脱氢表雄酮对高脂诱导的人脐静脉内皮细胞MMP-2表达的影响及CYP19在其中的作用

Effects of dehydroepiandrosterone and CYP19 on expression of MMP-2 in human umbilical vein endothelial cells induced by high lipid

目的观察脱氢表雄酮(DHEA)对高脂诱导的人脐静脉内皮细胞基质金属蛋白酶-2(MMP-2)表达的影响,以及细胞色素P450芳香酶(CYP19)在此过程中的作用。方法取健康新生儿脐带,分离并培养人脐静脉内皮细胞(HUVEC)。将HUVEC分为4组:正常对照组予以DMEM/F12培养基,ox-LDL组予以DMEM/F12培养基+30mg/L ox-LDL,DHEA组予以DMEM/F12培养基+1μmol/L DHEA,ox-LDL+DHEA组予以DMEM/F12培养基+30mg/L ox-LDL+1μmol/L DHEA。作用24 h后,采用实时荧光定量PCR和ELISA法检测各组细胞MMP-2 mRNA及蛋白表达。将HUVEC分为pcDNA3.1-CYP19-GFP转染组、pcDNA3.1-GFP空质粒组,通过脂质体介导的方法分别转染pcDNA3.1-CYP19-GFP真核表达质粒和pcDNA3.1-GFP空质粒。将pcDNA3.1-CYP19-GFP转染组再分为3组:CYP19组予以无血清DMEM/F12培养基,CYP19+ox-LDL组予以无血清DMEM/F12培养基+30 mg/L ox-LDL,CYP19+ox-LDL+DHEA组予以无血清DMEM/F12培养基+30 mg/L ox-LDL+1μmol/L DHEA。将pcDNA3.1-GFP空质粒组再分为3组:空质粒组予以无血清DMEM/F12培养基;空质粒+ox-LDL组予以无血清DMEM/F12培养基+30 mg/L ox-LDL,空质粒+ox-LDL+DHEA组予以无血清DMEM/F12培养基+30 mg/L ox-LDL+1μmol/L DHEA。均培养24 h后,采用实时荧光定量PCR和ELISA法检测各组细胞MMP-2 mRNA及蛋白表达。结果 oxLDL组MMP-2 mRNA及蛋白表达较正常对照组升高,ox-LDL+DHEA组较ox-LDL组降低(P均<0.05)。CYP19+ox-LDL+DHEA组MMP-2 mRNA及蛋白表达较空质粒+ox-LDL+DHEA组降低,CYP19+ox-LDL组较CYP19组升高,CYP19+ox-LDL+DHEA组较CYP19+ox-LDL组降低(P均<0.05)。结论 DHEA能够抑制高脂诱导的MMP-2表达,过表达CYP19能够增强DHEA的这一作用。

Objective To investigate the effects of dehydroepiandrosterone（ DHEA） on the expression of matrix metalloproteinase-2（ MMP-2） in the human umbilical vein endothelial cells（ HUVECs） induced by high lipid,and the effect of cytochrome P450 aromatase gene（ CYP19） in this procedure. Methods We took the umbilical cord from the healthy newborn to isolate and culture the HUVECs. In vitro cultured HUVECs were divided into four groups,（1） control group：which were cultured in the DMEM/F12 culture medium;（2）ox-LDL group： using DMEM/F12 culture medium ＋ 30 mg/L ox-LDL;（3） DHEA group： using DMEM/F12 culture medium ＋ 1 μmol/L DHEA;（4） ox-LDL ＋ DHEA group： using DMEM/F12 culture medium ＋ 30 mg/L ox-LDL ＋ 1 μmol/L DHEA. Cells of all groups were treated for 24 h. The expression of MMP-2 mRNA and protein was determined by RT-q PCR and ELISA. HUVECs were divided into the pcDNA3. 1-CYP19-GFP transfection group and pcDNA3. 1-GFP empty plasmid group. The eukaryotic expression plasmid pcDNA3. 1-CYP19-GFP and empty plasmid pcDNA3. 1-GFP were transfected into HUVECs of the corresponding groups,respectively.The HUVECs of the pcDNA3. 1-CYP19-GFP transfection group were subdivided into 3 groups,CYP19 group： using DMEM/F12 culture medium; CYP19 ＋ ox-LDL group： using DMEM/F12 culture medium ＋ 30 mg/L ox-LDL; CYP19 ＋ox-LDL ＋ DHEA group： using DMEM/F12 culture medium ＋ 30 mg/L ox-LDL ＋ 1 μmol/L DHEA. The HUVECs of pcDNA3. 1-GFP empty plasmid group were subdivided into 3 groups,empty plasmid group： using DMEM/F12 culture medium; empty plasmid ＋ ox-LDL group： using DMEM/F12 culture medium ＋ 30 mg/L ox-LDL; empty plasmid ＋ ox-LDL ＋DHEA group： using DMEM/F12 culture medium ＋ 30 mg/L ox-LDL ＋ 1 μmol/L DHEA. Cells of all groups were treated by corresponding reagents for 24 h. The expression of MMP-2 mRNA and protein of all groups were determined by RTq PCR and ELISA. Results The expression of MMP-2 mRNA and protein in the ox-LDL group increased significantly as compared with that of the control group; compared with the ox-LDL group,the expression of MMP-2 mRNA and protein in the ox-LDL ＋ DHEA group was lower（ all P〈0. 05）. The expression of MMP-2 mRNA and protein in the CYP19 ＋ ox-LDL＋ DHEA group decreased significantly as compared with that of the empty plasmid ＋ ox-LDL ＋ DHEA group; the expression of MMP-2 mRNA and protein in the CYP19 ＋ ox-LDL group increased significantly as compared with that of the CYP19 group; compared with the CYP19 ＋ ox-LDL group,the expression of MMP-2 mRNA and protein in the CYP19 ＋ ox-LDL ＋DHEA group were significantly lower（ all P〈0. 05）. Conclusion DHEA inhibits the high lipid-induced MMP-2 expression,which may be one of the mechanisms of anti-atherosclerotic effect of DHEA,and CYP19 over-expression can enhance the effect of DHEA.