Cathepsin D及Fas ligand在苦参碱诱导急性T淋巴母细胞白血病JM细胞株凋亡中的表达研究

THE EXPRESSION OF CATHEPSIN D AND FAS LIGAND ON PCD OF JM CELLS INDUCED BY MATRINE

为了检测苦参碱诱导JM细胞发生凋亡过程中Cathepsin D、Fas-L的表达情况,应用光镜及电镜观察加药及未加药组细胞形态学变化。免疫细胞化学染色观察Cathepsin D在细胞内的表达及定位。半定量PCR检测Cathepsin D mRNA在转录水平的变化并用Western Blot检测Cathepsin D、Fas-L蛋白表达。结果显示0.6mg/ml加药组细胞培养72h,出现凋亡形态学改变。免疫细胞化学染色CathepsinD阳性信号主要位于呈凋亡形态学改变的胞浆和胞核内。其阳性细胞率在处理组明显高于对照组,处理组Cathepsin D的mRNA转录上调。处理组前体Cathepsin D(52kD)的条带亮度较对照组强,而切割后产物(32kD)亮度较对照组弱;Fas-L在处理组表达上调。提示:苦参碱诱导JM细胞的凋亡伴随Cathepsin D及Fas-L的表达改变。

To study the expression of cathepsin D and Fas-L on PCD of JM cells induced by matrine, morphologic changes were observed under light microscope with Wright-Giemsa staining and electron microscope. Immunohistochemical staining was used to observe the expression and location of cathepsin D within the JM cells. RT-PCR and Western Blot were conducted to investigate the expression of cathepsin D and Fas-L in transcriptional and translational level of JM cells respectively with and without treatment by matrine. At the 72h after the treatment of matrine(0.6mg/ml), typical apoptosis features of cells were observed under light microscope and electron microscope, and those features are more prominent with the time prolonging. Immunohistochemical staining found that cathepsin D positive signal intensively located in cytoplasm and nucleolus of those cells with apoptotic characteristics in both treatment and control group. While the rate of positive cells to 1000 positive and negative cells in treatment group was significantly higher than that in control. RT-PCR indicated the transcription of cathepsin D mRNA was up-regulated gradually at the 96h and 120h comparing to control group. Western Blot showed that 52kD procathepsin D was stronger in treatment group than that in control, while the pattern of 32kD processed form was just the opposite. The expression of Fas-L in JM cells was up-regulated after matrine treatment. The above dada suggest that matrine can up-regulate the expression of cathepsin D in both transcriptional and translational level. While at the same time, this alkaloid can interfere the process of procathepsin D(52kD)to its 32kD mature form, and result in the arrest of 52kD form. PCD induced by matrine executes through Fas/Fas-L pathway, and 52kD cathepsin D maybe play important role in this process. Out of regard for the independence of RNA and protein synthesization in the Fas/Fas-L pathway, we suppose that up-regulation of cathepsin D precedes the activation of the Fas receptor.