【摘要】 目的探讨氧化低密度脂蛋白(ox-LDL)和尾加压素Ⅱ(UⅡ)对大鼠血管平滑肌细胞(VSMC)增殖的相互作用及ox-LDL对UⅡ受体表达的影响。方法应用MTT法测定不同浓度的ox-LDL(0.01、0.1、1、10、50μg/ml)和UⅡ(10、50nmol/L)对大鼠VSMC增殖的作用;应用竞争RT-PCR和放射性配体受体结合试验分别从mRNA和受体功能水平分析不同浓度ox-LDL孵育下VSMC表达GPR14的变化。并设生理盐水对照组。结果ox-LDL0.01μg/ml与UⅡ10nmol/L可协同促进VSMC增殖,使MTT值增至对照的162%±29%(0.528±0.036vs0.308±0.026,P<0.01);而在0.1μg/ml时MTT值减弱为对照的153%±22%(0.461±0.017vs0.308±0.026,P<0.05);至1μg/ml时二者协同作用消失,MTT值降为对照的123%±13%(0.400±0.015vs0.308±0.026,P>0.05)。在浓度为50μg/ml时ox-LDL则表现为细胞毒性,抑制细胞增殖,MTT值下降至对照的59%(0.195±0.017vs0.308±0.026,P<0.01),而UⅡ则可部分拮抗其细胞毒性,使MTT值增至对照的68%。竞争PCR显示ox-LDL0.1μg/ml可使VSMC GPR14mRNA表达上调25%(P<0.01)。而1~50μg/ml可浓度依赖性下调GPR14mRNA表达,最大效应于50μg/ml时可使GPR14mRNA下调73%(P<0.01),此效应具有时间依赖性,于12h达最大效应,并持续至24h。ox-LDL0.1μg/ml孵育12h可使大鼠125I-UⅡ与VSMC最大结合力(Bmax)升高18%(P<0.01),而ox-LDL50μg/ml可使Bmax下降低69%(P<0.01)。结论UⅡ和ox-LDL可在一定浓度范围内协同促进VSMC增殖;低浓度ox-LDL可使VSMC表达GPR14水平上调,而高浓度则可使该受体下调。更多还原

【Abstract】 Objective To investigate the effects of oxidized-low density lipoprotein (ox-LDL) and urotensin Ⅱ (U Ⅱ) on the proliferation of rat aortic smooth muscle cells (VSMCs) and the effect of ox-LDL on the mRNA expression of U Ⅱ receptor GPR14 in. Methods Rat VSMCs were incubated with UⅡ and ox-LDL at different concentrations. The viability of rat VSMCs was detected by MTT assay. Rat VSMCs were incubated with ox-LDL at the concentrations of 0, 0.1, 1, 10, and 50 μg/ml. Twelve hours later competitive RT-PCR was used to detect the effects of concentration of ox-LDP on the mRNA expression of GPR14 in the VSMCs. Another VSMCs were incubated with ox-LDL at the final concentration of 50 μg/ml, and 0, 2, 6, 12, and 24 hours later competitive RT-PCR was used to detect the mRNA expression of GPR14 in the VSMCs. 125 I-labelled U Ⅱ was added into the culture fluid of VSMCs, and radioligand binding assay was used to calculate the maximum binding (B_ max ). Results UⅡ of the concentration of 50 nmol/L significantly increased the proliferation of VSMCs. When the concentration of ox-LDL was 10 μg/ml the proliferation of VSMCs (P<0.01) was 0.678±0.061, increased by 121% compared with the control group (0.325±0.052, P<0.01). 0.01 μg/ml of ox-LDL and 10 nmol/L of UⅡ showed a synergistic effect on the proliferation of VSMCs with the MTT value increased to 162%±29%that of the control group (P<0.01). When the concentration of ox-LDL increase to 0.1 μg/ml the synergistic effect was attenuated with the MTT value of 153%±22% that of the control group (P<0.05) and when the concentration of ox-LDL increased to 1 μg/ml the synergistic effect diminished , with a MTT value of 123%±13% that of the control group (P>0.05). When the concentration of ox-LDL was 50 μg/ml the proliferation of VSMCs the MTT value decreased to 59% that of the of control group, however, ox-LDL of the concentration of 50 μg/ml combined with UⅡ of the concentration of 10 nmol/L the MTT value increased to 68% that of the control group, showing that UⅡ of that concentration significantly inhibited the cytotoxic effect of ox-LDL. Incubation of VSMCs with the ox-LDL at the concentration of 0.1 μg/ml the GPR14 mRNA expression of the VSMCs was 125.1% that of the control group (P<0.01) as assessed by competitive RT-PCR, however, when the concentration of ox-LDL increased over 1 μg/ml the GPR14 mRNA expression does-dependently decreased as much as 72.6% that of the control group (all P<0.01), with the maximal effect when the concentration of ox-LDL reached 50 μg/ml. ox-LDL at the concentration of 50 μg/ml time-dependently down-regulated the GPR14 mRNA expression with the maximal effect 12 hours after addition of ox-LDL and this effect was sustained for up to 24 hours. After incubation for 12 hours the binding of 125 I-UⅡ to VSMCs was 117.5% that of the control group (P<0.01) with the ox-LDL at the concentration of 0.1 μg/ml, and was 68.8% that of the control group (P<0.01) with the ox-LDL at the concentration of 50 μg/ml. Conclusion ox-LDL and with U-II. GPR14 at certain concentrations show synergetic effects on the proliferation of VSMCs. The GPR14 mRNA expression in VSMCs can be upregulated by low concentration of ox-LDL, while downregulated by high concentration of ox-LDL.更多还原