PTEN抑制剂对软脂酸引起脐静脉内皮细胞凋亡的保护作用

Protective effect of PTEN inhibitors on palmitate induced apoptosis in human umbilical vein endothelial cells

目的探讨抑癌基因PTEN(phosphatase and tensin homology deleted on chromosome ten)的抑制剂双过氧钒(bpV)对软脂酸(PA)引起人脐静脉内皮细胞(HUVEC)凋亡的保护作用。方法四甲基偶氮唑盐(MTT)测定细胞生存率;Hoechst-PI染色荧光显微镜观测细胞凋亡形态;Annexin-Ⅴ-PI流式细胞术测定细胞凋亡,Westernblotting检测总PTEN和磷酸化PTEN蛋白水平,实时定量PCR测定细胞PTEN mRNA的表达。结果 MTT显示,PA(0.2～0.6mmol/L)作用24h后可明显抑制HUVEC细胞的生长;Hoechst-PI染色荧光显微镜及Annexin-Ⅴ-PI双染流式细胞检测证实不同浓度的PA处理24h后,随着PA浓度(0.2～0.6mmol/L)的增高,其诱导HUVEC细胞的凋亡作用增强,实时定量PCR及Western blotting显示,PA浓度(0.2～0.6mmol/L)作用细胞后PTEN的转录及活性随浓度增高而增加,并呈现一定的剂量依赖性。bpV作用细胞可以增加细胞的生存率,抑制细胞的凋亡,PTEN的转录及蛋白表达降低。结论一定浓度范围内PA剂量依赖性诱导HUVEC细胞发生凋亡性损伤,在此过程中伴随细胞内PTEN表达上调和活性增强,PTEN抑制剂双过氧钒使细胞的凋亡作用减轻,在此过程中PTEN转录及蛋白表达降低,PTEN信号通路可能是PA诱导脐静脉内皮细胞凋亡的重要通路之一,PTEN抑制剂可抑制此通路起到对细胞的保护作用。

Objective To study the protective effect of PTEN inhibitors on palmitate induced apoptosis human umbilical vein endothelial cell（HUVEC）. Methods MTT was used to determine cell survival; Hoechst-PI staining fluorescence microscopy was carried out to observe the morphology of apoptosis ; Annexin- V -PI flow cytometry was used to determine cell apoptosis; Western blotting was used to analize total PTEN and phosphorylated PTEN protein levels; Real- time quantitative PCR was for the expression of PTEN mRNA. Results MTT results showed palmitic acid （PA） （0. 2- 0. 6mmol/L） for 24 hours significantly inhibited the growth of HUVEC cells; Hoechst-Pl staining fluorescence microscopy and Annexin-V-PI double staining flow cytometry confirmed that different concentrations of PA {0. 2-0. 6mmol/L） for 24 hours, with the PA concentration （0.2-0. 6mmol/L） increassed, enhanced cell apoptosis; Real-time quantitative PCR and Western blotting showed that, the transcriptional and activity of PTEN increased with the increasing concentration in a dose dependent manner; The role of PIC can increase cell survival, inhibit apoptosis, decrease PTEN transcription and protein expression. Conclusion PA induced apoptosis in HUVEC cells in certain concentration; In the process, PTEN expression and activity increased, PTEN inhibitor bpV reduced cell apoptosis, in the process PTEN transcription and protein expression decreased; PTEN signaling pathway might be an important signal pathway, PTEN inhibitor could inhibit this path and play a protective effect on ceils.