雷帕霉素缓释片防治兔高危角膜移植免疫排斥反应和新生血管增殖的研究

目的探讨雷帕霉素(RAPA)缓释片防治兔高危角膜移植术后免疫排斥反应和角膜新生血管增殖的疗效及其作用机制。方法RAPA缓释片制作:RAPA与载体乙交酯-丙交酯-己内酯三元共聚物(PGLC)制成RAPA缓释片(W/W=50/50),每粒缓释片含RAPA0·5mg。65只健康新西兰大白兔,其中45只兔(45只眼)采用缝线法诱导角膜新生血管生成,选择大于3个象限、新生血管长入角膜超过3mm的40只兔按照随机数字表法分为对照组(A组)、1mgPGLC载体前房植入组(B组)、1%RAPA滴眼液组(C组)及0·5mgRAPA缓释片前房植入组(D组),每组10只兔;余20只兔为供体。对4组兔行右眼同种异体穿透性角膜移植术,术后观察90d,记录排斥指数(RI,为植片水肿、混浊及血管长入评分合计)和新生血管指数(NI,为血管长入植片评分)。定期检测C、D组兔房水中RAPA浓度;术后3周,原位杂交法对4组兔角膜植片中白细胞介素(IL)-2R、单核细胞化学吸引蛋白质(MCP)-1、Fas/FasL进行检测,采用免疫组化法对肿瘤坏死因子(TNF)α和血管内皮细胞生长因子(VEGF)进行检测。术后90d,病理组织学检查视网膜、肝肾结构变化。结果(1)免疫排斥:A、B、C及D组兔发生免疫排斥反应的平均时间分别为(16·5±2·5)、(16·0±2·6)、(47·1±13·2)、(87·6±5·8)d(P=0·000)。术后2周,4组兔植片RI分别为4·9±2·2、3·9±0·9、0·8±0·4、0·3±0·6(P=0·000)。术后12周分别为10·4±0·8、10·0±0·0、7·2±2·2、2·0±3·3(P=0·000)。(2)角膜新生血管:术后2周,4组NI数分别为2·4±0·7、2·1±0·5、0·6±0·5、0·3±0·5(P=0·000)。术后12周分别为3·8±0·5、3·8±0·4、0·8±0·7、0·4±0·8(P=0·000)。(3)房水中RAPA浓度:术后第2、4、8、12周,D组房水中RAPA浓度分别10·7、12·0、9·2、7·0ng/ml,C组房水中RAPA浓度检测不出。(4)细胞因子表达:A、B组植片大量表达IL-2R、MCP-1、TNF-α、VEGF细胞因子,C、D组不表达上述细胞因子,4组植片中Fas/FasL均不表达。(5)组织病理:4组兔视网膜、肝肾组织结构正常。结论局部应用RAPA可以防治兔高危角膜移植术后免疫排斥反应和角膜新生血管增殖,RAPA缓释片疗效优于滴眼液。

Sustained rapamycin drug delivery system in prevention of high risk corneal allograft rejection and neovascularization in rabbits

OBJECTIVE: To evaluate the immunosuppressive and antiangiogenesis effects of rapamycin drug delivery system (RAPA DDS) in high risk rabbit model of penetrating keratoplasty (PK). METHODS: (1) RAPA DDS preparation: 50 mg of PGLC and 50 mg of RAPA were mixed as a RAPA drug delivery system. (2) High risk rabbit model: Corneal vascularization was induced in 45 New Zealand white rabbits (45 eyes) by passing 5 - 0 silk sutures in corneal stroma in each quadrant. (3) 40 rabbits with corneal neovascularization beyond three quadrants were received a unilateral 7 mm diameter central PK. The 40 were divided into four treatment groups: Group A, control group and received no therapy; Group B, 1 mg PGLC carrier was implanted in the anterior chamber; Group C, 1% RAPA eye drops was applied four times daily; Group D, 0.5 mg RAPA DDS was implanted in the anterior chamber. (4) Postoperative examination: The cornea allografts (opacity, edema and neovascularization) were examined by the slit-lamp biomicroscopy for ninety days. Rejection index (RI) and neovascularization index (NI) of these animal models were recorded. RAPA concentration in the aqueous humor was detected on 2, 4, 8 and 12 weeks in group C and D after surgery; the expressions of IL-2R, MCP-1, Fas/FasL in samples were detected with in situ hybridization; TNF-alpha and VEGF were detected with immuno-histochemical technique three weeks after the operation in all groups. Histochemical method was carried out on the procured specimens of cornea, retina, liver and kidney at ninety days. RESULTS: (1) Allografts rejection: Mean survival times in 4 trial groups were (16.5 +/- 2.5), (16.0 +/- 2.6), (47.1 +/- 13.2), (87.6 +/- 5.8) d respectively (P = 0.000). (2) Corneal neovascularization: Mean NI was 2.4 +/- 0.7, 2.1 +/- 0.5, 0.6 +/- 0.5, 0.3 +/- 0.5 (P = 0.000) 2 weeks after the operation, and the NI value was 3.8 +/- 0.5, 3.8 +/- 0.4, 0.8 +/- 0.7, 0.4 +/- 0.8 (P = 0.000) 12 weeks after the operation in groups A, B, C and D respectively. (3) RAPA concentration in aqueous humor: Mean RAPA concentration in aqueous humor was 10.7, 12.0, 9.2, 7.0 ng/ml in group D in the 2, 4, 8 and 12 weeks after the operation respectively. RAPA can not be detected in group C. (4) Cytokine expression: IL-2R, MCP-1, TNF-alpha and VEGF were overexpression in group A and B, and undetectable in group C and D. Fas and FasL were negative in all groups. (5) No inflammatory cell infiltration was found in retina, liver and kidney tissue ninety days after the surgery. CONCLUSIONS: Sustained RAPA DDS and eyedrops can prolong allograft survival and inhibit cornea neovascularization in rabbit model. However, RAPA DDS is better than eyedrops.