目的:观察酰基辅酶A:胆固醇酰基转移酶1(ACAT1)反义寡核苷酸对泡沫细胞(FC)形成的影响。方法:体外培养人THP-1单核细胞系,由佛波酯(PMA)作用使其分化为巨噬细胞;并建立过表达ACAT1基因的THP-1单核细胞系。在脂质体的介导下将针对ACAT1基因序列的反义和错义寡核苷酸分别作用于上述细胞6h后加入Ac—LDL进行脂质负荷,以Western blotting法检测ACAT1蛋白表达,放射性同位素标记底物法检测ACAT酶活性的变化,油红O染色法观察泡沫细胞的形成情况。结果:ACAT1反义寡核苷酸可明显抑制巨噬细胞和过表达ACAT1基因的THP-1单核细胞内的ACAT酶活性,而且可明显抑制Ac—LDL负荷后泡沫细胞的形成;错义寡核苷酸则无此作用。结论:ACAT1反义寡核苷酸可抑制ACAT的生物学活性及泡沫细胞的形成。
AIM : To study the effect of acyl coenzyme A : cholesteryl acyltransferase 1 ( ACAT1 ) antisense oligonucleotides on the formation of foam cells ( FC ). METHODS : THP - 1 cells were cultured and differentiated into macrophages (MP) by phorbol myfistate acetate (PMA). Over - expressing ACAT1 gene THP - 1 cells were constructed. The ACAT1 antisense and missense oligonucleotides conducted by LipofectamineTM 2000 were incubated with above cells. Ac - LDL was added 6 h later and incubated for 24 h. The expression of ACAT1 protein was detected by Western blotting. The ACAT activity was measured by quantifying the incorporation of [ 1 - 14C] oleoyl CoA into cholesteryl esters. The formation of foam cells was detected by oil red O staining. RESULTS : The ACAT1 antisense oligonucleotides inhibited the activity of ACAT in macrophages and over - expressing ACAT1 gene THP - 1 cells. It also inhibited the formation of foam cell in mac- rophages and over - expressing ACAT1 gene THP - 1 cells with lipid loading. The missense oligonucleotides did not show the inhibitory effects. CONCLUSION : The ACAT1 antisense oligonucleotides inhibit the activity of ACAT and the formation of foam cells.