【摘要】：目的比較不同Na~+濃度及其作用時間促大鼠血管平滑肌細(xì)胞(VSMC)增殖的差異,探討高鹽誘導(dǎo)VSMC增殖的可能機(jī)制。方法組織貼壁法培養(yǎng)原代VSMC,傳至第四代去血清同步化1d,分別用139、141、144、147、150、153、156、159、162和165mmol/L的Na~+干預(yù)1、2、3和4d后,MTT比色法和細(xì)胞增殖檢測試劑盒(CCK-8)檢測VSMC增殖情況。選取促增殖最顯著的鹽濃度和作用時間,以Na~+139mmol/L為對照進(jìn)行后續(xù)實(shí)驗(yàn)。CCK-8法檢測滲透壓對VSMC增殖的影響;流式細(xì)胞儀分析VSMC增殖周期;免疫熒光染色和Western blot檢測VSMC增殖細(xì)胞核抗原(PCNA)、血管緊張素Ⅱ(AngⅡ)、血管緊張素Ⅱ1型受體(AT_1R)的表達(dá)情況;實(shí)時熒光定量PCR檢測VSMC中血管緊張素原(AGT)、AT_1R mRNA的表達(dá)。結(jié)果MTT和CCK-8結(jié)果顯示,Na~+153~165mmol/L作用2、3d可促進(jìn)VSMC增殖(Na~+159mmol/L作用3d最明顯);CCK-8檢測滲透壓對VSMC增殖結(jié)果顯示,與Na~+139mmol/L組比較,Na~+159mmol/L細(xì)胞明顯增殖[(1.21±0.16)%比(1.00±0.25)%,P0.05],而甘露醇組與Na~+139 mmol/L組比較差異無統(tǒng)計(jì)學(xué)意義;流式細(xì)胞儀分析VSMC增殖周期結(jié)果顯示,Na~+159mmol/L組G_0/G_1期細(xì)胞比例降低,S期細(xì)胞比例增多(均P0.05);PCNA免疫熒光染色可見Na~+159mmol/L組VSMC中PCNA陽性細(xì)胞核增加,Western blot結(jié)果顯示Na~+159mmol/L組PCNA蛋白表達(dá)明顯增加(均P0.05);實(shí)時熒光定量PCR顯示,與Na~+139mmol/L組相比,Na~+159mmol/L組中AGT、AT_1R mRNA表達(dá)增加(均P0.05);Western blot顯示,與Na~+139 mmol/L組相比,Na~+159 mmol/L組中AngⅡ、AT_1R蛋白表達(dá)增多(均P0.05)。結(jié)論高Na~+(153~165 mmol/L)作用2~3d能誘導(dǎo)VSMC增殖,且Na~+159mmol/L作用3d增殖效應(yīng)最為顯著;作用機(jī)制可能與高Na~+促進(jìn)AngⅡ和AT_1R表達(dá)有關(guān)。
[Abstract]:Objective to compare the effects of different concentrations of Na~ and their time on (VSMC) proliferation of rat vascular smooth muscle cells (VSMCs), and to explore the possible mechanism of VSMC proliferation induced by high salt. Methods Primary VSMC, cultured by tissue adherent method was transferred to the fourth generation of serum for 1 day. The proliferation of VSMC was detected by MTT colorimetric assay and cell proliferation kit (CCK-8) after intervention with 139141144147150153156159162 and 165mmol/L Na~ for 2, 3 and 4 days, respectively. The most significant salt concentration and action time were selected to carry out the follow-up experiment with Na~ 139mmol/L as control. CCK-8 method was used to detect the effect of osmotic pressure on the proliferation of VSMC, flow cytometry was used to analyze the proliferation cycle of VSMC, and CCK-8 method was used to detect the effect of osmotic pressure on proliferation of VSMC. The expression of proliferating cell nuclear antigen (PCNA), angiotensin 鈪，

本文編號：2474089