中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
11期
965-969
,共5页
吴梦竹%邹悦%顾青%樊莹
吳夢竹%鄒悅%顧青%樊瑩
오몽죽%추열%고청%번형
胰岛素%信号转导%磷脂酰肌醇-3-羟基酶/苏氨酸激酶%视网膜色素上皮细胞%转化生长因子β2%近视%细胞培养
胰島素%信號轉導%燐脂酰肌醇-3-羥基酶/囌氨痠激酶%視網膜色素上皮細胞%轉化生長因子β2%近視%細胞培養
이도소%신호전도%린지선기순-3-간기매/소안산격매%시망막색소상피세포%전화생장인자β2%근시%세포배양
Insulin%Signal transduction%PI3K/Akt%Retinal pigment epithelial cell%Transforming growth factor-β2%Myopia%Cell culture
背景 胰岛素具有促进近视发生的作用,胰岛素受体已被证实存在于视网膜色素上皮(RPE)细胞上,转化生长因子β2(TGF-β2)是RPE细胞分泌的重要的近视信号因子之一.目的 研究胰岛素是否通过磷脂酰肌醇-3-羟基酶/苏氨酸激酶(PI3K/Akt)通路促进RPE细胞增生及分泌TGF-β2.方法 使用含质量分数10％胎牛血清的DMEM培养基常规培养人RPE细胞株ARPE-19细胞,分别将10×103 U(商品单位)/ml胰岛素、LY294002、10× 103 U/ml胰岛素+LY294002、Wortmanin和10× 103 U/ml胰岛素+Wortmanin20 μmol/L各20μl加入培养基,另设常规培养的细胞作为空白对照.作用后48 h,采用MTS法检测各组RPE细胞的增生情况,采用ELISA法检测各组RPE细胞上清液中TGF-β2质量浓度,以评估细胞分泌TGF-β2的能力;各种药物作用后1h和2h,采用逆转录PCR(RT-PCR)法检测各组细胞中TGF-β2mRNA的相对表达量.结果 MTS结果显示,胰岛素+LY294002组RPE细胞的增生值(A值)明显低于胰岛素组(0.75±0.03 versus0.98±0.04),差异有统计学意义(P＜0.05),而胰岛素+Wortmanin组RPE细胞增生与胰岛素组相比,差异无统计学意义(0.97±0.07 versus 0.98±0.04,P＞0.05).ELISA法检测结果显示,胰岛素+LY294002组和胰岛素+Wortmanin组RPE细胞上清液中TGF-β2质量浓度分别为(11.59±2.85)pg/ml和(49.16±10.94) pg/ml,明显低于胰岛素组的(548.50±35.18) pg/ml,差异均有统计学意义(P＜0.05),且胰岛素+LY294002组细胞上清液中TGF-β2质量浓度的下降值明显高于胰岛素+Wortmanin组,差异有统计学意义(t=8.131,P=0.000).RT-PCR结果显示,药物作用后1h和2h,胰岛素+LY294002组和胰岛素+Wortmanin组细胞中TGF-β2mRNA的相对表达量均明显低于胰岛素组,差异均有统计学意义(P＜0.05),胰岛素+ LY294002组细胞中TGF-β2mRNA相对表达量的下降程度稍大于胰岛素+Wortmanin组,作用后1h差异有统计学意义(t=4.176,P=0.014),但作用后2h差异无统计学意义(t=0.756,P=0.492).结论 胰岛素可以通过PI3 K/Akt途径促进RPE细胞增生,并促进RPE细胞分泌TGF-β2以进行信号传递,可能是胰岛素促进近视发生的机制之一.
揹景 胰島素具有促進近視髮生的作用,胰島素受體已被證實存在于視網膜色素上皮(RPE)細胞上,轉化生長因子β2(TGF-β2)是RPE細胞分泌的重要的近視信號因子之一.目的 研究胰島素是否通過燐脂酰肌醇-3-羥基酶/囌氨痠激酶(PI3K/Akt)通路促進RPE細胞增生及分泌TGF-β2.方法 使用含質量分數10％胎牛血清的DMEM培養基常規培養人RPE細胞株ARPE-19細胞,分彆將10×103 U(商品單位)/ml胰島素、LY294002、10× 103 U/ml胰島素+LY294002、Wortmanin和10× 103 U/ml胰島素+Wortmanin20 μmol/L各20μl加入培養基,另設常規培養的細胞作為空白對照.作用後48 h,採用MTS法檢測各組RPE細胞的增生情況,採用ELISA法檢測各組RPE細胞上清液中TGF-β2質量濃度,以評估細胞分泌TGF-β2的能力;各種藥物作用後1h和2h,採用逆轉錄PCR(RT-PCR)法檢測各組細胞中TGF-β2mRNA的相對錶達量.結果 MTS結果顯示,胰島素+LY294002組RPE細胞的增生值(A值)明顯低于胰島素組(0.75±0.03 versus0.98±0.04),差異有統計學意義(P＜0.05),而胰島素+Wortmanin組RPE細胞增生與胰島素組相比,差異無統計學意義(0.97±0.07 versus 0.98±0.04,P＞0.05).ELISA法檢測結果顯示,胰島素+LY294002組和胰島素+Wortmanin組RPE細胞上清液中TGF-β2質量濃度分彆為(11.59±2.85)pg/ml和(49.16±10.94) pg/ml,明顯低于胰島素組的(548.50±35.18) pg/ml,差異均有統計學意義(P＜0.05),且胰島素+LY294002組細胞上清液中TGF-β2質量濃度的下降值明顯高于胰島素+Wortmanin組,差異有統計學意義(t=8.131,P=0.000).RT-PCR結果顯示,藥物作用後1h和2h,胰島素+LY294002組和胰島素+Wortmanin組細胞中TGF-β2mRNA的相對錶達量均明顯低于胰島素組,差異均有統計學意義(P＜0.05),胰島素+ LY294002組細胞中TGF-β2mRNA相對錶達量的下降程度稍大于胰島素+Wortmanin組,作用後1h差異有統計學意義(t=4.176,P=0.014),但作用後2h差異無統計學意義(t=0.756,P=0.492).結論 胰島素可以通過PI3 K/Akt途徑促進RPE細胞增生,併促進RPE細胞分泌TGF-β2以進行信號傳遞,可能是胰島素促進近視髮生的機製之一.
