中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
5期
444-449
,共6页
陈春丽%申焕君%张宗端%周仲楼%陈晓燕%闫东升%宋宗明
陳春麗%申煥君%張宗耑%週仲樓%陳曉燕%閆東升%宋宗明
진춘려%신환군%장종단%주중루%진효연%염동승%송종명
表皮生长因子%Müller细胞%增生%迁移%缺氧%信号传导通路%增生性玻璃体视网膜病变
錶皮生長因子%Müller細胞%增生%遷移%缺氧%信號傳導通路%增生性玻璃體視網膜病變
표피생장인자%Müller세포%증생%천이%결양%신호전도통로%증생성파리체시망막병변
Epidermal growth factor%Müller cells%Proliferation%Migration%Hypoxia%Signal pathway%Proliferative vitreoretinopathy
背景 研究证实表皮生长因子(EGF)可促进鼠视网膜Müller细胞的增生和迁移,但EGF能否促进人眼Müller细胞的增生迁移尚未见报道.目的 探讨人眼Müller细胞能否自身表达和分泌EGF及EGF对Müller细胞增生迁移的影响及其作用机制.方法 同一代人永生株Müller细胞系MIO1MI细胞用高糖DMEM进行培养,利用MTT比色法观察不同质量浓度EGF作用24、48、72 h,不同浓度CoCl2作用12 h和24 h,加入导致Müller细胞半数致死量的CoCl2浓度前2 h加入不同质量浓度EGF的Müller细胞增生情况;Transwell小室观察正常及CoCl2缺氧条件下用不同质量浓度EGF作用24、48、72 h后Müller细胞的迁移情况;采用ELISA法观察Müller细胞表达和分泌EGF的情况;通过Western blot法检测体外不同条件培养下EGF对ERK1/2及Akt信号转导通路的作用.结果 正常培养条件下,不同质量浓度的EGF作用后Müller细胞的吸光度(A570)值的总体比较差异有统计学意义(F=123.765,P=0.000),100 mg/L EGF促Müller细胞增生作用最强,为对照组的1.6倍,10 mg/L EGF促Mliler细胞迁移的作用最强,为对照组的4.5倍.各EGF组Müller细胞的A570值均明显高于对照组,差异有统计学意义(P<0.01).缺氧条件下培养Müller细胞半数致死量的CoCl2浓度为600 nmol/L,提前2 h加入10～100μg/L EGF后,EGF对抗缺氧所致Müller细胞的损伤作用最明显.700 nmol/L CoCl2致Müller细胞损伤80%时其自身低分泌7.8 ng/L EGF.10～100 mg/L EGF作用Müller细胞促增生迁移作用信号最明显,600 nmol/L CoCl2作用Müller细胞24 h后ERK1/2及Akt信号强度减弱,提前2 h加入外源性EGF后,ERK1/2及Akt两条信号通路最明显.结论 EGF能以剂量依赖的方式促进体外培养的Müller细胞增生及迁移.正常培养条件下的Müller细胞自身不表达,缺氧条件下Müller细胞自身可少量分泌EGF.EGF可能通过ERK1/2及Akt信号转导通路介导Müller细胞的增生和迁移,并对CoCl2损伤的Müller细胞发挥保护作用.
揹景 研究證實錶皮生長因子(EGF)可促進鼠視網膜Müller細胞的增生和遷移,但EGF能否促進人眼Müller細胞的增生遷移尚未見報道.目的 探討人眼Müller細胞能否自身錶達和分泌EGF及EGF對Müller細胞增生遷移的影響及其作用機製.方法 同一代人永生株Müller細胞繫MIO1MI細胞用高糖DMEM進行培養,利用MTT比色法觀察不同質量濃度EGF作用24、48、72 h,不同濃度CoCl2作用12 h和24 h,加入導緻Müller細胞半數緻死量的CoCl2濃度前2 h加入不同質量濃度EGF的Müller細胞增生情況;Transwell小室觀察正常及CoCl2缺氧條件下用不同質量濃度EGF作用24、48、72 h後Müller細胞的遷移情況;採用ELISA法觀察Müller細胞錶達和分泌EGF的情況;通過Western blot法檢測體外不同條件培養下EGF對ERK1/2及Akt信號轉導通路的作用.結果 正常培養條件下,不同質量濃度的EGF作用後Müller細胞的吸光度(A570)值的總體比較差異有統計學意義(F=123.765,P=0.000),100 mg/L EGF促Müller細胞增生作用最彊,為對照組的1.6倍,10 mg/L EGF促Mliler細胞遷移的作用最彊,為對照組的4.5倍.各EGF組Müller細胞的A570值均明顯高于對照組,差異有統計學意義(P<0.01).缺氧條件下培養Müller細胞半數緻死量的CoCl2濃度為600 nmol/L,提前2 h加入10～100μg/L EGF後,EGF對抗缺氧所緻Müller細胞的損傷作用最明顯.700 nmol/L CoCl2緻Müller細胞損傷80%時其自身低分泌7.8 ng/L EGF.10～100 mg/L EGF作用Müller細胞促增生遷移作用信號最明顯,600 nmol/L CoCl2作用Müller細胞24 h後ERK1/2及Akt信號彊度減弱,提前2 h加入外源性EGF後,ERK1/2及Akt兩條信號通路最明顯.結論 EGF能以劑量依賴的方式促進體外培養的Müller細胞增生及遷移.正常培養條件下的Müller細胞自身不錶達,缺氧條件下Müller細胞自身可少量分泌EGF.EGF可能通過ERK1/2及Akt信號轉導通路介導Müller細胞的增生和遷移,併對CoCl2損傷的Müller細胞髮揮保護作用.
