-10-DC组CD83、CD86表达最低,而12-DC组表达最高。三组比较,差异具有统计学意义(P0.01)。 2.各组大鼠角膜植片存活情况:平均存活时间为A组(10.10±1.66)d,B组(17.80±1.81)d,C组(26.40±1.65)d。术后第14d,A组角膜植片均发生排斥,而B、C两组角膜植片仍保持透明。B、C两组植片存活时间与A组比较显著延长(P0.01);B、C组间比较,差异有统计学意义(P0.01)。植片组织病理学检查:A组角膜植片显著水肿、增厚,出现大量的淋巴细胞和单核细胞浸润,角膜基质排列紊乱。B、C两组植片炎性细胞浸润均显著减少,角膜厚度及结构基本正常。CD86及NF-κB,在对照组中呈高表达(P0.05),B,C组间比较无显著性差异(P0.05)。结论 1.直接分离诱生法联合应用细胞因子GM-CSF、IL-4可以从大鼠骨髓中获得大量较高纯度imDC,满足了进一步实验对细胞量的要求。IL-10可有效抑制DC成熟。 2.摄取供体的imDC能显著延长角膜植片的存活时间,成功诱导了同种异体大鼠角膜移植的免疫耐受。CD86及NF-κB参与了同种异体角膜移植排斥反应的调控。IL-10-DC可抑制CD86及NF-κB的表达水平,从而抑制角膜排斥反应的发生。
[Abstract]:Purpose. To investigate the role of CD86 and NF- 魏 B in IL-10-DC induced immune tolerance in rat corneal allograft. Method. 1. DC culture and identification: according to the classical Hermans protocol, the cells were cultured by direct isolation and induction, and were divided into three groups. 6-DC group (12-DC group) and IL-10-DC group (6-DC group) were cultured on the 6th day, the second group continued to culture to 12 days, the third group added IL-10 to 12 days, and the third group was cultured to 12 days after the addition of IL-10 on the 6th day, the second group continued to culture to 12 days, and the third group was cultured to 12 days after the addition of IL-10 on the 6th day. The morphology of DC was observed by light microscope, and the phenotype of DC was detected by flow cytometry (FCM). 2. The penetrating corneal transplantation model of Wistar-SD rats was established by preconditioning the recipients with 6-DC and IL-10-DC from cultured donors. 21 Wistar donor rats and 42 SD recipients were randomly divided into 3 groups, with 14 rats in each group. 3 days before corneal transplantation, each recipient rat was injected with different substances through caudal vein. Group A: group B: group B: group B: injected with 6-DC.The number of cells in group C was 2 脳 10 ~ (-6) cells injected with IL-10-DC2 脳 10 ~ (-6) cells per mouse. Edema and neovascularization were used as clinical evaluation criteria. The survival time of corneal grafts was observed and the expression of CD86 and NF- 魏 b were detected by immunohistochemistry and histopathological examination. Results. 1. The growth, morphology and phenotype of DC: the newly isolated cells were round and small. At the second day of culture, the colony began to grow, and on the 4th day, the colony was the largest, with the extension of culture time. The increase of cell volume showed typical dendritic cell morphology. The expression of CD83 + CD86 was the lowest in the DC group with flow cytometry and the highest in the 12-DC group. The difference among the three groups was statistically significant (P 0.01). 2. Survival status of corneal grafts in each group: the mean survival time was 10.10 卤1.66 days in group A and 17.80 卤1.81 days in group C, and the corneal graft rejection occurred in group A on the 14th day after operation. However, the survival time of corneal grafts in group B and C was significantly longer than that in group A, and there was a significant difference between group A and group A (P 0.01). Histopathological examination of grafts: in group 1, corneal grafts showed significant edema, thickening, large number of lymphocytes and monocytes infiltrating, and inflammatory cell infiltration in corneal grafts decreased significantly in both groups. The corneal thickness and structure were basically normal. CD86 and NF- 魏 B were almost normal. There was no significant difference between the two groups (P 0.05). Conclusion. 1. A large number of imDCs with high purity can be obtained from rat bone marrow by direct isolation and induction combined with cytokine GM-CSF- IL-4, which meets the requirements of further experiments. IL-10 can effectively inhibit DC maturation. 2. The survival time of corneal grafts was significantly prolonged by uptake of donor imDC, and the immune tolerance. CD86 and NF- 魏 B involved in the regulation of corneal allograft rejection. IL-10-DC could inhibit the expression of CD86 and NF- 魏 B. Thus inhibiting the occurrence of corneal rejection.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R779.65

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