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[1]鄭良良,張弛.神經(jīng)生長因子過表達(dá)的人臍帶血間充質(zhì)干細(xì)胞來源外泌體修復(fù)大鼠坐骨神經(jīng)慢性壓迫損傷的效果及作用機(jī)制研究[J].中醫(yī)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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神經(jīng)生長因子過表達(dá)的人臍帶血間充質(zhì)干細(xì)胞來源外泌體修復(fù)大鼠坐骨神經(jīng)慢性壓迫損傷的效果及作用機(jī)制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第33卷
期數(shù):
2021年09期
頁碼:
3-14
欄目:
基礎(chǔ)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:
鄭良良; 張弛
(金華市中心醫(yī)院,浙江 金華 321000)
Author(s):
ZHENG Liangliang; ZHANG Chi
Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China
關(guān)鍵詞:
坐骨神經(jīng);  大鼠;  慢性壓迫損傷;  干細(xì)胞;  神經(jīng)生長因子;  外泌體;  動(dòng)物實(shí)驗(yàn)
Keywords:
sciatic nerve;  rats;  chronic constriction injury;  stem cells;  nerve growth factor;  exosomes;  animal experimentation
摘要:
目的:探討神經(jīng)生長因子(nerve growth factor,NGF)過表達(dá)的人臍帶血間充質(zhì)干細(xì)胞(human umbilical cord blood-derived mesenchymal stem cells,hUCB-MSCs)來源外泌體(exosome,Exo)修復(fù)大鼠坐骨神經(jīng)慢性壓迫損傷(chronic constriction injury,CCI)的效果及作用機(jī)制。方法:①取培養(yǎng)至第2代的hUCB-MSCs,采用流式細(xì)胞儀對(duì)其進(jìn)行鑒定。②根據(jù)NGF基因序列,構(gòu)建NGF過表達(dá)的慢病毒載體,包裝后轉(zhuǎn)染hUCB-MSCs,并測定hUCB-MSCs轉(zhuǎn)染率。③采用超速離心法提取NGF過表達(dá)的hUCB-MSCs來源Exo(NGF-hUCB-MSCs-Exo),進(jìn)行Exo形態(tài)及標(biāo)志蛋白的鑒定。④采用不同的熒光染色試劑分別對(duì)hUCB-MSCs細(xì)胞核、細(xì)胞骨架及NGF-hUCB-MSCs-Exo進(jìn)行染色,在熒光顯微鏡下觀察hUCB-MSCs攝取內(nèi)化NGF-hUCB-MSCs-Exo。⑤從20只雄性Sprague Dawley大鼠中選取15只,采用手術(shù)建立坐骨神經(jīng)CCI模型,分別于術(shù)后第1天、第3天、第5天、第7天采用IITC動(dòng)物熱痛刺激儀測定大鼠機(jī)械刺激縮足反射閾值(paw withdrawal mechanical threshold,PWMT)和熱刺激縮足反射潛伏期(paw withdrawal thermal latency,PWTL),評(píng)價(jià)造模是否成功。⑥分別采用空載體慢病毒和NGF過表達(dá)慢病毒轉(zhuǎn)染hUCB-MSCs,制備空載體慢病毒轉(zhuǎn)染hUCB-MSCs來源Exo(hUCB-MSCs-Exo)和NGF-hUCB-MSCs-Exo。將15只坐骨神經(jīng)CCI模型大鼠隨機(jī)分為坐骨神經(jīng)CCI模型組、hUCB-MSCs-Exo注射組和NGF-hUCB-MSCs-Exo注射組,每組5只; 將剩余5只正常大鼠納入正常對(duì)照組。正常對(duì)照組大鼠不做任何處理,坐骨神經(jīng)CCI模型組大鼠于尾靜脈注射1 mL PBS,hUCB-MSCs-Exo注射組大鼠于尾靜脈注射1 mL hUCB-MSCs-Exo和hUCB-MSCs混合液,NGF-hUCB-MSCs-Exo注射組大鼠于尾靜脈注射1 mL NGF-hUCB-MSCs-Exo與hUCB-MSCs混合液,每周注射1次,連續(xù)注射3周。3周后處死大鼠,取L4～L5制備冷凍切片,進(jìn)行HE染色,采用Allen脊髓后角灰質(zhì)病變?cè)u(píng)分標(biāo)準(zhǔn)評(píng)價(jià)脊髓后角灰質(zhì)的病理變化; 進(jìn)行TUNEL染色,于顯微鏡下觀察,評(píng)價(jià)L4～L5脊髓組織的細(xì)胞凋亡情況; 提取大鼠脊髓組織總蛋白,采用蛋白印跡法檢測大鼠脊髓組織中NOD樣受體蛋白3(NOD-like receptor protein 3,NLRP3)、白細(xì)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的蛋白表達(dá)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.

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更新日期/Last Update: 1900-01-01