【作者】 史伟云；

【导师】 谢立信；

【作者基本信息】 青岛大学 ， 眼科学， 2005， 博士

【摘要】 目的: 1.通过对BALB/c小鼠和CD4、CD8基因敲除小鼠角膜移植术后免疫排斥反应特点的研究,探讨角膜移植术后免疫排斥反应的发生机制。2.观察基因重组蛋白免疫抑制药物CTLA4-Ig和FK506缓释系统防治高危角膜移植术后免疫排斥反应的效果,探讨防治角膜移植术后免疫排斥反应的用药新途径及其机理。 方法: 1、小鼠穿透性角膜移植术后免疫排斥反应的特征 建立BALB/C小鼠穿透性角膜移植动物模型。对照组:同种系移植;实验组:同种异系移植,供体为C57BL/6小鼠。术后用裂隙灯显微镜观察角膜移植片的存活情况;免疫组织化学检测观察CD11b~+、CD4~+、CD8~+细胞在眼前节的浸润情况;LYVE-1单克隆抗体检测虹膜-睫状体组织中新生淋巴管的变化;应用RT-PCR方法检测发生免疫排斥的角膜组织中IL-10,TNFα,IFNr,B7和CD40的表达情况。同时,通过迟发性超敏反应检测推测角膜免疫排斥时的全身免疫状态。 2、基因敲除小鼠穿透性角膜移植术后免疫排斥特征的研究 试验组:CD4和CD8基因敲除小鼠为受体;对照组:C57BL/6小鼠为受体,供体为BALB/c小鼠,建立穿透性角膜移植动物模型,每组各20只。术后裂隙灯显微镜观察角膜植片的存活情况,免疫组织化学检测术后不同时间眼前段CD4~+和CD8~+细胞的变化。术后2周,每组随机选10只小鼠接受来自BALB/c小鼠的皮肤移植,观察皮肤植片的存活情况。 3、FK506缓释系统(FK506 DDS)抑制兔高危角膜移植术后免疫排斥反应 建立兔高危角膜移植动物模型,随机分为对照组、空白DDS前房植入组、CsA DDS(含CsA 1mg)前房植入组、0.1%FK506滴眼液组和FK506 DDS(含FK506 0.5mg)前房植入组。观察各组角膜移植片存活情况;术后不同时间抽取各组兔房水和静脉血进行FK506药物浓度检测;术后4周及观察期结束时进行角膜的组织病理学检查,并利用原位杂交的方法检测各组角膜植片内IL-2R α、更多还原

