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

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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。结果:①hUCB-MSCs鉴定结果。流式细胞仪检测第2代hUCB-MSCs,CD34阳性率0.4%,CD45阳性率0.6%,CD90阳性率95.8%,CD105阳性率98.3%,符合hUCB-MSCs表面蛋白表达特征。②重组慢病毒转染结果。重组慢病毒转染72 h,hUCB-MSCs转染率(89.22±6.91)%。③NGF-hUCB-MSCs-Exo鉴定结果。透射电子显微镜结果显示,NGF-hUCB-MSCs-Exo为杯口状结构。蛋白印迹法检测结果显示,NGF-hUCB-MSCs-Exo标志蛋白CD63、CD9和CD81表达量高于hUCB-MSCs,表明Exo提取成功。④hUCB-MSCs摄取内化NGF-hUCB-MSCs-Exo观察结果。hUCB-MSCs与NGF-hUCB-MSCs-Exo共培养12 h后,观察到hUCB-MSCs摄取并内化NGF-hUCB-MSCs-Exo。⑤坐骨神经CCI模型建立结果。术后第1天、第3天、第5天、第7天,坐骨神经CCI模型大鼠的PWMT和PWTL均低于正常大鼠[PWMT:(3.05±0.06)g,(7.34±0.08)g,t=3.903,P=0.000;(3.07±0.04)g,(7.39±0.06)g,t=3.342,P=0.001;(3.11±0.05)g,(7.47±0.04)g,t=3.892,P=0.000;(2.89±0.02)g,(7.56±0.09)g,t=4.903,P=0.000;PWTL:(6.13±0.03)s,(18.12±0.45)s,t=3.562,P=0.000;(6.07±0.05)s,(18.09±0.36)s,t=3.769,P=0.000;(6.01±0.04)s,(18.77±0.44)s,t=3.809,P=0.000;(5.77±0.09)s,(18.37±0.39)s,t=4.651,P=0.000],表明造模成功。⑥病理学检查结果。正常对照组、坐骨神经CCI模型组、hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组大鼠Allen脊髓后角灰质病变评分比较,差异有统计学意义[(0.32±0.04)分,(4.21±0.11)分,(4.09±0.15)分,(1.89±0.17)分,F=11.714,P=0.000]。坐骨神经CCI模型组、hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组大鼠Allen脊髓后角灰质病变评分均高于正常对照组(q=2.783,P=0.015;q=3.921,P=0.000;q=3.784,P=0.000);hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组大鼠Allen脊髓后角灰质病变评分均低于坐骨神经CCI模型组(q=2.059,P=0.024;q=2.071,P=0.021);NGF-hUCB-MSCs-Exo注射组大鼠Allen脊髓后角灰质病变评分低于hUCB-MSCs-Exo注射组(q=2.809,P=0.013)。⑦细胞凋亡检测结果。正常对照组、坐骨神经CCI模型组、hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组大鼠脊髓组织细胞凋亡率比较,差异有统计学意义[(0.15±0.02)%,(8.98±0.37)%,(8.14±0.29)%,(3.46±0.31)%,F=9.908,P=0.012]。坐骨神经CCI模型组、hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组大鼠脊髓组织细胞凋亡率均高于正常对照组(q=2.142,P=0.017;q=3.287,P=0.000;q=2.275,P=0.025);hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组大鼠脊髓组织细胞凋亡率均低于坐骨神经CCI模型组(q=2.021,P=0.021;q=2.086,P=0.024);NGF-hUCB-MSCs-Exo注射组大鼠脊髓组织细胞凋亡率低于hUCB-MSCs-Exo注射组(q=3.008,P=0.011)。⑧炎症反应和细胞凋亡相关基因的蛋白表达分析结果。正常对照组、坐骨神经CCI模型组、hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组大鼠脊髓组织中NLRP3、IL-1β、Caspase-1和Caspase-3蛋白表达量比较,组间差异均有统计学意义(122.16±15.23,764.29±20.14,627.86±21.17,198.25±14.37,F=12.428,P=0.000;106.27±14.11,698.11±24.57,687.45±22.36,208.42±19.79,F=15.008,P=0.000;108.43±13.79,305.58±24.36,301.25±27.72,153.14±11.99,F=19.897,P=0.000;102.58±12.17,725.16±21.24,711.32±20.37,215.33±19.27,F=12.278,P=0.000)。坐骨神经CCI模型组、hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组大鼠脊髓组织中NLRP3、IL-1β、Caspase-1和Caspase-3蛋白表达量均高于正常对照组(坐骨神经CCI模型组:q=3.709,P=0.000;q=3.328,P=0.000;q=3.145,P=0.000;q=2.974,P=0.000;hUCB-MSCs-Exo注射组:q=2.893,P=0.019;q=2.944,P=0.013;q=3.008,P=0.009;q=3.852,P=0.000;NGF-hUCB-MSCs-Exo注射组:q=2.428,P=0.022;q=4.903,P=0.000;q=3.884,P=0.000;q=4.382,P=0.000);hUCB-MSCs-Exo注射组和NGF-hUCB-MSCs-Exo注射组大鼠脊髓组织中NLRP3、IL-1β、Caspase-1和Caspase-3蛋白表达量均低于坐骨神经CCI模型组(hUCB-MSCs-Exo注射组:q=3.609,P=0.000;q=3.811,P=0.000;q=3.476,P=0.000;q=3.889,P=0.000;NGF-hUCB-MSCs-Exo注射组:q=2.338,P=0.000;q=3.098,P=0.000;q=2.358,P=0.000;q=3.775,P=0.000);NGF-hUCB-MSCs-Exo注射组大鼠脊髓组织中NLRP3、IL-1β、Caspase-1和Caspase-3蛋白表达量均低于hUCB-MSCs-Exo注射组(q=3.798,P=0.000;q=3.573,P=0.000;q=2.998,P=0.000;q=3.208,P=0.000)。结论:NGF-hUCB-MSCs-Exo治疗大鼠坐骨神经CCI,能够抑制脊髓后角灰质病变和脊髓细胞凋亡,其作用机制可能与抑制NLRP3、IL-1β、Caspase-1和Caspase-3的表达有关。 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 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 L_(4) and L_(5) 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.Results:①The results of hUCB-MSCs identification.The positive rates of CD34,CD45,CD90 and CD105 in second-generation hUCB-MSCs were 0.4%,0.6%,95.8%and 98.3%respectively,which accorded with the surface protein expression characteristics of hUCB-MSCs.②The results of recombinant lentivirus transfection.The transfection rate of hUCB-MSCs was 89.22±6.91%after 72-h transfection with recombinant lentivirus.③The results of NGF-hUCB-MSCs-Exo identification.As demonstrated by transmission electron microscopy,NGF-hUCB-MSCs-Exo exhibited as a cup-like structure.The Western blotting results showed that the expression levels of NGF-hUCB-MSCs-Exo marker proteins CD63,CD9 and CD81 were higher than those of hUCB-MSCs,indicating success in the extraction of Exo.