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目的评价Cu2+对人脐静脉血管内皮细胞(human umbilical vein endothelial cell,HUVEC)增殖与分化的影响。方法体外培养并传代HUVEC。将HUVEC以5×103个/孔密度接种于96孔板,根据向孔板中加入溶液浓度的不同将细胞随机分成3组,A组5μmol/L CuSO4,B组25μmol/L CuSO4,C组为空白对照,每组4个复孔,基础培养基为MCDB131,采用MTT法检测细胞增殖并绘制生长曲线。另取HUVEC以2×105个/孔密度接种于6孔板,分组同前,荧光定量RT-PCR检测3组HUVEC的内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)基因和Tie-1基因表达。结果生长曲线示前3d为指数增长期,第4天开始进入平台期。A组细胞增殖能力从第3天开始明显高于B、C组,B组在2d内高于C组,但从第4天开始显著低于C组,比较差异均有统计学意义(P<0.05)。荧光定量RT-PCR检测示作用48h后,A、B、C组eNOS基因表达分别为7.294±1.488、0.149±0.044和1.000±0.253,Tie-1基因的表达分别为1.481±0.137、1.131±0.191和1.000±0.177。A组与B、C组比较,2个基因的表达均有上调(P<0.05);B、C组比较,eNOS基因表达下调(P<0.05),Tie-1基因表达差异无统计学意义(P>0.05)。结论5μmol/L Cu2+能有效促进HUVEC的增殖与分化。
Objective To evaluate the effect of Cu2 + on the proliferation and differentiation of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were cultured and passaged in vitro. HUVECs were seeded into 96-well plates at a density of 5 × 10 3 / well and cells were randomly divided into three groups according to the concentration of the solution added to the well plate. A group, 5 μmol / L CuSO 4, B group, 25 μmol / L CuSO 4, Blank control group with 4 replicate wells. The basal medium was MCDB131. Cell proliferation and growth curve were measured by MTT assay. In addition, HUVECs were inoculated into 6-well plates at a density of 2 × 10 5 / well, and divided into the same groups. Fluorescent quantitative RT-PCR was used to detect the expression of endothelial nitric oxide synthase (eNOS) and Tie- 1 gene expression. Results The growth curve showed an exponential growth phase before 3d, and began to enter the plateau on the 4th day. The cell proliferation ability of group A was significantly higher than that of group B and C from day 3, and group B was higher than group C within 2 days, but significantly lower than that of group C from day 4 (P < 0.05). The expression of eNOS gene in groups A, B and C were 7.294 ± 1.488, 0.169 ± 0.044 and 1.000 ± 0.253 respectively after 48 h of fluorescence quantitative RT-PCR. The expression of Tie-1 gene was 1.481 ± 0.137,1.131 ± 0.191 and 1.000 ± 0.177. The expression of both genes was up-regulated in group A and group B and C (P <0.05). The expression of eNOS gene was down-regulated in group B and C (P <0.05), and there was no significant difference in Tie- P> 0.05). Conclusion 5μmol / L Cu2 + can effectively promote the proliferation and differentiation of HUVECs.