배경 이도소구유촉진근시발생적작용,이도소수체이피증실존재우시망막색소상피(RPE)세포상,전화생장인자β2(TGF-β2)시RPE세포분비적중요적근시신호인자지일.목적 연구이도소시부통과린지선기순-3-간기매/소안산격매(PI3K/Akt)통로촉진RPE세포증생급분비TGF-β2.방법 사용함질량분수10％태우혈청적DMEM배양기상규배양인RPE세포주ARPE-19세포,분별장10×103 U(상품단위)/ml이도소、LY294002、10× 103 U/ml이도소+LY294002、Wortmanin화10× 103 U/ml이도소+Wortmanin20 μmol/L각20μl가입배양기,령설상규배양적세포작위공백대조.작용후48 h,채용MTS법검측각조RPE세포적증생정황,채용ELISA법검측각조RPE세포상청액중TGF-β2질량농도,이평고세포분비TGF-β2적능력;각충약물작용후1h화2h,채용역전록PCR(RT-PCR)법검측각조세포중TGF-β2mRNA적상대표체량.결과 MTS결과현시,이도소+LY294002조RPE세포적증생치(A치)명현저우이도소조(0.75±0.03 versus0.98±0.04),차이유통계학의의(P＜0.05),이이도소+Wortmanin조RPE세포증생여이도소조상비,차이무통계학의의(0.97±0.07 versus 0.98±0.04,P＞0.05).ELISA법검측결과현시,이도소+LY294002조화이도소+Wortmanin조RPE세포상청액중TGF-β2질량농도분별위(11.59±2.85)pg/ml화(49.16±10.94) pg/ml,명현저우이도소조적(548.50±35.18) pg/ml,차이균유통계학의의(P＜0.05),차이도소+LY294002조세포상청액중TGF-β2질량농도적하강치명현고우이도소+Wortmanin조,차이유통계학의의(t=8.131,P=0.000).RT-PCR결과현시,약물작용후1h화2h,이도소+LY294002조화이도소+Wortmanin조세포중TGF-β2mRNA적상대표체량균명현저우이도소조,차이균유통계학의의(P＜0.05),이도소+ LY294002조세포중TGF-β2mRNA상대표체량적하강정도초대우이도소+Wortmanin조,작용후1h차이유통계학의의(t=4.176,P=0.014),단작용후2h차이무통계학의의(t=0.756,P=0.492).결론 이도소가이통과PI3 K/Akt도경촉진RPE세포증생,병촉진RPE세포분비TGF-β2이진행신호전체,가능시이도소촉진근시발생적궤제지일.
Background Insulin can promote the occurrence of myopia.It has been proven that insulin receptor exists in human retinal pigment epithelial (RPE) cells and can promote RPE cells to secrete transforming growth factor-β2(TGF-β2),which is one of the most important myopic signal molecules.Objective This study was to investigate if PI3K/Akt mediates the promotive effects of insulin on proliferation of human RPE cells and secretion of TGF-β2.Methods Human RPE cell line,ARPE-19 cells,were regularly cultured using DMEM containing 10％ fetal bovine serum,and 10× 103 U/ml insulin,LY294002,10× 103 U/ml insulin+LY294002,Wortmanin,10× 103 U/ml insulin+Wortmanin were added into the medium respectively for 48 hours,and the regularly cultured cells served as blank controls.The proliferation value (absorbance,A) of the cells was evaluated by MTS,and the TGF-β2 level in the cell supernatant was detected by ELISA.The relative expression of TGF-β2 mRNA in the cells was assayed using reverse transcription PCR (RT-PCT) 1 hour and 2 hours after the addition of reagents.Results MTS showed that the proliferation value of the cells in the insulin+LY294002 group was 0.75±0.03,which was significantly lower than that in the insulin group (0.98± 0.04).No significant difference was seen in the proliferative value between the insulin+Wortmanin group and the insulin group (0.97±0.07 versus 0.98± 0.04,P＞0.05).ELISA revealed that the content of TGF-β2 in the the cell supernatant was (11.59±2.85) pg/ml and (49.16± 10.94) pg/ml in the insulin + LY294002 group and the insulin + Wortmanin group,respectively,showing a significant decline in comparison with (548.50±35.18) pg/ml in the insulin group (both at P＜0.05).A significant difference was found in the TGF-β2 content between the insulin+LY294002 group and the insulin+Wortmanin group (t =8.131,P =0.000).The RT-PCR showed that 1 hour and 2 hours after addition of the reagents,the expression levels of TGF-β2 mRNA in the cells were lower in both insulin+LY294002 group and insulin+Wortmanin group than those in the insulin group (P＜0.05).The decline range of TGF-β2 mRNA expression level was more significant in the insulin+LY294002 group than that in the insulin+Wortmanin group at 1 hour (t=4.176,P=0.014) rather than at 2 hours (t=0.756,P=0.492).Conclusions Insulin can promote the proliferation of human RPE cells and secretion of TGF-β2 through PI3K/Akt pathway.This may be one of the mechanisms of insulin causes myopia.
