IL-10基因修饰的树突状细胞在大鼠角膜移植中的作用

发布时间：2018-01-17 13:07

本文关键词：IL-10基因修饰的树突状细胞在大鼠角膜移植中的作用　出处：《辽宁医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的1.培养大鼠骨髓源树突状细胞，研究应用腺病毒转染IL-10基因后树突状细胞的表型及功能变化。2.建立同种异体大鼠角膜穿透性移植模型，探讨IL-10基因修饰的未成熟树突状细胞在大鼠角膜移植排斥反应中的作用。方法1.通过利用大鼠淋巴细胞分离液及细胞因子诱生方法，提取大鼠骨髓源树突状细胞的前体细胞并进行培养。培养至第6天时将培养的细胞分为3组：第1组原条件继续培养至第12天，即12-DC组；第2组转染滴度为107的GFP-Adenovirus，继续培养至第12天，即GFP-DC组；第3组转染滴度为107的IL-10-GFP-Adenovirus，继续培养至第12天，即IL-10-GFP-DC组。利用流式细胞仪（FCM）检测3组树突状细胞特异性成熟标志CD83以及细胞表面共刺激分子CD86的表达；利用MTT方法比较3组树突状细胞刺激同种异体T淋巴细胞增殖的能力；利用台盼兰检测方法，检测细胞活性；利用Western Blot方法，检测IL-10蛋白的表达。2.将受体SD大鼠随机分为4组：阳性对照组、GFP-DC组、8-DC组及IL-10-GFP-DC组，分别于角膜移植术前3天尾静脉注射等量的PBS、供体Wistar大鼠骨髓源8-DC（培养8天的DC）、转染48h的GFP-DC及IL-10-GFP-DC，建立大鼠穿透性角膜移植模型。术后每天在裂隙灯下对角膜植片进行临床观察，记录排斥反应指数及角膜植片存活时间。大鼠角膜移植术后第14天行各组角膜组织的病理学检查及免疫组织化学检查。结果1.新提取的树突状细胞前体细胞呈圆形，细胞小，形状规则；培养到第2天时细胞体积开始增大，并呈现出集落生长；培养至第4天时，细胞体积继续增大，细胞集落也增大，很多细胞开始出现大小不规则、形状不一的突起；第6天，细胞体积进一步增大，细胞突起更为明显。随着培养时间的延长，细胞形态变化更加明显，并开始呈现树突状细胞形态；第12天可见细胞不规则，细胞表面出现粗细不等、长短不一的树枝状或毛刺状突起，呈现典型的树突状细胞的形态。流式细胞仪检测结果显示： IL-10-GFP-DC表面分子CD83，CD86表达为约16%，20%，较GFP-DC组（44%，42%）及12-DC组（45%，48%）表达水平低。MTT结果显示：IL-10-GFP-DC刺激同种异体T细胞增殖能力最弱，12-DC刺激同种异体T细胞增殖能力最强，差异具有显著的统计学意义（P0.01）。台盼兰染色检测，显示细胞活性98%。Western Blot检测，观察到IL-10-GFP-DC组出现IL-10蛋白特征性条带表达。2.阳性对照组、GFP-DC组、8-DC组及IL-10-GFP-DC组角膜植片的存活时间分别为：10.29±1.11d、19.14±1.34d、18.57±1.27d、25.86±1.07d，GFP-DC组、8-DC组、IL-10-GFP-DC组与阳性对照组比较差异具有统计学意义（P0.01）；IL-10-GFP-DC组角膜植片存活时间较GFP-DC组、8-DC组比较显著延长（P0.01）；GFP-DC组与8-DC组比较无统计学意义（P0.05）。术后第14天阳性对照组、GFP-DC组、8-DC组以及IL-10-GFP-DC组的排斥反应指数分别为：6.71±0.49、3.71±0.49、3.86±0.38、1.71±0.49，GFP-DC组、8-DC组、IL-10-GFP-DC组与阳性对照组比较，差异具有统计学意义（P0.01）；IL-10-GFP-DC组与8-DC组、GFP-DC组相比较，有统计学意义（P0.01）；GFP-DC组与8-DC组比较，差异无统计学意义（P0.05）。病理组织学检查结果显示，阳性对照组角膜植片明显增厚，基质层结构紊乱，植片中可见大量炎细胞浸润，并可见新生血管；GFP-DC组、8-DC组、IL-10-GFP-DC组角膜植片的炎症反应较阳性对照组轻，植片中央未见明显新生血管。免疫组织化学结果显示：IL-10-GFP-DC组的CD4~+、CD8~+、CD25~+、IL-2~+、NK~+及NF-κB~+阳性细胞数量较阳性对照组、GFP-DC组、8-DC组减少，阳性对照组角膜植片中CD4~+、CD8~+、CD25~+、IL-2~+、NK~+及NF-κB~+阳性细胞数量最多。结论1.应用细胞因子诱生方法，成功培养出大鼠骨髓源树突状细胞。转染IL-10基因后的树突状细胞分泌IL-10细胞因子，大鼠骨髓源树突状细胞表面成熟特异性标志CD83及共刺激分子CD86的表达水平最低，，刺激T细胞增殖的能力弱，抑制树突状细胞的成熟。2.经过供体来源树未成熟突状细胞预处理的受体，角膜植片的存活时间显著延长，成功诱导角膜移植免疫耐受。CD4~+、CD8~+、CD25~+、IL-2~+、NK~+及NF-κB~+阳性细胞参与了同种异体角膜移植排斥反应的调控，IL-10-GFP-DC可降低CD4~+、CD8~+、CD25~+、IL-2~+、NK~+及NF-κB~+阳性细胞的浸润，抑制角膜移植排斥反应的发生。
[Abstract]:The purpose of the 1. cultured rat bone marrow-derived dendritic cells, research and application of adenovirus transfected IL-10 gene changes of.2. phenotype and function of dendritic cells in allogeneic rat corneal penetrating transplantation model of IL-10 gene modified immature dendritic cells in rat corneal allograft rejection in rats.
Methods 1. by induced by rat lymphocyte separation medium and cytokines, extraction of progenitor cells from rat bone marrow derived dendritic cells were cultured and cultured for sixth days culture. The cells were divided into 3 groups: the first groups of the original conditions up to the twelfth day, 12-DC group; second group transfection titer 107 GFP-Adenovirus, up to the twelfth day, GFP-DC group; third group transfection titer was 107 IL-10-GFP-Adenovirus, up to the twelfth day, IL-10-GFP-DC group. Using flow cytometry (FCM) detection of 3 groups of dendritic cell specific markers of cell surface expression of CD83 and costimulatory molecules CD86; using MTT method comparison of 3 groups of dendritic cells stimulate proliferation of allogeneic T lymphocytes; using trypan blue detection method, detection of cell activity; using Western Blot method to detect the expression of.2. IL-10 protein by SD Rats were randomly divided into 4 groups: positive control group, GFP-DC group, 8-DC group and IL-10-GFP-DC group, respectively, on the 3 day tail vein injected PBS before corneal transplantation, donor Wistar rat bone marrow derived 8-DC (cultured with DC for 8 days), GFP-DC and IL-10-GFP-DC transfected with 48h, the establishment of penetrating corneal transplantation model every day after operation. The rats under slit lamp of corneal graft were observed and recorded the rejection index and corneal graft survival time. Immunohistochemical examination and pathological examination of rat corneal transplantation after fourteenth days were corneal tissue.
