玻璃体内注射他克莫司对异种移植的视网膜色素上皮细胞存活的影响

Effect of intravitreal injection of FK506 on the survival of heteroplastic retinal pigment epithelial cells

目的 观察少量、多次玻璃体内注射免疫抑制剂他克莫司 (FK5 0 6 )对移植到兔眼视网膜下间隙内的人视网膜色素上皮 (retinal pigmentepithelium ,RPE)细胞存活情况的影响。 方法 采用绿色荧光蛋白 (green fluorescent protein,GFP)逆转录病毒感染人 RPE细胞。将 5 0μl(4× 10 3个 /μl)表达 GFP的人RPE细胞悬液注射到 18只白兔及 10只灰兔双眼视网膜下间隙 ,手术后所有兔左眼玻璃体内注射 5 μlFK5 0 6 (5μg/μl) ,每周 1次 ,连续 5周 ,然后间周一次至 2 0周 ;右眼不注射作为对照。眼球壁铺片倒置荧光显微镜观察移植细胞的存活状态。 结果 白兔于移植后 1、2、3、4、6、10、11、14、18、2 0、2 3、2 4、2 5、33、5 4周 ,灰兔于移植后 4、5、6、7、14、18、2 0、2 6周双眼视网膜下均可见表达的 GFP细胞 ,但移植术后 1～ 14周内玻璃体内注射了 FK5 0 6的左眼视网膜下 RPE- GFP细胞形态、细胞膜的完整程度均比未注射的右眼好。 18周后 ,7只白兔和 3只灰兔双眼视网膜下移植细胞的状态差异不明显。眼球切片苏木素 -伊红 (hema-toxylin- eosin,HE)染色观察结果显示 ,移植术后 1～ 6周 ,6只白兔和 3只灰兔右眼脉络膜小血管周围可见灶性或弥散的淋巴细胞 ,应用了 FK5 0 6的左眼淋巴细胞的浸润明显减少。

Objective To investigate effect of intravitreal injection of FK506 on the survival of human retinal pigment epithelial (RPE) cells heteroplastically transplanted into the subretinal space of rabbits. Methods The immortalized human RPE cells were genetically labeled by retrovirus vector carrying a green fluorescent protein (GFP). A total of 50 μl RPE cells suspension with 4×10 3 cells/μl which expressed GFP were injected into the subretinal space of both eyes of 18 white rabbits and 10 gray rabbits. The left eyes of all of the rabbits were injected of 5 μl FK506 (5 μg/μl) intravitreally once a week during the first 5 weeks, then once every other week until the 20 th week and the right eyes were as the control. The histological sections of heteroplastic RPE cells were observed by epifluorescent microscope. Results GFP-expressing cells could be seen after 1 week, 2, 3, 4, 6, 10, 11, 14, 18, 20, 23, 24, 25, 26, 33, and 54 weeks in white rabbits and after 4, 5, 6, 7, 14, 18, 20, and 26 weeks in gray rabbits. The configuration and integrality of the RPE-GFP cells in the left eyes which had been intravitreally injected of FK506 1-14 weeks after transplantation were better than those in the right eyes without injection. After 18 weeks, the condition of heteroplastic cells with few difference in both eyes in 7 white and 3 gray rabbits were found. After 1-6 weeks, focal and disseminated lymphocytes around the choroidal small vessles of right eyes in 6 white and 3 gray rabbits could be seen while the infiltration of the lymphocytes in the left eyes was much reduced. Conclusion Intravitreal injection of a small amount of FK506 at the first 3 months after transplantation may significantly improve the survival of heteroplastic RPE cells in the subretinal space of rabbits.