内皮前体细胞来源的微囊泡对新生大鼠缺氧缺血性脑损伤的保护作用研究

Protective effects of endothelial progenitor cells-derived microvesicles on hypoxic-ischemic brain damage in newborn rats

目的探讨内皮前体细胞来源的微囊泡(endothelial progenitor cells-derived microvesicles,EPC-MVs)对缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)的保护作用及机制。方法培养大鼠内皮前体细胞(endothelial progenitor cells,EPCs),使用超速离心法从EPCs培养液中提取微囊泡。选择健康7日龄新生SD大鼠60只,随机分为对照组、HIBD组、生理盐水组、EPC-MVs组,每组各15只。HIBD组、生理盐水组、EPC-MVs组制备HIBD模型,生理盐水组、EPC-MVs组分别在HIBD模型制备完成后予脑室内注射生理盐水.EPC-MVs,72h后处死大鼠取脑组织,TTC染色检测脑组织梗死情况、实时荧光定量聚合酶链反应检测血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA.蛋白免疫印迹检测VEGF蛋白表达变化。结果从12周龄SD大鼠脾脏提取及培养的细胞经形态学及流式细胞术检测确认为EPCs。EPCs培养上清中高速离心分离获得的EPC-MVs经透射电镜观察符合微囊泡的形态学特征。对照组未检测到梗死脑组织,HIBD组、生理盐水组xEPC-MVs组脑梗死体积比分别为(80.3±6.3)%,(77.9±8.9)%,(35.2±7.7)%,EPC-MVs组小于HIBD组和生理盐水组(P<O.001)。HIBD组、生理盐水组.EPC-MVs组脑组织VEGF mRNA及蛋白表达均高于对照组(P<0.001),EPC-MVs组高于HIBD组和生理盐水组(P<0.001),生理盐水组和HIBD组间差异无统计学意义(P>0.05)。结论脑室内注射EPC-MVs可减轻新生大鼠HIBD的程度,其机制可能与上调VEGF表达相关。

Objective To study the protective effects and preliminary mechanisms of endothelial progenitor cells-derived microvesicles(EPC-MVs)on hypoxic-ischemic brain damage(HIBD)in newborn rats by using the HIBD model.Method Rat endothelial progenitor cells(EPCs)were cultured and microvesicles were extracted from EPCs culture medium by ultracentrifugation.A total of 60 neonatal SD rats were randomly assigned into control group,HIBD group,saline group and EPC-MVs group.The HIBD model was prepared in HIBD group,saline group and EPC-MVs group.After the preparation of the HIBD model,rats in saline group and EPC-MVs group received intraventricular injection with saline and EPC-MVs,respectively.After 72 hours,the rats were sacrificed for brain tissue,cerebral infarction was detected by TTC staining,vascular endothelial growth factor(VEGF)mRNA was tested by real-time PCR,protein western blot was used to detect changes in VEGF protein expression.Result Cells extracted and cultured from the spleen of 12-week-old SD rats were confirmed as EPCs by morphology and flow cytometry.EPC-MVs isolated by high-speed centrifugation from EPCs culture supernatant met the morphological characteristics of microvesicles by transmission electron microscopy.The infarcted brain tissue was not detected in the control group.The cerebral infarction volume ratios of HIBD group,saline group and EPC-M Vs group were(80.3±6.3)%,(77.9±8.9)%,(35.2±7.7)%,respectively.The infarct volume of EPC-MVs group was significantly lower than that of HIBD group and saline group(P<0.001).The expression of VEGF mRNA and protein in HIBD group,saline group and EPC-MVs group were higher than those in control group(P<0.001).Among them,EPC-MVs group had the most significant increase,compared with the other three experimental groups,and the difference was statistically significant(P<0.001).There was no significant difference between saline group and HIBD group in the expression of VEGF mRNA and protein(P>0.05).Conclusion Intraventricular injection of EPC-MVs can attenuate HIBD in neonatal rats,and the mechanism may be related to up-regulation of VEGF expression.