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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。
In order to detect the expression of Cathepsin D and Fas-L in matrine-induced apoptosis of JM cells, the morphological changes of the cells were observed with light microscope and electron microscope. Immunocytochemistry staining was used to observe the expression and localization of Cathepsin D in cells. Semi-quantitative PCR was used to detect the change of Cathepsin D mRNA at transcriptional level. Cathepsin D and Fas-L protein expression was detected by Western Blot. The results showed that 0.6mg / ml dosing group cultured 72h, apoptosis morphological changes. Immunocytochemical staining of Cathepsin D positive signals mainly located in apoptotic morphological changes in the cytoplasm and nucleus. The positive rate of Cathepsin D mRNA in treatment group was significantly higher than that in control group. The brightness of the band of Cathepsin D (52 kD) was higher than that of the control, while the brightness of the product after cutting (32 kD) was weaker than that of the control. Fas-L was up-regulated in the treated group. Tip: Matrine-induced apoptosis of JM cells with Cathepsin D and Fas-L expression changes.