人胚胎干细胞来源间充质干细胞减轻心搏骤停猪复苏后脑损伤的作用机制研究  

Mechanism of human embryonic stem cell-derived mesenchymal stem cells on alleviating brain injury after cardiopulmonary resuscitation in swine with cardiac arrest

作　　者：葛风 徐杰丰[1] 朱锦江 曹光立 王旭光 周梅亚 陈铁江[2] 张茂[1] Ge Feng;Xu Jiefeng;Zhu Jinjiang;Cao Guangli;Wang Xuguang;Zhou Meiya;Chen Tiejiang;Zhang Mao(Department of Emergency Medicine,Second Affiliated Hospital,Zhejiang University School of Medicine,Zhejiang Provincial Key Laboratory of Severe Trauma and Burn Diagnosis and Treatment,Zhejiang Provincial Research Center for Clinical Medicine of Acute and Critical Illness,Hangzhou 310009,Zhejiang,China;Department of Emergency Medicine,Yiwu Central Hospital,Yiwu 322000,Zhejiang,China;Department of Emergency Medicine,Shaoxing Second Hospital,Shaoxing 312099,Zhejiang,China;Department of Emergency Medicine,Ningbo First Hospital,Ningbo 315000,Zhejiang,China;Hangzhou Emergency Center,Hangzhou 310003,Zhejiang,China)

机构地区：[1]浙江大学医学院附属第二医院急诊医学科/浙江省严重创伤与烧伤诊治重点实验室/浙江省急危重症临床医学研究中心,杭州310009 [2]义乌市中心医院急诊医学科,浙江义乌322000 [3]绍兴第二医院急诊医学科,浙江绍兴312099 [4]宁波市第一医院急诊医学科,浙江宁波315000 [5]杭州市急救中心,浙江杭州310003

出　　处：《中华危重病急救医学》2025年第2期133-139,共7页Chinese Critical Care Medicine

基　　金：浙江省重点研发计划项目(2024C04045,2021C03073);浙江省金华市重大(重点)科技计划项目(2023-3-005,2022-3-043)。

摘　　要：目的探讨人胚胎干细胞来源间充质干细胞(hESC-MSC)减轻心搏骤停(CA)猪复苏后脑损伤的作用机制。方法采用随机数字表法将29只健康雄性大白猪分为假手术(Sham)组(9只)、心肺复苏(CPR)组(10只)和hESC-MSC组(10只)。Sham组仅完成动物准备。CPR组和hESC-MSC组通过电刺激诱导心室纤颤10 min、CPR 6 min的方法制备猪CA-CPR模型;复苏成功后5 min,hESC-MSC组于1 h内经静脉微泵注射hESC-MSC 2.5×10^(6)/kg。于复苏前及复苏4、8、24、48、72 h采集静脉血标本,通过酶联免疫吸附试验(ELISA)检测血清神经元特异性烯醇化酶(NSE)和S100B蛋白质(S100B)水平。于复苏24、48和72 h,采用神经功能缺损评分(NDS)和脑功能表现分级(CPC)评估动物的神经功能。每组分别于复苏24、48、72 h随机选择3只动物进行安乐死,迅速获取大脑海马组织,采用免疫荧光染色检测hESC-MSC在海马组织中的分布情况;采用免疫组化染色检测海马组织中星形胶质细胞和小胶质细胞激活情况、神经元细胞存活情况;采用原位末端缺刻标记法(TUNEL)检测细胞凋亡程度。结果CPR组和hESC-MSC组复苏4 h脑损伤标志物血清NSE和S100B水平即显著高于Sham组,随后逐渐升高;但hESC-MSC组复苏各时间点血清NSE和S100B水平均显著低于CPR组〔NSE(μg/L):4 h为20.69±3.62比28.95±3.48,72 h为27.04±5.56比48.59±9.22;S100B(μg/L):4 h为2.29±0.39比3.60±0.73,72 h为2.38±0.15比3.92±0.50,均P<0.05〕。在神经功能方面,与Sham组比较,CPR组和hESC-MSC组复苏24 h NDS评分、CPC评分即显著升高,随后逐渐下降;但hESC-MSC组复苏24 h NDS评分、CPC评分即显著低于CPR组〔NDS(分):111.67±20.21比170.00±21.79,CPC(分):2.33±0.29比3.00±0.00,均P<0.05〕。荧光显微镜下观察显示,hESC-MSC组复苏24、48、72 h海马组织中均可见hESC-MSC阳性标志物CD73、CD90和CD105表达,表明hESC-MSC能归巢至受损海马组织。此外,与Sham组比较,CPR组复苏24、48、72 h海马组织星Objective To investigate the mechanism of human embryonic stem cell-derived mesenchymal stem cells(hESC-MSC)in alleviating brain injury after resuscitation in swine with cardiac arrest(CA).Methods Twenty-nine healthy male large white swine were randomly divided into Sham group(n=9),cardiopulmonary resuscitation(CPR)group(n=10)and hESC-MSC group(n=10).The Sham group only completed animal preparation.In CPR group and hESC-MSC group,the swine model of CA-CPR was established by inducing ventricular fibrillation for 10 minutes with electrical stimulation and CPR for 6 minutes.At 5 minutes after successful resuscitation,hESC-MSC 2.5×10^(6)/kg was injected via intravenous micropump within 1 hour in hESC-MSC group.Venous blood samples were collected before resuscitation and at 4,8,24,48 and 72 hours of resuscitation.The levels of neuron specific enolase(NSE)and S100B protein(S100B)were detected by enzyme linked immunosorbent assay(ELISA).At 24,48 and 72 hours of resuscitation,neurological deficit score(NDS)and cerebral performance category(CPC)were used to evaluate the neurological function of the animals.Three animals from each group were randomly selected and euthanized at 24,48,and 72 hours of resuscitation,and the hippocampus tissues were quickly obtained.Immunofluorescence staining was used to detect the distribution of hESC-MSC in hippocampus.Immunohistochemical staining was used to detect the activation of astrocytes and microglia and the survival of neurons in the hippocampus.The degree of apoptosis was detected by TdT-mediated dUTP nick end labeling(TUNEL).Results The serum NSE and S100B levels of brain injury markers in CPR group and hESC-MSC group were significantly higher than those in Sham group at 24 hours of resuscitation,and then gradually increased.The levels of NSE and S100B in serum at each time of resuscitation in hESC-MSC group were significantly lower than those in CPR group[NSE(μg/L):20.69±3.62 vs.28.95±3.48 at 4 hours,27.04±5.56 vs.48.59±9.22 at 72 hours;S100B(μg/L):2.29±0.39 vs.3.60±0.73

关 键 词：人胚胎干细胞来源间充质干细胞 心搏骤停 心肺复苏 脑损伤 猪 

分 类 号：R541.7[医药卫生—心血管疾病] R54[医药卫生—内科学]