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目的比较不同Na~+浓度及其作用时间促大鼠血管平滑肌细胞(VSMC)增殖的差异,探讨高盐诱导VSMC增殖的可能机制。方法组织贴壁法培养原代VSMC,传至第四代去血清同步化1d,分别用139、141、144、147、150、153、156、159、162和165mmol/L的Na~+干预1、2、3和4d后,MTT比色法和细胞增殖检测试剂盒(CCK-8)检测VSMC增殖情况。选取促增殖最显著的盐浓度和作用时间,以Na~+139mmol/L为对照进行后续实验。CCK-8法检测渗透压对VSMC增殖的影响;流式细胞仪分析VSMC增殖周期;免疫荧光染色和Western blot检测VSMC增殖细胞核抗原(PCNA)、血管紧张素Ⅱ(AngⅡ)、血管紧张素Ⅱ1型受体(AT_1R)的表达情况;实时荧光定量PCR检测VSMC中血管紧张素原(AGT)、AT_1R mRNA的表达。结果MTT和CCK-8结果显示,Na~+153~165mmol/L作用2、3d可促进VSMC增殖(Na~+159mmol/L作用3d最明显);CCK-8检测渗透压对VSMC增殖结果显示,与Na~+139mmol/L组比较,Na~+159mmol/L细胞明显增殖[(1.21±0.16)%比(1.00±0.25)%,P<0.05],而甘露醇组与Na~+139 mmol/L组比较差异无统计学意义;流式细胞仪分析VSMC增殖周期结果显示,Na~+159mmol/L组G_0/G_1期细胞比例降低,S期细胞比例增多(均P<0.05);PCNA免疫荧光染色可见Na~+159mmol/L组VSMC中PCNA阳性细胞核增加,Western blot结果显示Na~+159mmol/L组PCNA蛋白表达明显增加(均P<0.05);实时荧光定量PCR显示,与Na~+139mmol/L组相比,Na~+159mmol/L组中AGT、AT_1R mRNA表达增加(均P<0.05);Western blot显示,与Na~+139 mmol/L组相比,Na~+159 mmol/L组中AngⅡ、AT_1R蛋白表达增多(均P<0.05)。结论高Na~+(153~165 mmol/L)作用2~3d能诱导VSMC增殖,且Na~+159mmol/L作用3d增殖效应最为显著;作用机制可能与高Na~+促进AngⅡ和AT_1R表达有关。
OBJECTIVE: To compare the proliferation of rat vascular smooth muscle cells (VSMCs) with different concentrations of Na ~ + and its action time, and to explore the possible mechanism of high salt-induced VSMC proliferation. Methods The primary VSMCs were cultured by tissue adherent method, and then desynchronized into the fourth generation for 1 day. The interventions of 139,141,144,147,150,153,156,159,162 and 165mmol / L Na + After 2, 3 and 4 days, the proliferation of VSMCs was detected by MTT assay and cell proliferation assay kit (CCK-8). The most significant salt concentration and duration of action were selected, followed by Na ~ + 139 mmol / L as control. The effects of osmotic pressure on the proliferation of VSMCs were detected by CCK-8 assay. The proliferation of VSMCs was analyzed by flow cytometry. The expressions of proliferating cell nuclear antigen (PCNA), angiotensin Ⅱ (Angiotensin Ⅱ), angiotensin Ⅱ (AT_1R). Real-time fluorescent quantitative PCR was used to detect the expression of angiotensinogen (AGT) and AT_1R mRNA in VSMCs. Results The results of MTT and CCK-8 showed that the effect of Na ~ + 153 ~ 165mmol / L for 2 and 3 days could promote the proliferation of VSMC (Na ~ + 159mmol / L for 3d was the most obvious effect) Compared with Na ~ + 139 mmol / L group, Na ~ + 159 mmol / L cells significantly proliferated (1.21 ± 0.16% vs 1.00 ± 0.25%, P <0.05) The results of flow cytometry showed that the proportion of cells in G 0 / G 1 phase decreased and the proportion of S phase cells increased (all P <0.05) in Na ~ + 159 mmol / L group; PCNA immunofluorescence The number of PCNA positive cells in Na ~ + 159mmol / L group was increased by Western blot, and the expression of PCNA protein in Na ~ + 159mmol / L group was significantly increased (all P <0.05) Compared with Na ~ + 139 mmol / L group, the expression of AGT and AT_1R mRNA in Na ~ + 159 mmol / L group increased (all P <0.05) Ang Ⅱ, AT_1R protein expression increased (all P <0.05). Conclusion The proliferation of VSMC induced by high Na ~ + (153 ~ 165 mmol / L) for 2 ~ 3d can induce VSMC proliferation, and the effect of Na ~ + 159 mmol / L on the proliferation of VSMC was the most significant. The mechanism may be related to the high Na ~ + promoting the expression of Ang Ⅱ and AT_1R .