报告基因成像评价Ghrelin对骨髓间充质干细胞的缺氧保护作用

目的通过报告基因成像评价Ghrelin对骨髓间充质干细胞(MSCs)的缺氧保护作用。方法采用密度梯度离心法分离稳定表达萤火虫荧光素酶(Fluc)的MSCs,随机分为对照组、缺氧复氧组和10~(-9)、10~(-8)、10~(-7) Ghrelin组,后3组分别加入10~(-9)、10~(-8)、10~(-7)mol/L Ghrelin培养。运用Fluc报告基因成像、MTT法对MSCs的生物学活性进行定量评价,Western blot和ELISA法检测MSCs的作用机制。结果 MSCs稳定表达报告基因Fluc,其光学信号强度与细胞数量呈正相关(r~2=0.96)。缺氧复氧组MSCs生存活力较对照组显著下降;与缺氧复氧组比较,10~(-8)、10~(-7) Ghrelin组MSCs生存率和VEGF分泌水平明显升高,10~(-8) Ghrelin组磷酸化Akt表达明显升高(P<0.05)。结论 Ghrelin能够显著提高缺氧复氧后MSCs的存活能力,可能是MSCs的保护性因素。

Evaluation of the Ghrelin protective effect on bone marrow mesenchymal stem cells with hypoxia injury by imaging

Objective To evaluate the protective effect of Ghrelin on bone marrow derived mesenchymal stem cells(MSCs) under hypoxia-reoxygenation(H/R) injury by reporter gene imaging. Methods MSCs stably expressing firefly luciferase(Fluc) were isolated fromβ-actin-luc mice and characterized by flow cytometry and reporter gene imaging.MSCs were divided into Control,H/R and Ghrelin(10~9,10~(-8),10~(-7) mol/L,respectively) groups.MSCs' survival and proliferation were assessed by imaging and MTT assay.The mechanism of Ghrelin effect was analyzed by Western blot and ELISA.Results The Flue signal intensity of MSCs was well correlated with different cell number(r~2=0.96).Reporter gene and ELISA assay imaging indicated that the survival and VEGF level of MSCs in 10~8 and 10~(-7) mol/L Ghrelin group were significantly better than H/R group(P<0.05).Western blot revealed p-Akt level was higher in 10~(-8) mol/L Ghrelin group than that in H/R group(P<0.05).Conclusion Ghrelin has beneficial effect on the survival of MSCs under H/R environment and might promote the therapeutic action of implanted stem cells in ischemic heart disease.