CAJ | 학술논문

目的骨髓间充质干细胞移植治疗急性心肌梗死的效果受梗死后局部恶劣微环境限制,前期研究已证实他汀类药物可通过改良心肌微环境提高移植疗效,但具体机制不详.本研究旨在观察阿托伐他汀( Ator)对体外缺氧无血清(H/SF)诱导的猪骨髓间充质干细胞凋亡的影响并探索其作用机制及通路.方法猪骨髓间充质干细胞分正常对照组、H/SF组、浓度梯度(0.001～10 μmol/L)Ator处理组、AMP蛋白激酶(AMPK)通路抑制剂compound C组(CC组),Ator+ CC组、磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸激酶(AKt)通路拮抗剂LY294002组(LY组)、Ator+ LY组.流式细胞仪检测各组细胞凋亡比例,Western blot检测AMPK、Akt、内皮型一氧化氮合酶(eNOS)蛋白及其磷酸化水平,实时聚合酶链反应(Real Time-PCR)检测AMPK、Akt和eNOS基因表达水平.结果 Ator 0.01～10 μmol/L组间充质干细胞凋亡比例显著低于H/SF对照组(1.94％～6.10％比10.94％,P＜0.01或0.05),Ator+ CC组间充质干细胞凋亡比例显著高于1μmol/L Ator组(4.94％±0.98％比2.59％ +0.84％,P＜0.01),而Ator+ LY组细胞凋亡比例与1μmol/L Ator组比较差异无统计学意义(2.02％±0.45％比2.59％ +0.84％,P＞0.05).Ator浓度梯度组AMPK、Akt和eNOS基因表达增加伴AMPK和eNOS磷酸化水平提高(P＜0.01或0.05).eNOS的磷酸化水平与AMPK磷酸化显著相关(r =0.599,P=0.004),而与Akt磷酸化水平无显著相关(P =0.263).结论阿托伐他汀可抑制H/SF诱导的猪骨髓间充质于细胞凋亡,该效应主要与AMPK信号通路介导的eNOS活化有关,本实验条件下PI3K/Akt信号通路不起主要作用.
목적 골수간충질간세포이식치료급성심기경사적효과수경사후국부악렬미배경한제,전기연구이증실타정류약물가통과개양심기미배경제고이식료효,단구체궤제불상.본연구지재관찰아탁벌타정( Ator)대체외결양무혈청(H/SF)유도적저골수간충질간세포조망적영향병탐색기작용궤제급통로.방법 저골수간충질간세포분정상대조조、H/SF조、농도제도(0.001～10 μmol/L)Ator처리조、AMP단백격매(AMPK)통로억제제compound C조(CC조),Ator+ CC조、린지선기순3격매(PI3K)/사안산소안산격매(AKt)통로길항제LY294002조(LY조)、Ator+ LY조.류식세포의검측각조세포조망비례,Western blot검측AMPK、Akt、내피형일양화담합매(eNOS)단백급기린산화수평,실시취합매련반응(Real Time-PCR)검측AMPK、Akt화eNOS기인표체수평.결과 Ator 0.01～10 μmol/L조간충질간세포조망비례현저저우H/SF대조조(1.94％～6.10％비10.94％,P＜0.01혹0.05),Ator+ CC조간충질간세포조망비례현저고우1μmol/L Ator조(4.94％±0.98％비2.59％ +0.84％,P＜0.01),이Ator+ LY조세포조망비례여1μmol/L Ator조비교차이무통계학의의(2.02％±0.45％비2.59％ +0.84％,P＞0.05).Ator농도제도조AMPK、Akt화eNOS기인표체증가반AMPK화eNOS린산화수평제고(P＜0.01혹0.05).eNOS적린산화수평여AMPK린산화현저상관(r =0.599,P=0.004),이여Akt린산화수평무현저상관(P =0.263).결론 아탁벌타정가억제H/SF유도적저골수간충질우세포조망,해효응주요여AMPK신호통로개도적eNOS활화유관,본실험조건하PI3K/Akt신호통로불기주요작용.
Objective The effect of mesenchymal stem cells (MSCs) transplantation is poor because of the harsh environment post infarction.Our previous studies have proven that Statins could enhance the implanted bone marrow MSCs survival,but the exact mechanism remained to be clarified.We hypothesized that atorvastatin (Ator) could protect MSCs from hypoxia and serum-free (H/SF) induced apoptosis and investigated the potential mechanisms.Methods Chinese mini-swine′s bone marrow derived MSCs were cultured in vitro and exposed to hypoxia and H/SF,Ator of various concentrations (0.001 - 10μmol/L),AMPK inhibitor-compound C ( CC),PI3K inhibitor-LY294002 (LY),Ator + CC and Ator +LY.Cell apoptosis was assessed using Annexin V/Propidine Iodine kit by flow cytometry.Phosphorylation of AMPK,Akt,endothelial nitric oxide synthase (eNOS) level and phosphorylation were tested with Western blot.Real Time-PCR was performed to analyze the gene expression of AMPK,Akt and eNOS.Results MSCs apoptosis in Ator (0.01 - 10 μmol/L) treated H/SF groups was significantly reduced compared with H/SF group ( 1.94％ - 6.10％ vs.10.94％,P ＜ 0.01 or 0.05).Apoptosis was higher in Ator + CC group than in 1 μmol/L Ator group (4.94％ + 0.98％ vs.2.59％ ± 0.84％,P ＜ 0.01 ) and similar between Ator + LY and 1 μmol/L Ator group ( 2.02％ ± 0.45％ vs.2.59％ ± 0.84％,P ＞ 0.05 ).The gene expressions of AMPK,Akt and eNOS were significantly upregulated in atorvastatin treated groups.Meanwhile,phosphorylation of AMPK and eNOS increased in MSCs treated with atorvastatin (P ＜0.01 or0.05).Phosphorylation of eNOS significantly correlated with AMPK phosphorylation (r = 0.599,P =0.004),but not with Akt phosphorylation ( P = 0.263).Conclusions Atorvastatin can protect MSCs from H/SF induced apoptosis through AMPK pathway,which resulting in activation of eNOS.