内皮素-1对大鼠骨髓内皮前体细胞增殖、细胞周期及NO分泌功能的影响

Effect of endothelin-1 on proliferation,cell cycle and NO secreting function of endothelial progenitor cells from rat bone marrow

目的探讨内皮素-1(ET-1)对大鼠骨髓内皮前体细胞(EPC)增殖、细胞周期及一氧化氮(NO)分泌功能的影响。方法采用密度梯度离心法获取大鼠骨髓单核细胞,在M199培养液中培养扩增EPC并进行鉴定。不同浓度ET-1(A组:0mol/L、B组:10-9mol/L、C组:10-8mol/L、D组:10-7mol/L、E组:10-6mol/L)作用于EPC,MTT法检测ET-1对EPC增殖能力的影响,流式细胞仪检测ET-1对EPC细胞周期的影响,硝酸还原酶法检测细胞培养液中NO含量的变化,观察ET-1对EPC的NO分泌功能的影响。结果与对照组(A组,0.405±0.017)相比,ET-1浓度C组(0.434±0.016)、D组(0.463±0.016)、E组(0.473±0.015)EPC增殖能力显著增高(n=8,P<0.01)。EPC的G0/G1期细胞百分数C组(57.28±3.65)%、D组(44.99±3.19)%、E组(40.29±3.74)%较对照组(70.55±1.37)%明显降低(n=5,P<0.01),EPC的S期细胞百分数C组(26.75±2.87)%、D组(32.79±2.41)%、E组(35.74±2.94)%较对照组(20.04±1.64)%明显升高(n=5,P<0.01),EPC的G2/M期细胞百分数C组(15.96±1.71)%、D组(22.22±2.22)%、E组(23.64±2.86)%较对照组(9.41±0.81)%明显升高(n=5,P<0.01)。EPC培养液中NO含量(μmol/L)ET-1浓度C组(11.70±1.80)、D组(15.69±1.86)、E组(16.89±1.55)较对照组(7.45±2.41)明显增加(n=8,P<0.01)。B组检测结果与对照组相比,各项指标均无统计学差异。结论ET-1能够促进EPC的增殖,促进细胞向S期和G2期的转化,并提高细胞NO分泌功能,可能参与缺血性疾病的血管再生过程。

AIM To study the effects of endothelin-1 on proliferation, cell cycle and NO secreting function of endothelial progenitor cells from rat bone marrow. METHODS Mononuclear cells were collected from rat bone marrow by density gradient centrifugation, cultured with medium 199 and were identified to be EPC on day 6 by Di I -ac-LDL and FITC-UEA- I double staining. Different concentrations of ET-1 （group A, 0 mol/L; group B, 10^-9 mol/L; group C, 10^-8 mol/L; group D, 10^-7 mol/L; group E, 10^-6 mol/L） were added into culture medium to detect the effect on EPC. MTr assay was performed to detect the effect of ET-1 on the multiplication ability of EPC and Flow cytometry was employed to detect the cell cycle. NO content was measured in the cell culture medium by nitrate reductase method to find the promotion of ET-1 on cell functions. RESULTS Compared with that in the control group （ group A, 0. 405 ±0. 017）, the proliferation of EPC greatly enhanced in ET-1 group （group C, 0. 434 ±0. 016;group D, 0. 463± 0. 015 ; group E, 0. 473 ± 0. 015） （ n = 8, P 〈 0.01 ）. The amount of EPC in ET-1 group in G0/G1 phase （group C, 57.28%±3.65% ; group D, 44.99%±3.19% ; group E, 40. 29%± 3.74% ） was significantly lower than those in control group （ group A, 70. 55% ± 1.37% ） （ n =5, P 〈0. 01 ）. Compare with that in the control group （20. 04%± 1.64% ） （9.41%±0. 81% ）, the amount of EPC in ET-1 group in S phase （group C, 26.75%±2.87% ; group D, 32.79% ±2.41% ; group E, 35.74%± 2.94%） and in G2/M phase （group C, 15.96%±1.71%; group D, 22.22%±2.22%; group E, 23.64%± 2.86% ） increased （ n = 5, P 〈 0. 01 ）. Compare with that in the control group （group A, 7.45 ± 2.41 ）, NO secreting function of EPC increased in ET-1 group （ group C, 11.70 ± 1.80 ; group D, 15.69 ± 1.86 ; group E, 16.89 ± 1.55） （n = 8, P 〈 0. 01 ）. Group B showed no or only minor difference as compared with control group. CONCLUSION ET-1 stimulates EPC proliferation, promotes the division and multiplication of EPC and enhances the NO secretion capacity.