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

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β2(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、40×10-6 mol/L)作用于豚鼠RPE细胞,24 h后用MTY法检测细胞增殖情况.第2组加入10×10-6 mol/L的ATRA.分别于2、4、6、8、16 h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10×10-6 mol/L的ATRA作用于豚鼠RPE细胞,分别于0 min、5 min、30 min、2 h,6 h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、10×10-6、40×10-6 mol/L ATRA作用RPE细胞24 h后,OD值分别为0.099±0.008、0.117±0.008、0.0871±0.011.与对照组(0.103±0.017)比较,40×10-6 mol/L ATRA作用时OD值显著降低,差异有统计学意义(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、6 h时较对照组明显升高,差异均有统计学意义(P<0.05).8 h时无明显变化,16 h时降低(P<0.05).10×10-6 mol/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30 min和2 h时升高,其他时同变化不明显.结论 浓度为40×10-6 mol/L的ATRA对豚鼠RPE细胞的增殖有抑制作用.10×10-6 mol/L的ATRA作用RPE细胞后,TGF-β2的分泌量在6 h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.

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 cells

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 cells 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 6f the drug ATRA (5×10-6, 10×10-6, 40×10-6 mol/L). After 24 hours, cell proliferation was analyzed using an MTT assay. ②The medium was changed to DMEM/F12 containing 10×10-6 mol/L ATRA. The TGF-β2 secreted by RPE cells was tested using an ELISA kit at 2, 4, 6, 8, and 16 hours. ③ Intracellular 1, 4, 5 -trisphosphate (IP3) and cyclic adenosine monophosphate (cAMP) were extracted at 0 min, 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 0.099±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