神经生长因子过表达的人脐带血间充质干细胞来源外泌体修复大鼠坐骨神经慢性压迫损伤的效果及作用机制研究  

Efficacy and mechanism of exosomes derived from nerve growth factor-overexpressing human umbilical cord blood-derived mesenchymal stem cells against chronic constriction injury of sciatic nerve in rats

作　　者：郑良良 张弛 ZHENG Liangliang;ZHANG Chi(Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China)

机构地区：[1]金华市中心医院,浙江金华321000

出　　处：《中医正骨》2021年第9期3-14,共12页The Journal of Traditional Chinese Orthopedics and Traumatology

摘　　要：目的:探讨神经生长因子(nerve growth factor,NGF)过表达的人脐带血间充质干细胞(human umbilical cord blood-derived mesenchymal stem cells,hUCB-MSCs)来源外泌体(exosome,Exo)修复大鼠坐骨神经慢性压迫损伤(chronic constriction injury,CCI)的效果及作用机制。方法:①取培养至第2代的hUCB-MSCs,采用流式细胞仪对其进行鉴定。②根据NGF基因序列,构建NGF过表达的慢病毒载体,包装后转染hUCB-MSCs,并测定hUCB-MSCs转染率。③采用超速离心法提取NGF过表达的hUCB-MSCs来源Exo(NGF-hUCB-MSCs-Exo),进行Exo形态及标志蛋白的鉴定。④采用不同的荧光染色试剂分别对hUCB-MSCs细胞核、细胞骨架及NGF-hUCB-MSCs-Exo进行染色,在荧光显微镜下观察hUCB-MSCs摄取内化NGF-hUCB-MSCs-Exo。⑤从20只雄性Sprague Dawley大鼠中选取15只,采用手术建立坐骨神经CCI模型,分别于术后第1天、第3天、第5天、第7天采用IITC动物热痛刺激仪测定大鼠机械刺激缩足反射阈值(paw withdrawal mechanical threshold,PWMT)和热刺激缩足反射潜伏期(paw withdrawal thermal latency,PWTL),评价造模是否成功。⑥分别采用空载体慢病毒和NGF过表达慢病毒转染hUCB-MSCs,制备空载体慢病毒转染hUCB-MSCs来源Exo(hUCB-MSCs-Exo)和NGF-hUCB-MSCs-Exo。将15只坐骨神经CCI模型大鼠随机分为坐骨神经CCI模型组、hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组,每组5只;将剩余5只正常大鼠纳入正常对照组。正常对照组大鼠不做任何处理,坐骨神经CCI模型组大鼠于尾静脉注射1 mL PBS,hUCB-MSCs-Exo注射组大鼠于尾静脉注射1 mL hUCB-MSCs-Exo和hUCB-MSCs混合液,NGF-hUCB-MSCs-Exo注射组大鼠于尾静脉注射1 mL NGF-hUCB-MSCs-Exo与hUCB-MSCs混合液,每周注射1次,连续注射3周。3周后处死大鼠,取L_(4)~L_(5)制备冷冻切片,进行HE染色,采用Allen脊髓后角灰质病变评分标准评价脊髓后角灰质的病理变化;进行TUNEL染色,于显微镜下观察,评价L_(4)~L_(5)脊髓组织的细�Objective:To explore the effects and mechanism of exosome(Exo)derived from nerve growth factor(NGF)-overexpressing(OE)human umbilical cord blood-derived mesenchymal stem cells(hUCB-MSCs)in repairing chronic constriction injury(CCI)of sciatic nerve in rats.Methods:①The second-generation hUCB-MSCs were identified by flow cytometry(FCM).②According to NGF gene sequence,a NGF-OE lentiviral vector was constructed,packaged and then transfected into hUCB-MSCs,followed by the determination of its transfection rate.③The NGF-OE hUCB-MSCs-derived Exo(NGF-hUCB-MSCs-Exo)was extracted by ultracentrifugation for identifying its morphology and marker protein.④The nuclei and cytoskeletons of hUCB-MSCs and NGF-hUCB-MSCs-Exo were stained with different fluorescent dyes and then the uptake and internalization of NGF-hUCB-MSCs-Exo by hUCB-MSCs were observed under a fluorescence microscope.⑤Among the 20 male Sprague Dawley(SD)rats,15 ones were selected and subjected to surgery for inducing CCI of sciatic nerve.The paw withdrawal mechanical threshold(PWMT)and paw withdrawal thermal latency(PWTL)were measured on days 1,3,5 and 7 after the surgery respectively with a tail flick analgesia meter to evaluate whether the modeling was successful.⑥The empty vector lentivirus-and NGF-OE lentivirus-transfected hUCB-MSCs were separately used to prepare hUCB-MSCs-Exo and NGF-hUCB-MSCs-Exo.The 15 rats were randomly divided into CCI model group,hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group,5 cases in each group.The remained 5 normal rats were assigned into the normal control group without any treatment,while those in CCI model group,hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group were injected with PBS(1 mL),a mixture of hUCB-MSCs-Exo and hUCB-MSCs(1 mL)and a mixture of NGF-hUCB-MSCs-Exo and hUCB-MSCs(1 mL)respectively via the tail vein,once a week for consecutive 3 weeks.Afterwards,the rats were sacrificed and sampled from L_(4) and L_(5) tissues for preparing frozen sections,which were stained w

关 键 词：坐骨神经 大鼠 慢性压迫损伤 干细胞 神经生长因子 外泌体 动物实验 

分 类 号：R745[医药卫生—神经病学与精神病学]