腺苷酸环化酶对泡沫细胞胆固醇流出的影响

Effect of adenylate cyclase on the cholesterol efflux from foam cells

目的观察腺苷酸环化酶AC1和AC4对泡沫细胞胆固醇流出的影响。方法以人AC1和AC4基因为靶点,设计合成sh RNA寡核苷酸片段,插入慢病毒载体p GMLV-SC1,构建重组慢病毒载体。用构建的AC-sh RNA慢病毒感染THP-1细胞,q PCR检测干扰载体对AC1和AC4基因的干扰效果。慢病毒感染泡沫细胞后,采用液体闪烁计数法测定apo A-1介导的泡沫细胞胆固醇流出率。结果测序结果表明,AC1和AC4基因的sh RNA寡聚核苷酸序列已成功插入慢病毒载体。荧光观察显示,两种慢病毒的感染效率达80%。q PCR分析表明,AC1-sh RNA和AC4-sh RNA干扰载体对靶基因有明显的抑制效果(P<0.01),其抑制效率分别为83.9%和80.1%。AC1和AC4-sh RNA慢病毒感染泡沫细胞后,胆固醇流出明显降低(P<0.01)。结论本实验初步证实AC1和AC4可降低泡沫细胞胆固醇流出,提示其可能在胆固醇代谢中起作用。

Objective To study the effect of adenylate cyclase on the cholesterol efflux from foam cells. Methods The human adenylate cyclase AC1 and AC4 targeted oligonucleotides were designed and synthesized,then inserted into the p GMLVSC1 vector to construct recombinant vector. The inhibitory effect of AC-sh RNA on AC1 and AC4 gene were analyzed by q PCR after THP-1 cells were infected with packaged AC1-sh RNA and AC4-sh RNA lentivirus. Cholesterol efflux from foam cells was determined using liquid scintillation counting. Results Sequencing confirmed that lentiviral p GMLV-SC1 vector had been successfully inserted into AC1 and AC4 gene specific oligonucleotide sequences. Fluorescence observation showed that infection efficiency of two lentiviruses was all above 80%. The m RNA expression of AC1 and AC4 were significantly decreased（P〈0.01）,and the interference effect of two AC-sh RNA was 83.9% and 80.1%,respectively by q PCR analysis. After foam cells were infected with AC1 and AC4-sh RNA lentivirus,the ratio of cholesterol efflux were decreased significantly（P〈0.01）. Conclusion This experiment preliminarily confirmed that AC1 and AC4 can decrease cholesterol efflux from foam cells,indicating that it may be related to cholesterol metabolism.