神经酰胺诱导药物敏感型和耐药型细胞凋亡机制的探讨

The Mechanism of Ceramide Induced Apoptosis in Drug sensitive and Drug resistant Cell Line

以药物敏感型细胞株Ｋ５６２／Ｓ和耐药型细胞株Ｋ５６２／Ａ０２为对象．观察原癌基因Ｂｃｌ－２的表达量在两种细胞中的差异，以及神经酰胺作为一个新的脂质第二信使诱导细胞凋亡的能力，并利用酪氨酸激酶抑制剂ｇｅｎｉｓｔｅｉｎ，酪氨酸磷酸酯酶抑制剂ｖａｎａｄａｔｅ，观察酪氨酸可逆磷酸化与细胞凋亡间的关系．结果显示：在Ｋ５６２／Ａ０２中Ｂｃｌ－２的表达量明显高于Ｋ５６２／Ｓ；外源性神经酰胺能成功地诱导Ｋ５６２／Ｓ，Ｋ５６２／Ａ０２细胞凋亡，凋亡细胞具有典型的形态学改变和ＤＮＡ“Ｌａｄｄｅｒ”形成，ＦＣＭ检测出现凋亡细胞峰，但在同样的诱导条件下，Ｋ５６２／Ｓ细胞凋亡明显高于Ｋ５６２／Ａ０２细胞．ＦＣＭ检测ｇｅｎｉｓｔｅｉｎ能显著改变这两种细胞生长周期，但细胞阻滞于Ｇ２／Ｍ期，便对神经酰胺诱导的细胞凋亡无明显作用，ｖａｎａｄａｔｅ单独对细胞地明显作用，但与神经酰胺共同作用能明显提高细胞凋亡率．以上结果表明在药物诱导的细胞调亡中Ｂｃｌ－２基因起重要作用，神经酰胺能诱导Ｋ５６２／Ｓ和Ｋ５６２／Ａ０２细胞调亡．

Drug sensitive cell line K 562 /S and drug resistant cell line K 562 /A02 were expose to ceramide which is a new kind of lipid second messenger in signal transduction.Protein tyrosine kinase inhibitor genistein and protein tyrosine phosphatase inhibitor vanadate were used to observe the effect of phosphorylation and dephosphoryaltion of cellular protein on apoptosis.The result showed that the level of Bcl 2 protein in K 562 /A 02 cells is much higher than that in K 562 /S cells.Natural ceramide could induce apoptosis on 562 /A 02 and K 562 /S cells.The apoptosis cells were characterized by typical morphologic changes,DNA"ladder"formation in gel electophoresis and pre G1 peak in the FCM DNA histogram.The reactions of K 562 /A 02 and K 562 /S cells with genistein resulted in cell cycel arrest(G2/M)without triggring apoptosis in these cells.Although vanadae itself had no obvious effect on K 562 /A 02 and K 562 /S cells,pretreatment of cells with vanadate accelerated apoptosis induced by ceramide.It was indicated that Bcl 2gene played an important role in drug resistant cells,and ceramide could induce apoptosis in K 562 /S and K 562 /A 02 cells.Protein tyrosine phosporylation was implicated in apoptosis.