中华眼视光学与视觉科学杂志
中華眼視光學與視覺科學雜誌
중화안시광학여시각과학잡지
CHINESE JOURNAL OF OPTOMETRY OPHTHALMOLOGY AND VISUAL SCIENCE
2010年
3期
189-193
,共5页
张未娟%邓志宏%赵少贞%赵小云%焦春娜
張未娟%鄧誌宏%趙少貞%趙小雲%焦春娜
장미연%산지굉%조소정%조소운%초춘나
维甲酸%豚鼠%视网膜%色素上皮,眼%近视%转化生长因子β2%第二信使系统
維甲痠%豚鼠%視網膜%色素上皮,眼%近視%轉化生長因子β2%第二信使繫統
유갑산%돈서%시망막%색소상피,안%근시%전화생장인자β2%제이신사계통
Retinoic acid%Guinea pig%Retina%Pigment epithelial of eye%Myopia%Transforming growth factor β2%Second messenger systems
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.
目的 觀察全反視黃痠(ATRA)對培養的豚鼠視網膜色素上皮(RPE)細胞增殖以及分泌轉化生長因子β2(TGF-β2)的影響,併觀察細胞內第二信使cAMP和IP3的含量變化.方法 培養原代豚鼠RPE細胞,傳2代後用于實驗.實驗分3組進行.第1組用不同濃度ATRA(5×10-6、10×10-6、40×10-6 mol/L)作用于豚鼠RPE細胞,24 h後用MTY法檢測細胞增殖情況.第2組加入10×10-6 mol/L的ATRA.分彆于2、4、6、8、16 h後收集培養液,用ELISA方法檢測RPE細胞TGF-β2的分泌量.第3組以10×10-6 mol/L的ATRA作用于豚鼠RPE細胞,分彆于0 min、5 min、30 min、2 h,6 h後收集細胞裂解液,行放射免疫和ELISA方法檢測細胞內cAMP和IP3的含量變化.每組均以等量溶劑DMSO作為對照.對不同濃度ATRA組間MTT結果行方差分析,其餘實驗組與對照組間比較行配對t檢驗.結果 5×10-6、10×10-6、40×10-6 mol/L ATRA作用RPE細胞24 h後,OD值分彆為0.099±0.008、0.117±0.008、0.0871±0.011.與對照組(0.103±0.017)比較,40×10-6 mol/L ATRA作用時OD值顯著降低,差異有統計學意義(P<0.05),其他濃度組與對照組相比差異無統計學意義(P>0.05).10×10-6 mol/L的ATRA作用RPE細胞後,TGF-β2的分泌量在作用2、4、6 h時較對照組明顯升高,差異均有統計學意義(P<0.05).8 h時無明顯變化,16 h時降低(P<0.05).10×10-6 mol/L的ATRA作用于豚鼠RPE細胞後,IP3的含量在各時間點均較對照顯著下降,且隨時間的延長下降越明顯;cAMP的含量隻有在作用30 min和2 h時升高,其他時同變化不明顯.結論 濃度為40×10-6 mol/L的ATRA對豚鼠RPE細胞的增殖有抑製作用.10×10-6 mol/L的ATRA作用RPE細胞後,TGF-β2的分泌量在6 h內增加,之後隨時間延長而降低.ATRA對RPE細胞的作用可能與IP3的下降有關.
목적 관찰전반시황산(ATRA)대배양적돈서시망막색소상피(RPE)세포증식이급분비전화생장인자β2(TGF-β2)적영향,병관찰세포내제이신사cAMP화IP3적함량변화.방법 배양원대돈서RPE세포,전2대후용우실험.실험분3조진행.제1조용불동농도ATRA(5×10-6、10×10-6、40×10-6 mol/L)작용우돈서RPE세포,24 h후용MTY법검측세포증식정황.제2조가입10×10-6 mol/L적ATRA.분별우2、4、6、8、16 h후수집배양액,용ELISA방법검측RPE세포TGF-β2적분비량.제3조이10×10-6 mol/L적ATRA작용우돈서RPE세포,분별우0 min、5 min、30 min、2 h,6 h후수집세포렬해액,행방사면역화ELISA방법검측세포내cAMP화IP3적함량변화.매조균이등량용제DMSO작위대조.대불동농도ATRA조간MTT결과행방차분석,기여실험조여대조조간비교행배대t검험.결과 5×10-6、10×10-6、40×10-6 mol/L ATRA작용RPE세포24 h후,OD치분별위0.099±0.008、0.117±0.008、0.0871±0.011.여대조조(0.103±0.017)비교,40×10-6 mol/L ATRA작용시OD치현저강저,차이유통계학의의(P<0.05),기타농도조여대조조상비차이무통계학의의(P>0.05).10×10-6 mol/L적ATRA작용RPE세포후,TGF-β2적분비량재작용2、4、6 h시교대조조명현승고,차이균유통계학의의(P<0.05).8 h시무명현변화,16 h시강저(P<0.05).10×10-6 mol/L적ATRA작용우돈서RPE세포후,IP3적함량재각시간점균교대조현저하강,차수시간적연장하강월명현;cAMP적함량지유재작용30 min화2 h시승고,기타시동변화불명현.결론 농도위40×10-6 mol/L적ATRA대돈서RPE세포적증식유억제작용.10×10-6 mol/L적ATRA작용RPE세포후,TGF-β2적분비량재6 h내증가,지후수시간연장이강저.ATRA대RPE세포적작용가능여IP3적하강유관.
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 the short term but there was a time-related decrease. And the ATRA influence on RPE cells may correlate with a decreased second messenger IP3.
