[1]郑良良,张弛.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[J].中医正骨,2021,33(09):3-14.
　ZHENG Liangliang,ZHANG Chi.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[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2021,33(09):3-14.
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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()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第33卷
期数:
2021年09期
页码:
3-14
栏目:
基础研究
出版日期:
2021-09-20

文章信息/Info

Title:
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
(金华市中心医院,浙江 金华 321000)
Author(s):
ZHENG Liangliang; ZHANG Chi
Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China
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
Keywords:
sciatic nerve;  rats;  chronic constriction injury;  stem cells;  nerve growth factor;  exosomes;  animal experimentation
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L4～L5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L4～L5脊髓组织的细胞凋亡情况; 提取大鼠脊髓组织总蛋白,采用蛋白印迹法检测大鼠脊髓组织中NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)、白细胞介素(interleukin,IL)-1、兔抗鼠半胱氨酸天冬氨酸蛋白酶(cysteine aspartic acid specific protease,Caspase)-1和Caspase-3的蛋白表达量。
Abstract:
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 L4 and L5 tissues for preparing frozen sections,which were stained with HE and used for evaluating the pathological changes of gray matter of spinal dorsal horn(SDH)based on Allen SDH gray matter lesion scoring criterion.Followed by TUNEL staining,the section were observed under the microscope for evaluating the apoptosis of L4 and L5 myoloid tissue.The total protein was extracted from myoloid tissue of rats,and the protein expression levels of NOD-like receptor protein 3(NLRP3),interleukin(IL)-1,rabbit anti-mouse cysteine aspartic acid specific protease(Caspase)-1 and Caspase-3 were assayed by Western blotting.

参考文献/References:

[1] 陈向荣,寇红伟,包德明,等.表儿茶酸激活Nrf2/ARE通路在小鼠脑外伤中的神经保护作用[J].中华实验外科杂志,2019,36(12):2224-2226.
[2] 彭艳.神经节苷脂对早产儿颅内出血引起的神经系统损伤的临床效果及神经、运动功能的影响分析[J].中外医学研究,2019,17(3):40-42.
[3] 于敏,李承鑫,张素蕊,等.高频电疗法促进周围神经损伤后再生研究进展[J].亚太传统医药,2014,10(9):56-57.
[4] 季航宇,顾军,谢凌寒,等.干细胞、组织工程支架与神经营养因子在脊髓损伤治疗中的应用[J].中国组织工程研究,2020,24(25):4088-4093.
[5] 于海微,李佩玲,庄如锦,等.人脐带血和骨髓来源间充质干细胞的体外分离、培养、分化及生物学特性比较[J].中国组织工程研究,2009,13(6):1021-1024.
[6] 吴奕江,章何陋,高炀,等.白藜芦醇影响骨髓间充质干细胞体外分化的研究进展[J].中医正骨,2020,32(10):47-51.
[7] LI Z J,LIU F,HE X,et al.Exosomes derived from mesenchymal stem cells attenuate inflammation and demyelination of the central nervous system in EAE rats by regulating the polarization of microglia[J].Int Immunopharmacol,2019,67:268-280.
[8] CHAU M J,DEVEAU T C,GU X,et al.Delayed and repeated intranasal delivery of bone marrow stromal cells increases regeneration and functional recovery after ischemic stroke in mice[J].BMC Neurosci,2018,19(1):20.
[9] YANG F,LIAO X Z,TIAN Y,et al.Exosome separation using microfluidic systems:size-based,immunoaffinity-based and dynamic methodologies[J].Biotechnol J,2017,12(4):11-19.
[10] MILANE L,SINGH A,MATTHEOLABAKIS G,et al.Exosome mediated communication within the tumor microenvironment[J].J Control Release,2015,219:278-294.
[11] WORTZEL I,DROR S,KENIFIC C M,et al.Exosome-mediated metastasis:communication from a distance[J].Dev Cell,2019,49(3):347-360.
[12] YAMASHITA T,TAKAHASHI Y,TAKAKURA Y.Possibility of exosome-based therapeutics and challenges in production of exosomes eligible for therapeutic application[J].Biol Pharm Bull,2018,41(6):835-842.
[13] 林平,陈毅,梁淑霞,等.NGF和PTEN双基因诱导大鼠骨髓间充质干细胞向神经元样细胞分化效果观察[J].浙江医学,2020,42(10):1000-1005.
[14] ALLEN A R.Surgery for experimental lesions of spinal cord equivalent to crush injury of fracture dislocation of spinal column:a preliminary report[J].JAMA,1911,57(4):878-880.
[15] 蔡伟森,尹传瑞,田宗光.梨状肌综合征的影像学与临床特点分析[J].中国骨与关节损伤杂志,2018,33(8):840-841.
[16] 赵睿,丛锐,田通,等.肘管综合征一期手术失败原因探讨及肘管翻修术的临床疗效评估[J].中华手外科杂志,2019,35(5):347-350.
[17] RICCIO M,MARCHESINI A,PUGLIESE P,et al.Nerve repair and regeneration:Biological tubulization limits and future perspectives[J].J Cell Physiol,2019,234(4):3362-3375.
[18] YANNAS I V,TZERANIS D S,SO P T C.Regeneration of injured skin and peripheral nerves requires control of wound contraction,not scar formation[J].Wound Repair Regen,2017,25(2):177-191.
[19] 朱斌,关炳瑜,贺元,等.电针调控大鼠脊髓损伤后灰质中BDNF,NGF和NT3的表达[J].上海针灸杂志,2019,38(10):1188-1194.
[20] 刘菲菲.NGF和BDNF联合诱导NSC分化及其在192-IgG-saporin致阿尔茨海默病模型鼠中的应用[D].广州:广州医科大学,2014.
[21] 陈亚军,韩焕芝,陈红光,等.NLRP3炎症小体在神经病理性痛大鼠脊髓小胶质细胞激活中的作用[J].中华麻醉学杂志,2019,39(12):1464-1467.
[22] 王丽蓉,朱进飘,方媛媛,等.大鼠炎性痛形成时脊髓神经元P2X7受体与NLRP3/IL-1β信号通路的关系[J].中华麻醉学杂志,2020,40(4):437-441.
[23] BECKWITH K S,BECKWITH M S,ULLMANN S,et al.Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection[J].Nat Commun,2020,11(1):2270.
[24] SUN X D,TANG Y,JIANG C H,et al.Oxidative stress,NF-κB signaling,NLRP3 inflammasome,and caspase apoptotic pathways are activated in mammary gland of ketotic Holstein cows[J].J Dairy Sci,2021,104(1):849-861.
[25] 刘朝圣,申梦洁,韩晓凤,等.红景天苷对UVB辐射HaCat细胞活性及Caspase-3/9蛋白表达的影响[J].湖南中医药大学学报,2019,39(8):952-956.

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更新日期/Last Update: 1900-01-01