人脂联素重组腺病毒对内皮细胞增殖和一氧化氮合酶的影响

目的探讨人脂联素重组腺病毒(Ad-apM1)对人脐静脉内皮细胞(HUVEC)的增殖和总一氧化氮合酶(NOS)、诱导型一氧化氮合酶(iNOS)和结构型一氧化氮合酶(eNOS)活性的影响,为动脉粥样硬化的基因治疗奠定基础。方法将Ad-apM1感染HUVEC,观察细胞形态,绘制生长曲线,四甲基偶氮唑盐(MTT)法检测HUVEC的增殖。双抗体夹心ELISA法检测培养上清中人脂联素的表达水平,比色法检测总NOS、iNOS、eNOS活力。结果重组腺病毒感染后的HUVEC和未受感染的HUVEC形态无明显改变,体外增殖能力也无显著差异。用感染复数(MOI)为50的人脂联素基因重组腺病毒感染HUVEC,24、48、72h后取上清检测脂联素浓度分别为(125±12)ng/ml、(160±14)ng/ml、(150±8)ng/ml。感染了Ad-apM1的HUVEC总NOS和eNOS的活力显著升高,而iNOS的活力显著降低。结论我们制备的重组腺病毒能有效感染HUVEC并分泌表达人脂联素,对HUVEC的形态和增殖无明显影响,可增强HUVEC总NOS和eNOS活性,抑制iNOS活性,为Ad-apM1用于干预动脉粥样硬化奠定基础。

Effects of recombinant adenovirus encoding human apM1 gene on proliferation and nitric oxide synthase activity in human umbilical vein endothelial cells

Objective To evaluate the effects of recombinant adenovirus encoding human apM1 gene on proliferation and nitric oxide synthase(NOS)activity in human umbilical vein endothelial cells(HUVECs).Methods Protein expression of apM1 in cell culture supernatant of HUVECs transfected with human Ad-apM1 was detected by double antibody sandwich ELISA.The effect of human adiponectin on cell proliferation was assessed by MTT assay.The total NOS and iNOS expressions were measured by chromatometry.Results Human adiponectin protein level and total NOS and eNOS expressions were significant increased and iNOS expression significantly reduced in culture supernatant of HUVECs infected with Ad-apM1 compared to that in control HUVECs.The recombinant adenovirus had no influence on HUVECs growth as determined by MTT assay.Conclusions Human Ad-apM1 can be effectively expressed in HUVECs and do not influence HUVECs growth.Increased total NOS and eNOS expressions and decreased iNOS expression in HUVECs transfected with Ad-apM1 gene suggest a potential role of Ad-apM1 gene transfer for the prevention and treatment of arteriosclerosis.