不同胎龄早产儿脐带血中内皮祖细胞水平及迁移能力研究

Research of number and migration ability of endothelial progenitor cells isolated from umbilical cord blood in preterm infants with different gestational ages

目的探讨不同胎龄早产儿脐带血中内皮祖细胞（EPC）水平及迁移能力的差异。方法选择2014年10月至2015年7月,于四川大学华西第二医院、四川省医学科学院·四川省人民医院、成都市第一人民医院收集的47份新生儿的脐带血为研究对象。根据收集脐带血的新生儿胎龄,将其分为极早产儿组（胎龄为28-32孕周,n=12）,早产儿组（胎龄为32-36孕周,n=20）,以及足月儿组（胎龄〉37孕周,n=15）。将3组新生儿脐带血于诱导分化前,采用流式细胞仪检测脐带血中EPC水平;于诱导分化后,采用荧光探针标记的乙酰化低密度脂蛋白（Dil-Ac-LDL）与异硫氰酸荧光素标记的荆豆凝集素（FITC-UEA-1）对单个核细胞进行孵育、染色,荧光显微镜下观察3组单个核细胞染色情况,判断EPC是否诱导分化成功。采用Transwell迁移实验,检测3组新生儿脐带血EPC迁移能力。统计学比较3组新生儿脐带血诱导分化前EPC水平,诱导分化后EPC迁移数量。本研究遵循的程序符合收集脐带血的上述3家医院伦理委员会所制定的伦理学标准,得到批准,脐带血获取前,均征得产妇的知情同意,并与之签署临床研究书知情同意书。3组收集脐带血的新生儿性别构成比、分娩方式及出生时Apgar评分等基本资料比较,差异均无统计学意义（P〉0.05）。结果 1流式细胞仪检测结果显示,脐带血诱导分化前,3组新生儿脐带血中,EPC水平比较,差异无统计学意义（F=0.302,P=0.654）。2Dil-Ac-LDL、FITC-UEA-1双荧光染色结果显示,3组新生儿脐带血中EPC均诱导分化成功,并且随着胎龄增加,单个核细胞诱导分化为EPC的数量逐渐增多。3Transwell迁移实验结果显示,极早产儿组、早产儿与足月儿组新生儿脐带血中EPC迁移数量分别为（13.8±21.7）个/视野、（18.8±12.4）个/视野与（39.9±23.8）个/视野,3组比较,差异有统计学意义（F=3.826,P=0.027）。其中,极早产儿组与早产儿组EPC迁移数量,均显著低于足月儿组,差异均有统计学意义（P=0.016、0.035）;极早产儿组EPC迁移数量,亦显著低于早产儿组,二者比较,差异亦有统计学意义（P=0.042）。结论随着胎龄增加,早产儿脐带血中EPC水平虽然无明显增高,但其迁移能力逐渐提高。

Objective To investigate the differences of the number and migration ability of endothelial progenitor cell（EPC）isolated from umbilical cord blood among preterm infants with different gestational ages.Methods From October 2014 to July 2015,a total of 47 cases of umbilical cord blood of neonates in West China Second University Hospital,Sichuan University,Sichuan Academy of Medical Sciences Sichuan Provincial People′s Hospital,and Chengdu First People′s Hospital were chosen into this study.According to the gestational age of newborns whose umbilical cord blood was collected,they were divided into very preterm group（the gestational ages were 28to32 weeks,n=12）,preterm group（the gestational ages were 32 to 36weeks,n=20）,and full-term group（the gestational ages were 37 weeks,n=15）.Before the induced differentiation of umbilical cord blood in the three groups,flow cytometry was used to detect the level of EPC.After the induced differentiation of umbilical cord blood in the three groups,the mononuclear cells were incubated and stained by fluorescent probes labeled Dil-acetylated low-density lipoprotein（Dil-Ac-LDL）and fluorescein isothiocyanate-labeled ulex europaeus agglutinin-1（FITC-UEA-1）.The stainings of mononuclear cells in the three groups were observed under fluorescence microscope to detect whether the EPC was successfully induced or not.Transwell migration assay was used to detect the migration ability of EPC in the three groups.The levels of EPC before the induced differentiation of umbilical cord blood in three groups and the migration numbers of EPC after the induced differentiation of umbilical cord blood in three groups were compared by statistical methods.The study protocol was approved by the Ethical Review Boards of three hospitals from which the umbilical cord blood was collected.Informed consent was obtained from each pregnant woman before obtaining umbilical cord blood.There were no significant differences in gender ratio,delivery method,Apgar scores and other basic data among the newborns from which the umbilical cord blood was collected in the three groups（P〈0.05）.Results 1Flow cytometry results showed that,there was no statistical difference in the level of EPC before the induced differentiation of umbilical cord blood in the three groups（F=0.302,P=0.654）.2Dil-Ac-LDL and FITC-UEA-1double fluorescent staining results showed that,EPC in the three groups all were successfully induced to differentiate from the umbilical cord blood,and with the increase of gestational age,the number of EPC induced from mononuclear cells increased gradually at the same time.3Transwell migration experimental results show that,the migration number of EPC in very preterm group,preterm group and full-term group was（13.8±21.7）cells/field,（18.8±12.4）cells/field,（39.9±23.8）cells/field,respectively,and the difference was statistically significant（F=3.826,P=0.027）.The migration number of EPC in very preterm group and preterm group were both significantly lower than that in full-term group,and both the differences were statistically significant（P=0.016,0.035）.The migration number of EPC in very preterm group was significantly lower than that in preterm group,and the difference was statistically significant（P=0.042）.Conclusions With the increasing of gestational age,the level of EPC in preterm infants′umbilical cord blood does not increase obviously,but the migration ability of EPC improves gradually as the gestational age increases.