PDE5抑制剂伐地那非预处理对人羊膜间充质干细胞生物学特性的影响

Effects of preconditioning with phosphodiesterase-5 inhibitor on the Biological Properties of human amniotic mesenchymal stem cells

目的:观察特异性5型磷酸二酯酶抑制剂(PDE5)伐地那非对人羊膜间充质干细胞(hAMSCs)生物学特性的影响。方法:分离培养hAMSCs,以终浓度为10μmol/L伐地那非预处理hAMSCs,观察伐地那非处理hAMSCs组(Vard-hAMSCs)生长形态,采用流式细胞术分析比较hAMSCs细胞免疫表型、增殖能力及抗氧化损伤能力;免疫双荧光染色计数阳性表达细胞数,计算hAMSCs向神经细胞诱导分化百分率。结果:(1)流式细胞术结果显示,hAMSCs、Vard-hAMSCs均阳性表达CD90、CD105、CD73,不表达CD45、CD34、CD19、CD11b及HLA-DR;Vard-hAMSCs组细胞S期细胞比率及增殖指数为(0.57±0.40)%和(2.20±1.60)%,与hAMSCs组[分别是(0.62±1.33)%,(1.90±1.40)%]比较,无统计学差异(P>0.05);(2)Annexin V/PI双染色结果显示,经250μmol/L H2O2作用4 h后,Vard-hAMSCs组细胞凋亡率为(7.67±0.82)%,明显低于hAMSCs组(18.72±2.92)%和特异性Vardenafil阻断剂组(15.90±1.69)%(均P<0.05);(3)免疫双荧光染色结果显示,在相同诱导条件下,Vard-hAMSCs组细胞MAP-2及GFAP阳性细胞百分率分别是(49.8±6.42)%和(55.2±6.10)%,均高于hAMSCs组[分别为(29±3.94)%,(32.2±3.03)%,P<0.05]。结论 :伐地那非处理一定时间可明显提高hAMSCs抗氧化损伤效应和hAMSCs向神经细胞分化的能力,但短时间内不明显影响hAMSCs生长形态、增殖能力和免疫表型。提示伐地那非处理可能作为hAMSCs移植治疗神经系统疾病的优选预处理方案。

Objective To investigate the biological properties of human amniotic mesenchymal stem cells （hAMSCs） which were preconditioned with phosphodiesterase-5 inhibitor （Vardenfil）. Methods hAMSCs were in vitro isolated and cultured, hAMSCs were pre-treated with vardenfil in final concentration of 10 μmol/L. The morphology of Vard-hAMSCs was observed, and the immunological characteristics, proliferative capacity, and ability of anti-oxidative damage of hAMSCs and Vard-hAMSCs were analyzed by flow cytometry. Double labeling immunofluorescent staining was used to count the differences of differential potential between neural cells of hAMSCs and Vard-hAMSCs. Results （1）Flow cytometry revealed that both hAMSCs and Vard-hAMSCs positively expressed CD90,CD105 and CD73, and negatively expressed CD34,CD45,CDllb and HLA-DR. The SPF and PI in Vard- hAMSCs group were （0.57 ± 0.40）% and （2.20± 1.60）% respectively, there was no statistical significance compared with hAMSCs group; （2）After 4 hours treated by H202, the apoptosis rate in Vard-hAMSCs group were （7.67± 0.82）%,which were markedly lower than that in the hAMSCs group and specific blocker group; （3）Under the same induction condition, positive rates of MAP-2 and GFAP in Vard-hAMSCs group were （49.8 ± 6.42）% and （55.2 ± 6.10）% respectively detected by double labeling immunofluorescent staining, which were significantly higher than the control group. Conclusion The strategy that hAMSCs are treated with vandenfil can enrich the ability of anti-oxidative damage and the differential potential for neural cells in a certain time, and the morphology, immunological characteristics, proliferative capacity of Vard-hAMSCs have no significant change. It suggests that pre-treatment with vandenfil may provide a optimized experimental strategey for hAMSCs which were used to treat nervous system disease.