体外培养的猪眼色素上皮细胞屏障功能的研究

Barrier function of porcine retinal pigment epithelial cells cultured in vitro

目的研究体外培养的猪眼视网膜色素上皮(RPE)细胞的血视网膜外屏障功能。方法对猪眼RPE细胞进行原代培养,第3代细胞接种于微孔滤器,微孔滤器的滤膜为无聚乙烯吡咯酮(PVP)聚碳酸脂膜(polycarbonate),分别于培养1、2、3、4周对滤膜表面行光学显微镜观察,于培养后2周对膜切面行光学显微镜和透射电子显微镜观察;行跨膜电阻测定并用荧光素钠和辣根过氧化物酶(HRP)进行通透性检测。结果成功原代培养了猪眼RPE细胞。光学显微镜观察结果显示,RPE细胞接种1周后,细胞基本汇合,接种后2、3周时RPE细胞密度无明显变化,接种后4周时细胞密度下降;滤膜及细胞标本切面观察结果显示,RPE细胞可在滤膜表面表现出单层有极性生长的特性;接种于微孔滤器2周后,RPE细胞跨上皮细胞电阻达(97.44±11.36)Ω/cm2,并至少持续到接种后3周;通透性检测结果显示,孵育30min后只有0.27%的荧光素钠和0.17%的HRP从滤膜上方到达下方,分子量小的荧光素钠比分子量大的HRP通透率高。结论应用无PVP聚碳酸脂膜的微孔滤器可以建立体外RPE细胞有极性单层生长的模型,并用于屏障功能的研究。

Objective To investigate the blood-retinal barrier （BRB）function of porcine retinal pigment epithelial （RPE ）cells cultured in vitro. Methods Primary porcine RPE cells were cultured,and the third generation were inoculated in a microporous filter with the filter membrane of polyvinylpyrrolidone （PVP）-free polycarbonate membrane. After 1,2,3 and 4 weeks of culture, the surface of filter membrane was observed by light microscope,and after 2 weeks of culture, the section of filter membrane was observed by light microscope and transmission electron microscope. Transepithelial electrical resistance （TER）was detected and the permeability was measured with fluorescein sodium（FS） and horseradish peroxidase（HRP）. Results Primary porcine RPE cells were cultured successfully. RPE cells converged lweek after inoculations 2 and 3 weeks after inoculation,the density of RPE cells did not changed obviously; 4 weeks after inoculation, the density of RPE cells decreased. The characteristics of polarized growth of monolayer were found in RPE cells on the surface of filter membrane; 2 weeks after inoculation, the TER of RPE cells was（97. 44± 11. 36）Ω/cm^2 which maintained till the 3rd week after inocubation. After incubated for 30 minutes,only 0. 27% of FS and 0.17 % HRP reached the inferior filter membrane,and the permeability rate of SF with low molecular weight was higher than which of HRP with high molecular weight. Conclusions The filter with PVP-free polycarbonate membrane may be used to set up the model of RPE cells with polarized growth of monolayer and investigate the barrier function of RPE cells.