人脐带间充质干细胞单次静脉注射NOG小鼠组织分布及其分析方法学研究  

Tissue distribution and analysis of human umbilical cord mesenchymal stem cells in NOG mice following single intravenous injection

作　　者：叶志超 陈国玉 潘若浪 史煜华 顾利强 夏丽娟 林晓波 张强 徐莎莎 邵金金 张立将 YE Zhichao;CHEN Guoyu;PAN Ruolang;SHI Yuhua;GU Liqiang;XIA Lijuan;LIN Xiaobo;ZHANG Qiang;XU Shasha;SHAO Jinjin;ZHANG Lijiang(Key Laboratory of Drug Safety Evaluation and Research of Zhejiang Province,Center of Safety Evaluation and Research,Hangzhou Medical College,Hangzhou 310053,China;S-Evans Biosciences Co.,Ltd,Hangzhou 311100)

机构地区：[1]杭州医学院安全性评价研究中心,浙江省药物安全性评价技术研究重点实验室,杭州310053 [2]杭州易文赛生物技术有限公司,杭州311100

出　　处：《中国实验动物学报》2023年第12期1573-1580,共8页Acta Laboratorium Animalis Scientia Sinica

基　　金：浙江省重点研发计划项目(2021C03077);杭州医学院青年科研基金项目(KYQN202106)。

摘　　要：目的建立并验证小鼠组织中人源SRY基因DNA RT-qPCR分析方法学,研究人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUCMSCs)单次静脉注射后在重度免疫缺陷NOG小鼠体内的组织分布情况。方法建立小鼠组织中人源SRY基因DNA的RT-qPCR分析方法,进行标准曲线与线性范围、准确度与精密度、稳定性等方法学验证。取NOG小鼠36只,雌雄各半,单次静脉注射给予HUCMSCs 3.5×10^(7)cells/kg,于6、12、24、72 h、1周、2周各取6只小鼠麻醉,采集血液(EDTA抗凝)后解剖,取肺、肾、心脏、肝、大脑、脊髓、胃、小肠、脂肪、皮肤、脾、睾丸(子宫、卵巢)提取DNA,用已验证的小鼠组织中人源SRY基因DNA RT-qPCR分析方法测定各组织中HUCMSCs的分布情况。另选取NOG小鼠18只,雌雄各半,分为对照组6只、给药组12只,分别静脉注射0.9%氯化钠注射液、HUCMSCs 3.5×10^(7)cells/kg,给药期间观察小鼠急性毒性反应情况,给药组于给药后72 h、2、4周各取4只动物剖检观察脏器组织大体观情况,取肺组织石蜡切片采用免疫组化法检测线粒体蛋白表达,分析HUCMSCs在肺组织中的定植情况。结果建立的小鼠组织中人源SRY基因DNA的RT-qPCR分析方法各考察指标均符合验证标准;NOG小鼠单次静脉注射HUCMSCs后,主要分布于肺和血液中(给药后1周内),肺组织浓度高于血液,肺组织、血液中HUCMSCs浓度分别在6~24 h、6~72 h之内维持相对平稳水平,之后随时间推移下降,其他组织各采样点均未测得HUCMSCs分布;定植结果显示静脉注射HUCMSCs后72 h可在肺部检测到其存在,2周和4周时则未检测到;单次静脉注射HUCMSCs后NOG小鼠未见明显急性毒性反应。结论建立的HUCMSCs小鼠组织中分布分析方法可靠、可行;NOG小鼠单次静脉注射HUCMSCs后主要于给药后1周内分布于肺和血液,72 h主要定植于肺组织;单次静脉注射HUCMSCs具有良好的安全性。Objective To establish a quantitative polymerase chain reaction(PCR)method for the analysis of human-derived SRY DNA in mouse tissues,and to study the tissue distribution of human umbilical cord mesenchymal stem cells(HUCMSCs)in immunodeficient NOG mice after a single intravenous injection.Methods We established a quantitative PCR method for the analysis of human SRY DNA in mouse tissues,and validated the standard curve,linear range,accuracy,precision,and stability.Thirty-six NOG mice(18 male,18 female)were administered 3.5×10^(7)HUCMSCs/kg by single intravenous injection.Six mice were then anesthetized and dissected after blood collection(EDTA anticoagulation)at 6,12,24,and 72 h,and at 1 and 2 weeks,respectively.DNA was extracted from lung,kidney,heart,liver,brain,spinal cord,stomach,small intestine,fat,skin,spleen,testis,uterus,and ovary tissues,and the distribution of HUCMSCs in each tissue was determined by the validated quantitative PCR method for detecting the humanderived SRY gene in mouse tissues.In addition,18 NOG mice(9 male,9 female)were divided into control(n=6)and treatment groups(n=12)injected intravenously with 0.9%sodium chloride and 3.5×10^(7)cells/kg,respectively.Acute toxic reactions were observed during the administration period,and four animals were dissected at 72 h and at 2 and 4 weeks after administration to observe the gross organs.Mitochondrial protein expression was detected in paraffin sections of lung tissues by immunohistochemistry to analyze the colonization of HUCMSCs in lung tissues.Results The established RT-qPCR method for human-derived SRY DNA in mouse tissues met the validation criteria for each index.After a single intravenous injection in NOG mice,HUCMSCs were mainly distributed in the lungs and blood within 1 week after administration,with higher concentrations in lung tissues than in blood.The concentrations of HUCMSCs in lung tissue and blood remained relatively stable within 6~24 h and 6~72 h,respectively,and then decreased over time.The distribution of HUCMSCs in othe

关 键 词：人脐带间充质干细胞 NOG小鼠 定植分析 组织分布 

分 类 号：Q95-33[生物学—动物学]