Protective effects of neurotrophic factors on RPE physiology

用定量RT-PCR方法检测CNTF、CT1、OSM及其受体亚基在人RPE上的基因表达。用免疫印迹和免疫荧光方法检测受体亚基在原代培养的人胎儿RPE中的定位。用磷酸化实验检测CNTF、CT1和OSM在RPE中激活的信号转导通路。采用BrdU掺入法检测这些神经营养因子对细胞增殖和细胞存活的影响。采用电容探针技术检测CNTF对视网膜下间隙至脉络膜跨RPE的液体转运的影响。 与CNTF和OSM相比，hfRPE中LIF和CT1的mRNA表达水平较高。这些神经营养因子的受体亚基(CNTFR、LIFR、gp130和OsMR)在原代培养的hfRPE中均可检测到，尽管CNTFR的蛋白水平低于免疫印迹检测水平。在原代培养的hfRPE和成人RPE(ARPE-19)中，CNTF、CT1和OSM与其受体结合激活了JAK/STAT3信号通路。OSM可显著激活p44/p42(ERK)MAPK通路，而CNTF和CT1对ERK的磷酸化无明显影响。CNTF对hfRPE的增殖有促进作用，但作用不明显(P&lt；0.05)。CT1对RPE的增殖有剂量反应刺激作用，100 ng/ml时刺激作用最强(25%)，10-80 ng/ml的OSM对hfRPE的增殖呈剂量依赖性抑制作用，CNTF可显著增加跨RPE的液体转运(JV)，从8.7b0.7增加到20.7b3.3Lcm-2hr-1(n=3；P&lt；0.05)。

Gene Expression of CNTF, CT1, OSM and their receptor subunits were analyzed on human RPE by quantitative RT-PCR. Membrane Localization of the receptor subunits were determined by immunoblot and immunofluorescence analysis using primary culture of human fetal RPE (hfRPE). Phosphorylation assays were used to check the signal transduction pathway activated by CNTF, CT1 and OsM in RPE. BrdU incorporation assay was used to test the cell proliferation and cell survival effects of these neurotrophic factors. Capacitance probe technique was used to test the effect of CNTF on fluid transport (JV) across RPE from the subretinal space to choroid. The mRNA of LIF and CT1 are highly expressed in hfRPE compared to CNTF and OsM. The receptor subunits of these neurotrophic factors (CNTFR, LIFR, gp130 and OsMR) were all detected in primary cultures of hfRPE, although the protein level of CNTFR is under the detection level of immunoblots. Binding of CNTF, CT1 and OsM to their receptors activate the JAK/STAT3 signaling pathway in primary culture of hfRPE and adult RPE (ARPE-19). While OsM significantly activated P44/P42 (ERK) MAP kinase pathway, both CNTF and CT1 has no apparent effects on the phosphorylation of ERK. CNTF has small but significant stimulatory effect on hfRPE proliferation (P < 0.05). CT1 show dose response stimulatory effect on RPE proliferation and the maximum stimulatory effect (25%) was observed at 100 ng/ml; OsM show dose-dependent inhibitory effect on hfRPE proliferation from 10-80 ng/ml. Furthermore, CNTF significantly increased fluid transport (JV) across RPE from 8.7 b 0.7 to 20.7 b 3.3 lcm-2 hr-1 (n= 3; P < 0.05).