Results 1. new extracted dendritic cell precursor cells were round, small cell, regular shape; up to second days when the cell volume began to increase, and showed colony growth; after fourth days of culture, the cell volume increases, cell colony is also increasing, many cells began to appear irregular size, shape a process; Sixth days, cell volume increased, the more obvious cellularprocesses. With prolonged incubation time, the more obvious changes in cell morphology, and began to show the morphology of dendritic cells; twelfth days cells showed irregular, cell surface thickness ranging from different lengths of dendritic or burr like protrusions, typical dendritic cell morphology. Flow cytometry results showed that: the surface molecules of IL-10-GFP-DC CD83, the expression of CD86 was about 16%, 20%, compared with the GFP-DC group (44%, 42%) and 12-DC group (45%, 48%).MTT showed low levels of expression The weakest: IL-10-GFP-DC stimulation of allogeneic T cells proliferation, 12-DC to stimulate allogeneic T cell proliferation ability was the strongest, the difference was statistically significant (P0.01). Trypan blue staining showed that the cell activity of 98%.Western detection, Blot detection, were observed in the IL-10-GFP-DC group the IL-10 protein expression of.2. characteristic bands of the positive control group, GFP-DC group, 8-DC group and IL-10-GFP-DC group of corneal graft survival time was 10.29 + 1.11d, 19.14 + 1.34d, 18.57 + 1.27d, 25.86 + 1.07d, GFP-DC group, 8-DC group, IL-10-GFP-DC group and positive control group with significant difference (P0.01); group IL-10-GFP-DC, corneal graft survival time compared with GFP-DC group, 8-DC group significantly increased (P0.01); GFP-DC group and 8-DC group had no statistical significance (P0.05). After fourteenth days of positive control group, GFP-DC group, 8-DC group and IL-10-GFP-DC group respectively the rejection index : 6.71 + 0.49,3.71 + 0.49,3.86 + 0.38,1.71 + 0.49, GFP-DC group, 8-DC group, IL-10-GFP-DC group compared with the positive control group, the difference was statistically significant (P0.01); IL-10-GFP-DC group and 8-DC group, GFP-DC group compared with statistical significance (P0.01); GFP-DC group compared with 8-DC group, the difference was not statistically significant (P0.05). Histopathological examination showed that the positive control group of corneal stromal layer thickening, structural disorder, the graft showed large amounts of inflammatory cell infiltration, neovascularization and visible; GFP-DC group, 8-DC group, IL-10-GFP-DC group, inflammatory reaction corneal graft compared with the positive control group, the grafts had no obvious neovascularization. The immunohistochemistry results showed that: IL-10-GFP-DC group of CD4~+, CD8~+, CD25~+, IL-2~+, NK~+ and NF- K B~+ the number of positive cells compared with the positive control group, GFP-DC group, 8-DC group, positive control group corneal graft in CD4~+, CD8~+, CD The number of 25~+, IL-2~+, NK~+ and NF- kappa B~+ positive cells was the most.
Conclusion the application of 1. cytokines inducedmethod, successfully cultured rat bone marrow derived dendritic cells. The secretion of IL-10 cytokines after IL-10 gene transfection of dendritic cells derived from rat bone marrow dendritic cells maturation specific marker expression of CD83 and costimulatory molecule CD86 is the lowest, the ability to stimulate the proliferation of T cells is weak, inhibition of dendritic cells the mature.2. after donor derived immature dendritic tree cell pretreatment receptors, survival time of corneal grafts was prolonged and successfully induced corneal transplantation immune tolerance of.CD4~+, CD8~+, CD25~+, IL-2~+, NK~+ and NF- K B~+ positive cells participate in the regulation of corneal allograft rejection, IL-10-GFP-DC can reduce CD4~+, CD8~+. CD25~+, IL-2~+, NK~+ and NF- K infiltration of B~+ positive cells, inhibit the occurrence of corneal allograft rejection.

【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.65

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