【Abstract】 Objective1 To testify mechanism of immune rejection by means of BALB/c mice and knock-out mice model after corneal transplantation.2 To investigate therapeutic effect and underlying action mechanism of FK506 drug delivery system and CTLA4 Ig, further to demonstrate new methods to prevent immune rejection after corneal transplantation.Methods1 Properties of immune rejection after penetrating keratoplasty of miceControl group: BALB/c mice as both donors and recipients;experimental group: C57BL/6 mice as donor and BALB/c mice as recipients. To investigate migrationroute of CD11b~+ 、 CD4~+、 CD8~+ cells in anterior segment, iris lymphagenesis byimmunostaining LYVE-1 mAb, cytokines expression by reverse transcription polymerase circle reaction, systemic immune condition by delayed type hypersensitivity (DTH).2 Properties of immune rejection after penetrating keratoplasty of knock-out miceExperimental group, either CD4 knock-out mice or CD8 knock-out mice as receptors;Control group, C57BL/6 mice as receptors and BALB/c mice as donors. To investigate survival time of corneal allografts by slit-lamp biomicroscopy and migration of CD4~+ and CD8~+ cells by immunohistology. Ten mice each group received skin transplantation from BALB/c mice in the second week after penetrating keratoplasty. The skin grafts rejection was recorded and the corneal grafts also were inspected when the skin grafts were rejected.3 FK506 drug delivery system to suppress immune rejection of high-risk keratoplasty in rabbitsSixty-eight New Zealand rabbits were divided into five groups at random, control group, empty DDS implantation group, CsA DDS implantation group (containing lmg CsA), 0.1% FK506 drops group and FK506 DDS (containing 0.5mg FK506)implantation group. To observe survival time of corneal allografts and detect FK506 concentration by drawing aqueous humour and vein blood in groups in varied time point postoperatively. To examine IL-2R a -. MCP-K Fas and FasL mRNA of corneal grafts by in situ hybridization technique and pathological results at postoperative 4 weeks and at the end of observation in different groups. 4 CTLA4 Ig for prevention of immune rejection of high-risk keratoplastyThe mice corneal transplantation models were established. Experimental group which the corneal donors from C57BL/6 strain of mouse were incubated in corneal storage medium containing 10 ug/ml of CTLA4-Ig for 24 hours and then transplanted orthotopically into BALB/ c strain recipients. Control group, the corneal donors haven’t given any treatment before the surgery. To observe survival time of corneal allografts and detect migration of inflammatory cells with histological and immunohistochemical staining weekly. Some mice with corneal allografts survived beyond 6 weeks in experimental group were selected as recipients of skin grafts from C57BL/6 donors and performed DTH assay after 12 days of skin transplantation. Results 1 Properties of immune rejection after penetrating keratoplasty of miceThe median survival time of corneal grafts was 14 + 3 days in experimental group, beyond 100 days in control group. Histopathologic and immuneohistochemical results showed some different cells migration features. In experimental groupCD\ \b+, CZ)4+ > CDS* cells attacked corneal grafts from two different paths. Incontrol group CDWb* cells infiltrated into suture knots in the early phase ofoperation and nearly disappeared at 3-5 days, while no CD4 and CD8 cells were observed. Followed by the time passing, the number of lymphatic vessels increased. When immune rejection occurred, lymphangiognesis amounted to two folds compared with that in normal circumstance. But there is no hypersensitive condition observed in systemic immune system by DTH assay. 2 Properties of immune rejection after penetrating keratoplasty of knock-out miceThe median survival time of corneal grafts was over 90 days in CD4 knock-outmice, (28 ± 3 ) days in CD8 knock out mice, (14 + 2) days in control group (P<0.01). There are a few CD8+ and few CD4+ cells around limbus and ciliary body in CD4 knock-out mice. CD4+ cells appeared in 2 weeks and remarktably increased on anterior segment in 4 weeks, but no CD8+ cells were detected in CD8 knock-out group. A large amounts of CD4+ and CD8+ cells infiltrated into anterior segment in control group on postoperative 4 weeks. The survival time of skin grafts was 14+2 days in CD4 knock-out mice, 12+1 days in CD8 knock-out mice, 10 + 1 days in control groups (PO.001).3 FK506 drug delivery system to suppress immune rejection of high-risk keratoplasty in rabbitsThe survival time of coraeal allografts was beyond 180 days in FK506 DDS group which obviously excelled over other groups (/^O.OOOO) . FK506 concentration in anterior chamber and cornea was higher than that in FK506 drops group (P=0.0022) . Over lOng/ml FK506 levels in aqueous humor was effective for prevention of immune rejection. There are multiple inflammatory cells infiltrating and expression of IL-2R a % MCP-1 mRNA in control group and empty DDS implantation groups. No IL-2Ra fP MCP-1 mRNA were examined in CsA DDS group^ FK506 drops group and FK506 DDS group. Fas and FasL mRNA were not detected in all groups. 4 Properties of immune rejection after penetrating keratoplasty of miceThe survival time of corneal allografts was beyond 100 days in CTLA4-Ig treated group, not more than 14 days in control group. Few inflammatory cells migrated into grafts in CTLA4 Ig treated group, compared with large amounts of cells in rejected grafts in control group on postoperative 2 weeks. The cytokines including of IL10, TNF a, IFNy, B7-1 and CD 40 were expressed in the rejected corneas in control group. Median survival time of skin grafts was about 11 days. Rejected skin simultaneously induced ear swelling which referred to DTH array. There was a significant difference compared with negative control mice. Conclusion 1 Immune rejection is possibly ocular topical immune activity mediated by Tlymphocytes. Activated T cells attack corneal grafts through keratolimbus and ciliary body-iris-aqueous humor pathway respectively. Lymphagiogenesis in iris maybe associate with immune rejection response after keratoplasty.2 Immune rejection response after corneal transplantation is primarily mediated by CD4+ T lymphocytes, also involved in CD8+ cells.3 FK506 DDS implantation into anterior chamber is able to effectively prevent immune rejection of high-risk corneal transplantation. Sustained FK506 concentration in aqueous humor is the key factor to prevent immune rejection response after keratoplasty.4 CTLA4-Ig effectively protect corneal allografts from acute cell-mediated rejection and prolong graft survival time by means of competitive inhibition of the interaction between co-receptor blocker CD28/B7 and blockade of T cells activation.更多还原