全反视黄酸对豚鼠视网膜色素上皮细胞分2泌TGF-β2及细胞内第二信使IP3和cAMP的影响

Influence of all-trans retinoic acid on the secretion of TGF-β2 and the intracellular second messengers IP3 and cAMP in guinea pig retinal pigment epithelium ceils

目的观察全反视黄酸（ATRA）对培养的豚鼠视网膜色素上皮（RPE）细胞增殖以及分泌转化生长因子p2（TGF—β2）的影响，并观察细胞内第二信使cAMP和IP3的含量变化。方法培养原代豚鼠RPE细胞，传2代后用于实验。实验分3组进行。第1组用不同浓度ATRA（5×10^-6、10×10^6、40×10^-6mol／L）作用于豚鼠RPE细胞，24h后用MTT法检测细胞增殖情况。第2组加入10×10^-6mol／L的ATRA．分别于2、4、6、8、16h后收集培养液，用ELISA方法检测RPE细胞TGF—β2的分泌量。第3组以10×10^-6mol／L的ATRA作用于豚鼠RPE细胞．分别于0min、5min、30min、2h、6h后收集细胞裂解液。行放射免疫和ELISA方法检测细胞内cAMP和IP3的含量变化。每组均以等量溶剂DMSO作为对照。对不同浓度ATRA组间MTT结果行方差分析，其余实验组与对照组间比较行配对t检验。结果5×10^-6、10×100、40×100mol／LATRA作用RPE细胞24h后，DD值分别为0．099±0．008、0．117±0．008、0．087±0．011。与对照组（0．103±0．017）比较，40×10^-6mol／LATRA作用时OD值显著降低，差异有统计学意2（P〈0．05），其他浓度组与对照组相比差异无统计学意义（p〉0．05）。10×10^-6／mol／L的ATRA作用RPE细胞后，TGF—β2的分泌量在作用2、4、6h时较对照组明显升高，差异均有统计学意义（P〈0．05）．8h时无明显变化，16h时降低（P〈O．05）。10×10^-4mol／L的ATRA作用于豚鼠RPE细胞后，IP3的含量在各时间点均较对照组显著下降，且随时间的延长下降越明显；cAMP的含量只有在作用30min和2h时升高．其他时间变化不明显。结论浓度为40×10^-6mol／L的ATRA对豚鼠RPE细胞的增殖有抑制作用。10×10^-6mol／L的ATRA作用RPE细胞后．TGF-β2的分泌量在6h内增加，之后随时间延长而降低。ATRA对RPE细胞的作用可能与IP3的下降有关。

Objective To evaluate the role of all-trans retinoic acid （ATRA） on proliferation and function in the secretion of TGF-β2 and in the related signal cascades in cultured guinea pig retinal pigment epithelium cells （RPE）. Methods RPE ceils were taken from 3-week-old guinea pig eyes. The 2-3 passage cells in the logarithmic growth phase were used for the experiment. Cells were verified by keratin immunohistochemistry. When cells almost attained confluence, the medium was changed to a DMEM/F12 medium without FBS （fetal bovine serum） for 24 hours before being used. The cells were then divided into 3 groups. ①The medium was changed to a DMEM-F12 medium without FBS but contained different concentrations of the drug ATRA （5×10^-6, 10×10^-6, 40×10^-6 mol/L）. After 24 hours, cell proliferation was analyzed using an MTF assay. ②The medium was changed to DMEM/F12 containing 10×10^-4 mol/L ATRA. The TGF-β2 secreted by RPE cells was tested using an EI,ISA kit at 2. 4. 6. 8. and 16 hours. （3）Intracellular 1. 4. 5-trisnhosnhate （IP3） and cvclic adenosine monophosphate （cAMP） were extracted at 0 rain, 5 min, 30 min, 2 h and 6 h, and their concentrations were measured with the ELISA method and radioimmunoassay. The same dose of DMSO was added to all of the control groups. Results Twenty-four hours after ATRA concentrations of 5×10^-6 mol/L, 10×10^-6 mol/L, and 40×10^-6 mol/L were added to RPE cells, the respective OD values were 0.09±0.008, 0.117±0.008, and 0.087±0.011. Compared to the controls, 0.103±0.017, cell proliferation was inhibited by 40×10^-6 mol/L ATRA （P〈0.05）. After 10×10^-6 mol/L ATRA was added, the secretion of TGF-β2 increased in the first 6 hours （P〈0.05）, and then decreased. And the decrease was time related. Intracellular IP3 was inhibited at each time point. However, the amount of cAMP production increased at 30 min and 2 h. Conclusion A concentration of 40×10^-6 mol/L ATRA can inhibit the proliferation of guinea pig RPE cells. But the RPE cells can grow well in lower concentrations. The secretion of TGF-β2 increased in the short term but there was a time-related decrease. And the ATRA influence on RPE ceils may correlate with a decreased second messenger IP3.