배경 연구증실표피생장인자(EGF)가촉진서시망막Müller세포적증생화천이,단EGF능부촉진인안Müller세포적증생천이상미견보도.목적 탐토인안Müller세포능부자신표체화분비EGF급EGF대Müller세포증생천이적영향급기작용궤제.방법 동일대인영생주Müller세포계MIO1MI세포용고당DMEM진행배양,이용MTT비색법관찰불동질량농도EGF작용24、48、72 h,불동농도CoCl2작용12 h화24 h,가입도치Müller세포반수치사량적CoCl2농도전2 h가입불동질량농도EGF적Müller세포증생정황;Transwell소실관찰정상급CoCl2결양조건하용불동질량농도EGF작용24、48、72 h후Müller세포적천이정황;채용ELISA법관찰Müller세포표체화분비EGF적정황;통과Western blot법검측체외불동조건배양하EGF대ERK1/2급Akt신호전도통로적작용.결과 정상배양조건하,불동질량농도적EGF작용후Müller세포적흡광도(A570)치적총체비교차이유통계학의의(F=123.765,P=0.000),100 mg/L EGF촉Müller세포증생작용최강,위대조조적1.6배,10 mg/L EGF촉Mliler세포천이적작용최강,위대조조적4.5배.각EGF조Müller세포적A570치균명현고우대조조,차이유통계학의의(P<0.01).결양조건하배양Müller세포반수치사량적CoCl2농도위600 nmol/L,제전2 h가입10～100μg/L EGF후,EGF대항결양소치Müller세포적손상작용최명현.700 nmol/L CoCl2치Müller세포손상80%시기자신저분비7.8 ng/L EGF.10～100 mg/L EGF작용Müller세포촉증생천이작용신호최명현,600 nmol/L CoCl2작용Müller세포24 h후ERK1/2급Akt신호강도감약,제전2 h가입외원성EGF후,ERK1/2급Akt량조신호통로최명현.결론 EGF능이제량의뢰적방식촉진체외배양적Müller세포증생급천이.정상배양조건하적Müller세포자신불표체,결양조건하Müller세포자신가소량분비EGF.EGF가능통과ERK1/2급Akt신호전도통로개도Müller세포적증생화천이,병대CoCl2손상적Müller세포발휘보호작용.
Background Researches demonstrated that epidermal growth factor (EGF) can pmmote the proliferation and migration of Mailer cells of rat.However,whether EGF plays the role on human Müller cells is unknown.Objective Present study was to address if Müller ells express and secret EGF and explore the effects of EGF on the proliferation and migration of Müller cells under the normal and low oxygen conditions.Methods Human Müller cell line MIO-M1 cells were cultured and incubated in DMEM with high glucose and different concentrations of EGF in the presence or absence of varied amounts of GoCl2 for indicated time points.The preliferation and migration of Müller cells were analyzed with MTT and Transwell assay respectively.The expression and secretion of EGF in Müller cells were measured by using ELISA.Western blot was performed to detect and assess the protective role of ERKI／2 and Akt signal pathway under the presence of EGF.Results EGF stimulated the proliferation and migration of Müller cells in a concentration-dependent manner with a significantly different A570 values in the Müller cells among the various groups (F=123. 765, P = 0. 000). The maximum proliferation rate of Müller cells was found in 100 mg/L EGF group with the 1.6 times of elevation more than the free-EGF cells. Also,the 4. 5 times of increase was reached in migration rate in 10 mg/L EGF group more than free-EGF cells. In hypoxia condition,the median lethal concentration of CoCl2 was 600 nmoL/L. After pretreated with 10-100 mg/L EGF,the A570 values of Müller cells were obviously elevated with the statistical difference among various concentrations of EGF groups (F=22. 900 ,P=0. 000). The ELISA results showed that Müller cells secreted 7.8 μg/L EGF in the presence condition of 700 nmoL/L CoCl2. Western blot revealed that the presence of CoCl2 reduced the expression of ERK1/2 and Akt in Müller ells,however,pretreatment with EGF antagonized the harmful effect of CoCl2 on Müller cells.Conclusion EGF can induce the proliferation and migration of Müller cells at the dose-dependent pattern. Müller cells do not express and secrete EGF by themselves in normal condition, but hypoxia induce the expression and secretion of EGF. It is indicated that EGF promote the proliferation and migration of Müller cells through activating ERK1/2 and Akt signal pathways. External EGF can protect Müller cells against CoCl2 -induced cell damage.
