CAJ | 학술논문

为研究鞘脂激活蛋白原(Prosaposin)对细胞增殖、细胞凋亡的调控及其可能的分子机制,以pcDNA3.1 in NIH3T3阴性对照细胞株和过表达prosaposin的Psap-Myc in NIH3T3细胞株为模型,噻唑草稿蓝(MTT)比色法检测prosaposin对细胞增殖的影响;Annexin V联合碘化丙啶(Propidium iodide,PI)法检测血清饥饿状态下prosaposin 对细胞凋亡的影响:Western blotting检测P13K/Akt信号通路中蛋白磷酸化水平的变化;Real-time PCR检测P13K/Akt信号通路下游靶分子表达水平的改变.结果表明prosaposin可活化P13K/Akt信号通路,提高Akt~(Ser473)的磷酸化水平,抑制细胞周期抑制基因P27~(KIP1)的表达,上调细胞周期蛋白Cyclin Dl的表达,促进细胞周期从G_1→S期进展;诱导survival基因cIAP1、cIAP2的表达,促进细胞存活.这些结果提示,prosaposin对细胞增殖和凋亡的调控可能是通过P13K/Akt信号通路及其下游靶分子进行的.
위연구초지격활단백원(Prosaposin)대세포증식、세포조망적조공급기가능적분자궤제,이pcDNA3.1 in NIH3T3음성대조세포주화과표체prosaposin적Psap-Myc in NIH3T3세포주위모형,새서초고람(MTT)비색법검측prosaposin대세포증식적영향;Annexin V연합전화병정(Propidium iodide,PI)법검측혈청기아상태하prosaposin 대세포조망적영향:Western blotting검측P13K/Akt신호통로중단백린산화수평적변화;Real-time PCR검측P13K/Akt신호통로하유파분자표체수평적개변.결과표명prosaposin가활화P13K/Akt신호통로,제고Akt~(Ser473)적린산화수평,억제세포주기억제기인P27~(KIP1)적표체,상조세포주기단백Cyclin Dl적표체,촉진세포주기종G_1→S기진전;유도survival기인cIAP1、cIAP2적표체,촉진세포존활.저사결과제시,prosaposin대세포증식화조망적조공가능시통과P13K/Akt신호통로급기하유파분자진행적.
pcDNA3.1 in NIH3T3 and Psap-Myc in NIH3T3 cell strains were used as cell models in order to study the effect of prosaposin on cell proliferation,cell apoptosis and its possible molecular mechanism.MTr assay and Annexin V/PI apoptosis kit were used to detect the effect of prosaposin on cell proliferation and cell apoptosis induced by serum-starvation stress,respectively.Western blotting was conducted to detect the phosphorylative level of P13K/Akt pathway,and real-time PCR was carried out to explore the expression of the genes regulated by P13K/Akt pathway.Prosaposin protein was proved to activate the P13K/Akt signal pathway,upregulate the phosphorylative activity of Akt at Sefine 473,downregulate the expression of P27~(Kip1) gene,upregulate the expression of Cyclin D1 gene and then promote the G_1/S transition,and upregulate the expression of survival genes cIAP1 and cIAP2 and then prevent cell apoptosis.These findings suggest that the growth promotion and anti-apoptotic activity of prosaposin may be partly through the P13K/Akt signal pathway and its downstream targeted genes.