人脐带间充质干细胞对呼吸机相关性肺炎新生儿气道分泌物细菌抑制作用的体外研究

In vitro effects of human umbilical cord mesenchymal stem cells on airway secretions of ventilator associated pneumonia in newborns

目的 探讨人脐带间充质干细胞对呼吸机相关性肺炎（ventilator associated pneumonia,VAP）新生儿支气管肺泡灌洗液中细菌生长的抑制作用及机制.方法 选取2012年6月1日至2012年12月31日在广东省妇幼保健院新生儿重症监护病房住院,支气管肺泡灌洗液培养阳性、需气管插管机械通气并诊断为VAP的患儿为研究对象.每例患儿收集支气管肺泡灌洗液标本2份,随机分入研究组和对照组.研究组中加入人脐带间充质干细胞悬液,对照组中加入等量细胞培养基,共培育6h后采用平板计数法对2组共培育后的细菌进行菌落数（colony-forming unit,CFU）计数.采用酶联免疫吸附法检测内源性抗菌肽（Cathelicidin/LL-37）及人β-防御素-2（human β-defensin-2,HBD-2）的水平,应用Western印迹法检测Cathelicidin/LL-37及HBD-2的特异性蛋白表达.采用配对t检验对菌落数及抗菌肽数据进行统计学分析.结果 31例患儿的支气管肺泡灌洗液中,培养出肺炎克雷伯菌及嗜麦芽窄食单胞菌各6例（各占19.3％）,溶血葡萄球菌5例（16.1％）,大肠埃希菌、脑膜败血伊丽沙白金菌和鲍曼不动杆菌各3例（各占9.7％）,恶臭假单胞菌2例（6.4％）,铜绿假单胞菌、阴沟肠杆菌和金黄色葡萄球菌各1例（各占3.2％）.研究组菌落数目低于对照组_分别为（2.60±0.67）×104 CFU/ml与（1.18±0.32）×105 CFU/ml,t=-20.19,P＜0.01].研究组共培育液中Cathelicidin/LL-37和HBD-2的水平高于对照组[Cathelicidin/LL-37：（8.98±3.22） ng/ml与（3.18±1.57） ng/ml,t=17.79;HBD-2：（379.87±11.74） pg/ml与（39.89±2.86） pg/ml,t=37.62;P均＜0.01].Western印迹法检测到Cathelicidin/LL-37的特异性蛋白表达,并发现研究组中的Cathelicidin/LL-37表达高于对照组.结论 人脐带间充质干细胞可能通过分泌抗菌肽Cathelicidin/LL-37及HBD-2抑制VAP患者支气管肺泡灌洗液中的细菌生长.

Objective To explore the effect and its possible mechanism of human umbilical cord mesenchymal stem cells （hUCMSCs） on bacterial growth in bronchoalveolar lavage fluid （BALF） of newborns with ventilator associated pneumonia （VAP）.Methods Newborns admitted to neonatal intensive care unit （NICU） in Guangdong Women and Children Hospital Affiliated to Guangzhou Medical University from June 1,2012 to December 31,2012 were reviewed.The inclusion criteria were：（1）Positive BALF culture results.（2） Requirement of mechanical ventilation with tracheal intubation.（3） Diagnosed as ventilator-associated pneumonia.Two pieces of BALF samples of newborn were collected and randornaly divided into experimental and control group.hUCMSCs were added into the experimental group,while the same volume of conditioned medium was added into the control group.Both groups were incubated for six hours in humidified CO2 incubator at 37 ℃,then,bacterial growth was assessed by colony forming unit （CFU） counts.Levels of the antimicrobial peptides （Cathelicidin/LL-37 and human HBD-2） were determined by enzyme-linked immunosorbent assay and Western blot.Paired t-test was used for statistical analysis.Results Among the culture results of 31 newborns,there were Klebsiella pneumoniae （6 cases,19.3％）,Stenotrophomonas narrow food aeromonas （6 cases,19.3％）,Hemolytic staphylococci （5 cases,16.1％）,Escherichia coli （3 cases,9.7％）,Bacterial meningitis septicemia Elizabeth Platinum （3 cases,9.7％）,Acinetobacter baumannii （3 cases,9.7％）,Pseudomonas putida （2 cases,6.4％）,Pseudomonas aeruginosa （1 case,3.2％）,Staphylococcus aureus （1 case,3.2％） and Enterobacter cloacae （1 ease,3.2％）.The CFU counts in experimental group were much less than those in control group [（2.60±0.67） ×104] CFU/ml vs [（1.18±0.32） ×105] CFU/ml,（t=-20.19,P＜0.01）.Levels of Cathelicidin/LL-37 and HBD-2 in experimental group were higher than those in control group [Cathelicidin/LL-37：（8.98 ± 3.22） ng/ml vs （3.18 ± 1.57） ng/ml,t =17.79,P ＜ 0.01 ;HBD-2：（379.87±11.74） pg/ml vs （39.89±2.86） pg/ml,t=37.62,P＜0.01].Conclusions hUCMSCs had antimicrobial effect on bacterial growth in BALFs from ventilator associated pneumonia possibly by the secretion of antimicrobial peptides （Cathelicidin/LL-37 and HBD-2）.