香烟烟雾提取物对外周血内皮祖细胞衰老的影响及其机制研究

Study on Effects of Cigarette Smoke Extract on Senescence of Endothelial Progenitor Cells from Peripheral Blood and Its Mechanisms

目的观察香烟烟雾提取物(CSE)对外周血内皮祖细胞(EPC)衰老的影响,探讨其可能机制。方法密度梯度离心法获取人外周血单核细胞,培养4d后,收集贴壁细胞,分别加入2.5%,5%,10%和15%的CSE干预。采用SA-β-半乳糖苷酶染色试剂盒检测衰老细胞;MTT比色法检测EPC的增殖能力;端粒重复序列扩增法(TRAP)-ELISA定量检测EPC端粒末端转移酶活性;逆转录PCR法检测人端粒酶逆转录因子(human telomerase reverse transcriptase,h TERT)mRNA水平;Western蛋白印迹检测EPC Akt Ser473磷酸化水平。结果 CSE能显著增加SA-β-半乳糖苷酶阳性细胞数量,15%CSE影响最为明显,同时显著抑制EPC增殖能力;CSE能显著抑制端粒酶活性及h TERT mRNA表达;另外,CSE能抑制EPC Akt磷酸化。结论 CSE诱导EPC衰老,伴随EPC增殖能力的损害,提示细胞衰老也许是CSE影响EPC功能的机制之一;CSE诱导EPC衰老可能与抑制EPC端粒末端转移酶活性、减少h TERT mRNA表达及抑制Akt磷酸化有关。

Objective To investigate whether cigarette smoke extract（ CSE） might be able to accelerate the onset of endothelial progenitor cell（ EPC） senescence as well as its underlying mechanisms. Methods Total mononuclear cells（ MNCs） were isolated from peripheral blood by density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes. After 4 days,adherent cells were collected and treated with 2. 5,5,10 and 15% CSE respectively. Senescence-associated β-galactosidase（ SA-β-Gal） staining kit was utilized to monitor EPC senescence. The proliferation of EPC was assessed by 3-（ 4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（ MTT） assay. Telomerase activity was measured by quantitative analysis of telomeric repeat amplification protocol（ TRAP）-ELISA in EPC. The mRNA levels of human telomerase reverse transcriptase（ h TERT） were measured by reverse transcription- polymerase chain reaction（ PCR）. Phosphorylation of EPC Akt Ser473 was detected by Western blot. Results CSE significantly increased the number of SA-β-Gal positive cells. It reached to the maximum at 15%（ 14. 17 ± 5. 42 vs 55. 50 ± 9. 40）. Moreover,CSE significantly decreased EPC proliferation（ 0. 22 ± 0. 04 vs 0. 04 ± 0. 01）. CSE down-regulated the expression of h TERT mRNA in a concentration-dependent manner,with a maximal decrease in 15%. CSE also significantly decreased telomerase activity（ 0. 44 ± 0. 09 vs 0. 21 ± 0. 06）. In addition,CSE treatment of EPC concentration-dependently diminished Akt phosphorylation. Conclusions CSE induced EPC senescence with the damage to the proliferation of EPC. This indicates that cellular senescence may be one of the mechanisms by which CSE affects EPC function. CSE-induced EPC senescence could be associated with the inhibition of EPC telomerase activity,the decrease of mRNA h TERT expression and the inhibition of Akt phosphorylation.