Protective effects of neurotrophic factors on RPE physiology

用定量RT-PCR方法检测人RPE细胞中CNTF、CT 1、OSM及其受体亚单位的基因表达。用原代培养的人胎儿视网膜色素上皮细胞（hfRPE），通过免疫印迹和免疫荧光分析确定受体亚基的膜定位。用磷酸化法检测CNTF、CT 1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对视网膜下腔至脉络膜的液体转运（JV）的影响。 与CNTF和OsM相比，LIF和CT 1的mRNA在hfRPE中高表达。这些神经营养因子的受体亚基（CNTFR、LIFR、gp 130和OsMR）在hfRPE原代培养物中均被检测到，尽管CNTFR的蛋白水平低于免疫印迹的检测水平。CNTF、CT 1和OsM与其受体的结合激活hfRPE和成人RPE（ARPE-19）的原代培养物中的JAK/STAT 3信号通路。OsM可显著激活P44/P42（ERK）MAP激酶通路，而CNTF和CT 1对ERK磷酸化无明显影响。CNTF对hfRPE细胞增殖有明显的促进作用（P < 0.05）。CT 1对RPE增殖有剂量依赖性刺激作用，在100 ng/ml时刺激作用最大（25%），OsM在10-80 ng/ml范围内对hfRPE增殖有剂量依赖性抑制作用。此外，CNTF显著增加了穿过RPE的液体转运（JV），从8.7 B 0.7增加到20.7 B 3.3 lcm-2 hr-1（n= 3; P < 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).