GPI-PLD调节CEA的释放对白血病HL-60细胞的增殖和凋亡的影响

Effects of GPI-PLD on the Proliferation and Apoptosis of Leukemia HL-60 Cells by Hydrolyzing CEA

糖基化磷脂酰肌醇特异性磷脂酶D(glycosyl phosphatidyl inositol specific phospholipase D,GPI-PLD)是人体内唯一可水解细胞膜表面GPI结构、调节GPI锚定蛋白释放的酶.将GPI-PLD转染入急性粒细胞白血病(AGL)的HL-60细胞株,采用实时荧光定量PCR法和Western blot法确定转染后HL-60细胞内GPI-PLD的表达水平;并检测GPI-PLD活性;噻唑蓝(MTT)检测HL-60细胞的增殖;流式细胞仪检测HL-60细胞的凋亡.ELISA检测GPI锚定癌胚抗原(CEA)的表达和释放情况.转染GPI-PLD后,HL-60细胞株中GPI-PLD表达量与活性增加;MTT检测显示,GPI-PLD过表达后HL-60细胞株增殖生长受到抑制;流式检测证实HL-60细胞凋亡增加;且GPI锚定的蛋白质CEA释放增加.该结果提示GPI-PLD基因有抗肿瘤的作用,过表达GPI-PLD后能抑制HL-60细胞增殖且促进其凋亡,所涉机制可能与GPI-PLD释放GPI锚定蛋白,增强白血病细胞对补体杀伤的敏感性有关.

The eukaryotic expression of GPI-PLD gene vector was transfected into HL-60 cells taken from a- cute granulocytic leukemia （AGL） cell line. The expression levels of GPI-PLD in post-transfected HL-60 cells were identified by quantitative fluorescence PCR and Western blot. GPI-PLD activities were analyzed quantitatively by TX-114 partition, using GPI anchored placental alkaline phosphatase（PLAP） as a substrate. MTY was used to detect the proliferation in each groups, and the apoptosis of HL-60 cells was estimated by flow cytometry. The GPI anchored carcinoembryonic antigen （CEA） was detected by ELISA. After GPI-PLD gene was transfected into HL-60 cells, the expressions and activities of GPI-PLD in HL-60 cell line greatly increased. Their proliferative capacity following the over-expression of GPI-PLD was obviously depressed and the apoptosis in HL-60 ceils increased. The supernatant CEA levels in HL-60 cells transfected with pcDNA3.1/ GPI-PLD elevated significantly, as compared with those in HL-60 cells of negative control group and non- transfected group. These studies suggested that GPI-PLD gene exhibits anti-tumor effect, overexpression of GPI-PLD gene can inhibit the proliferation of HL-60 cells and promote apoptosis. The possible mechanism involved is related with the release of GPI anchored protein by GPI-PLD, intensifying the sensitivity of leukemic cells towards the complement mediated killing.