④The results of uptake and internalization of NGF-hUCB-MSCs-Exo by hUCB-MSCs.NGF-hUCB-MSCs-Exo was took up and internalized by hUCB-MSCs after hUCB-MSCs were co-cultured with NGF-hUCB-MSCs-Exo for 12 hours.⑤The results of sciatic nerve CCI modeling.The PWMT and PWTL of sciatic nerve CCI model rats declined as compared with those of the normal rats on days 1,3,5 and 7 after the surgery(PWMT:3.05±0.06 vs 7.34±0.08 g,t=3.903,P=0.000;3.07±0.04 vs 7.39±0.06 g,t=3.342,P=0.001;3.11±0.05 vs 7.47±0.04 g,t=3.892,P=0.000;2.89±0.02 vs 7.56±0.09 g,t=4.903,P=0.000;PWTL:6.13±0.03 vs 18.12±0.45 seconds,t=3.562,P=0.000;6.07±0.05 vs 18.09±0.36 seconds,t=3.769,P=0.000;6.01±0.04 vs 18.77±0.44 seconds,t=3.809,P=0.000;5.77±0.09 vs 18.37±0.39 seconds,t=4.651,P=0.000),which suggested the models were successfully built.⑥The results of pathological examination.There was statistical difference in Allen SDH gray matter lesion scores among normal control group,CCI model group,hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group(0.32±0.04,4.21±0.11,4.09±0.15,1.89±0.17 points,F=11.714,P=0.000).The Allen SDH gray matter lesion scores were all higher in CCI model group,hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group compared to normal control group(q=2.783,P=0.015;q=3.921,P=0.000;q=3.784,P=0.000),and were lower in hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group compared to CCI model group(q=2.059,P=0.024;q=2.071,P=0.021);and were lowest in NGF-hUCB-MSCs-Exo injection group(q=2.809,P=0.013).⑦The results of apoptosis detection.There was statistical difference in apoptosis rate of the rat myoloid tissue among normal control group,CCI model group,hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group(0.15±0.02,8.98±0.37,8.14±0.29,3.46±0.31%,F=9.908,P=0.012).The apoptosis rate of rat myoloid tissue were all higher in CCI model group,hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group compared to normal control group(q=2.142,P=0.017;q=3.287,P=0.000;q=2.275,P=0.025),and were lower in hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group compared to CCI model group(q=2.021,P=0.021;q=2.086,P=0.024);and was lowest in NGF-hUCB-MSCs-Exo injection group(q=3.008,P=0.011).⑧The results of analysis on protein expression of genes related to inflammation and apoptosis.There was statistical difference in the protein expression levels of NLRP3,IL-1β,Caspase-1 and Caspase-3 in myoloid tissue of rats among normal control group,CCI model group,hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group(122.16±15.23,764.29±20.14,627.86±21.17,198.25±14.37,F=12.428,P=0.000;106.27±14.11,698.11±24.57,687.45±22.36,208.42±19.79,F=15.008,P=0.000;108.43±13.79,305.58±24.36,301.25±27.72,153.14±11.99,F=19.897,P=0.000;102.58±12.17,725.16±21.24,711.32±20.37,215.33±19.27,F=12.278,P=0.000).The protein expression levels of NLRP3,IL-1β,Caspase-1 and Caspase-3 in myoloid tissues of rats were all higher in CCI model group,hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group compared to normal control group(CCI model group:q=3.709,P=0.000;q=3.328,P=0.000;q=3.145,P=0.000;q=2.974,P=0.000;hUCB-MSCs-Exo injection group:q=2.893,P=0.019;q=2.944,P=0.013;q=3.008,P=0.009;q=3.852,P=0.000;NGF-hUCB-MSCs-Exo injection group:q=2.428,P=0.022;q=4.903,P=0.000;q=3.884,P=0.000;q=4.382,P=0.000);and were lower in hUCB-MSCs-Exo injection group and NGF-hUCB-MSCs-Exo injection group compared to CCI model group(hUCB-MSCs-Exo injection group:q=3.609,P=0.000;q=3.811,P=0.000;q=3.476,P=0.000;q=3.889,P=0.000;NGF-hUCB-MSCs-Exo injection group:q=2.338,P=0.000;q=3.098,P=0.000;q=2.358,P=0.000;q=3.775,P=0.000);and were lowest in NGF-hUCB-MSCs-Exo injection group(q=3.798,P=0.000;q=3.573,P=0.000;q=2.998,P=0.000;q=3.208,P=0.000).Conclusion:NGF-hUCB-MSCs-Exo can inhibit SDH gray matter lesion and apoptosis in repairing CCI of sciatic nerve in rats,its mechanisms may be that it can inhibit the expression of NLRP3,IL-1β,Caspase-1 and Caspase-3.

