蛋白酪氨酸磷酸酶抑制剂对大鼠RPE细胞α-平滑肌肌动蛋白表达的影响  

Influence of protein tyrosine phosphatases inhibitor on α-SMA expressionin rat RPE cell

作　　者：杜兆东[1] 李颖[1] 胡运韬[1] 罗莉莎[1] 马志中[1] 

机构地区：[1]北京大学第三医院北京大学眼科中心,100191

出　　处：《眼科研究》2010年第4期319-323,共5页Chinese Ophthalmic Research

摘　　要：目的探讨蛋白酪氨酸磷酸酶(PTP)在大鼠视网膜色素上皮(RPE)细胞中的表达及其抑制剂原钒酸钠(SOV)对α-平滑肌肌动蛋白(α-SMA)表达的影响。方法免疫荧光染色法观察PTP的3种典型亚型PTP1B、PTP-LAR及PTEN在大鼠RPE细胞中的表达;SOV与培养液共培养RPE细胞为实验组,正常RPE细胞为对照组。实时定量聚合酶链反应(real-time PCR)检测PTP1B、PTP-LAR及PTEN基因表达的变化,免疫荧光染色法观察α-SMA在细胞中表达的改变。结果PTP1B在非融合和融合大鼠RPE细胞的细胞质和细胞核中均有明显表达,PTP-LAR在非融合细胞中以细胞核膜表达为主,而在融合细胞中主要表达于细胞质,PTEN未见明显表达。SOV孵育后PTP1B、PTP-LAR mRNA的表达量均明显下降(P<0.01),PTEN mRNA表达量很少,实验组与对照组相比差异无统计学意义(P>0.05);免疫荧光检测发现,随着SOV浓度的增加α-SMA阳性细胞比例明显增加(P<0.01),表达明显增强,其在细胞内的分布状态也有明显改变。结论PTP1B、PTP-LAR在体外培养的大鼠RPE细胞中有明显表达,SOV可明显抑制其作用并调节α-SMA的表达和分布。Background Tyrosine phosphorylation,controlled by the coordinated actions of protein tyrosine phosphatase（PTP） and protein tyrosine kinase（PTK）,is a critical control mechanism for numerous physiological processes,including growth,differentiation,metabolism,motility,cell cycle regulation and cytoskeletal function.ObjectiveThe aim of the present study was to determine whether PTPs are expressed in rat retinal pigment epithelium（RPE） cells or not and evaluate the influence of sodium orthovanadate（SOV） on α-smooth muscle actin（α-SMA） expression.MethodsThe RPE cells of SD rat from our laboratory were frozen-thaw and cultured in DMEM/F12 containing 10 fetal bovine serum.The expression of candidate PTPs,including PTP1B,PTP-LAR,and PTEN in cultured rat RPE cells were evaluated by immunofluorescence assay.SOV dissolved by PBS was prepared with free-serum DMEM/F12 into the concentration of 25μmol/L and 50μmol/L and used in the culture of cells.The expression of PTP1B,PTP-LAR,and PTEN in RPE cells was detected using real-time polymerase chain reaction（PCR） after treated with different doses of SOV.The expression of α-SMA was examined by immunofluorescence staining.ResultsPTP1B was intensely expressed in both the nucleus and cytoplasm of unconfluent and confluent cells,and stronger staining was found in the unconfluent cells.PTP-LAR was mainly expressed in nuclear membrane of the unconfluent cells,and in confluent cells,PTP-LAR was expressed mainly in the cytoplasm within the entire RPE cells.The absent positive response for PTEN was seen in both unconfluent and confluent cells.Real-time PCR revealed that the level of PTP1B and PTP-LAR mRNA was significantly declined as the increase of dose of SOV（FPTP1B=1211.05,P=0.00;FPTP-LAR =968.87,P=0.00）.Less expression of PTEN mRNA in RPE cells was found and showed an insignificant difference among control group and experimental groups（FPTEN mRNA=0.12,P=0.89）,and the levels of PTP1B and PTP-LAR mRNA in 25μmol/L and 50μmol/L SOV groups were signif

关 键 词：蛋白酪氨酸磷酸酶 视网膜色素上皮 原钒酸钠 α-平滑肌肌动蛋白 

分 类 号：R774.1[医药卫生—眼科] Q81[医药卫生—临床医学]