CAJ | 학술논문

背景：曾有报道雷帕霉素可以对内皮祖细胞的增殖能力、迁移能力、黏附能力产生影响，但是没有提到自噬在其中所起到的不可忽视的作用，以及自噬与凋亡之间的相互关系。目的：通过雷帕霉素激活自噬探讨自噬激活对大鼠内皮祖细胞增殖、凋亡和周期的影响。方法：采用密度梯度离心法从骨髓获得单个核细胞，将其接种在人纤维连接蛋白包被的培养板上，培养7 d后收集贴壁细胞，即内皮祖细胞。加入不同质量浓度雷帕霉素(0.01，0.1，1，10μg/ L)分别培养24 h。Western blot检测LC3-Ⅱ蛋白表达监测自噬的诱导情况，流式细胞仪检测细胞凋亡和细胞周期进程的变化，MTT比色法观察其增殖能力的变化，同时在透射电镜下观察其超微结构的变化。结果与结论：雷帕霉素质量浓度为0.01μg/L时，内皮祖细胞的LC3-Ⅱ蛋白表达与对照组相比并没有明显的增高，当质量浓度为0.1μg/L时LC3-Ⅱ蛋白表达处在一个较高的水平，质量浓度为1μg/L和10μg/L时LC3-Ⅱ蛋白表达虽然也高于对照组，但却明显低于0.1μg/L时，据此推断雷帕霉素在质量浓度为0.1μg/L时自噬尤为活跃。内皮祖细胞的凋亡率呈现随着雷帕霉素质量浓度的升高而增加的趋势，增殖率呈现随雷帕霉质量浓度增加而降低的趋势。结果说明雷帕霉素激活自噬后能够促进细胞的凋亡，明显改变细胞的周期进程，抑制内皮祖细胞的增殖。
배경：증유보도뢰파매소가이대내피조세포적증식능력、천이능력、점부능력산생영향，단시몰유제도자서재기중소기도적불가홀시적작용，이급자서여조망지간적상호관계。목적：통과뢰파매소격활자서탐토자서격활대대서내피조세포증식、조망화주기적영향。방법：채용밀도제도리심법종골수획득단개핵세포，장기접충재인섬유련접단백포피적배양판상，배양7 d후수집첩벽세포，즉내피조세포。가입불동질량농도뢰파매소(0.01，0.1，1，10μg/ L)분별배양24 h。Western blot검측LC3-Ⅱ단백표체감측자서적유도정황，류식세포의검측세포조망화세포주기진정적변화，MTT비색법관찰기증식능력적변화，동시재투사전경하관찰기초미결구적변화。결과여결론：뢰파매소질량농도위0.01μg/L시，내피조세포적LC3-Ⅱ단백표체여대조조상비병몰유명현적증고，당질량농도위0.1μg/L시LC3-Ⅱ단백표체처재일개교고적수평，질량농도위1μg/L화10μg/L시LC3-Ⅱ단백표체수연야고우대조조，단각명현저우0.1μg/L시，거차추단뢰파매소재질량농도위0.1μg/L시자서우위활약。내피조세포적조망솔정현수착뢰파매소질량농도적승고이증가적추세，증식솔정현수뢰파매질량농도증가이강저적추세。결과설명뢰파매소격활자서후능구촉진세포적조망，명현개변세포적주기진정，억제내피조세포적증식。
BACKGROUND:Previous studies have reported that rapamycin can affect the proliferation, migration and adhesion abilities of endothelial progenitor cels, but there is no report on the effect of autophagy, as wel as the interaction between autophagy and apoptosis. OBJECTIVE: To observe the effect of rapamycin activated autophagy activation on the proliferation, apoptosis, and cycle of endothelial progenitor cels. METHODS:Density gradient centrifugation was used to obtain mononuclear cels from bone marrow, and the mononuclear cels were inoculated on human fibronectin-coated culture plate.Then after cultured for 7 days the adherent cels colected were the endothelial progenitor cels. Different concentrations of rapamycin (0.01, 0.1, 1 and 10 μg/L) were added and cultured for 24 hours. Western blot was used to detect the LC3-II protein expression and monitor the induction of autophagy, flow cytometry was used to observe the cel cycle progression and apoptosis changes, and methylthiazolyldiphenyl-tetrazolium bromide colorimetric assay was used to observe the proliferation ability. Meanwhile, the ultrastructural changes were observed under transmission electron microscope. RESULTS AND CONCLUSION:Compared with the control group, there was no significant increasing of LC3-II protein expression of endothelial progenitor cels in 0.01 μg/L rapamycin group, and the LC3-II protein expression was in the high level. The LC3-IIprotein expression in the 1 μg/L and 10 μg/L rapamycin groups was higher than that in the control group, but lower than that in the 0.01 μg/L rapamycin group, which indicated that autophagywas particularly active when the concentration of rapamycin was 0.01 μg/L. The apoptosis of endothelial progenitor cels was increased with the increasing of concentration of rapamycin, and the proliferation rate was decreased with the increasing of concentration of rapamycin. The results indicate that activation of autophagy by bapamycin can promote the cel apoptosis, change the cel cycle significantly, and can inhibit the proliferation of endothelial progenitor cels.