视网膜下腔注射视网膜色素上皮细胞对小鼠视网膜色素上皮层的影响

Effect of subretinal injection of retinal pigment epithelial cells for retinal pigment epithelium in mice

目的观察视网膜下腔注射视网膜色素上皮（RPE）细胞对小鼠RPE层的影响。方法采用随机数字表法将30只7日龄C57BL／6J新生小鼠分成正常组、氧诱导视网膜病变（OIR）模型组及OIR模型细胞移植组，每组10只小鼠。OIR模型组及OIR模型细胞移植组建立OIR模型。OIR模型细胞移植组小鼠建模后经外路视网膜下腔移植RPE细胞。注射20d后，脊髓脱臼法处死各组小鼠并摘除双眼眼球。行视网膜冰冻切片，荧光显微镜下观察小鼠RPE层厚度变化；采用蛋白免疫印迹法（Westernblot）检测小鼠RPE层RPE65、斑萎蛋白（Bestrophin）、紧密连接蛋白（ZO）-1的蛋白相对表达量；采用实时荧光定量PCR（RT-PCR）检测小鼠RPE层RPE65、Bestrophin、ZO-1的mRNA相对表达量。结果荧光显微镜观察发现，OIR模型组小鼠RPE层较正常组明显变薄，OIR模型细胞移植组小鼠RPE层较OIR模型组明显增厚。Westernblot检测结果显示，OIR模型组、OIR模型细胞移植组小鼠RPE层RPE65、Bestrophin、ZO-1蛋白相对表达量比较，差异均有统计学意义（F=8．597、18．864、25．69i，P〈0．05）。RT—PCR检测结果显示，OIR模型组、OIR模型细胞移植组小鼠RPE层RPE65、Bestrophin、ZO-1mRNA相对表达量比较，差异均有统计学意义（F=39．458、11．461、34．796，P〈0．05）。结论视网膜下腔注射RPE细胞可促进小鼠RPE层增厚；RPE65、Bestrophin蛋白相对表达量增加，ZO-1蛋白相对表达量降低；RPE65、Bestrophin、Z（YlmRNA相对表达量增高。

Objective To observe the effect of subretinal injection of retinal pigment epithelium （RPE） cells for RPE in mice. Methods A total of 30 postnatal day 7 C57BL/6J mice were randomly divided into normal mice group, OIR model group and OIR model cell transplanted group, 10 mice in each group. The OIR model was induced in mice of OIR model group and OIR model cell transplanted group. The RPE cells were subretinal injected into the RPE of mice in OIR model cell transplanted group. At 20 days after the injection, the RPE thickness was evaluated by fluorescence microscope. The expression of RPE65, Bestrophin and zonula occludens-I （ZO-1） were estimated by Western blot and real-time quantitative PCR （RT-PCR）. Results The thickness of RPE in OIR model mice was thinner than that in normal mice~ the thickness of RPE in OIR model cell transplantation mice was significantly thicker than that in the OIR model mice. The results of Western blot and RT-PCR indicated that the differences of protein （F= 8. 597, 18. 864, 25. 691） and mRNA expression （F=39. 458, 11. 461, 34. 796） of RPE65, Bestrophin, ZO-1 were statistically significant between OIR model group and OIR model cell transplanted group （P〈0.05）. Conclusions Subretinal injection of RPE cells can promote RPE thickening. RPE65 and Bestrophin protein relative expression levels increased, ZO-1 protein relative expression levels reduced; mRNA expression levels of RPE65, Bestrophin and ZO-1 genes increased.