作者郑良良张弛 ZHENG Liangliang;ZHANG Chi(Jinhua Municipal Central Hospital,Jinhua 321000,Zhejiang,China)

机构地区金华市中心医院

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

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 sciatic nerve rats chronic constriction injury stem cells nerve growth factor exosomes animal experimentation

分类号 R745 [医药卫生—神经病学与精神病学]

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1周云帆,杨波,胡祥,焦洪亮,周长辉,田毅,雷宁静,谷晨熙,许予明,戴建武,关方霞.经血源性子宫内膜间充质干细胞的分离、培养与鉴定[J].中国组织工程研究与临床康复,2010,14(32):5952-5956. 被引量：14
2陈桂秀,冯滨,刘涛.脐带血干细胞：组织工程人工心脏研究种子细胞的新来源[J].中国组织工程研究与临床康复,2010,14(32):6027-6030.
3何晓婷,李怀芳.人脐血间充质干细胞分离培养的研究进展[J].同济大学学报（医学版）,2011,32(1):120-124. 被引量：2
4徐萍,王玥,熊燕,陈大明,王小理,于建荣.干细胞研究国际发展态势分析[J].科学观察,2011,6(2):1-15. 被引量：3
5任思坡,韩光宇,吴秀娟,张薇,拾莉,谭昆,王健,耿跃春,杨钦湘.人脐血间充质干细胞分离培养方法的优化[J].实用预防医学,2011,18(10):1958-1959.
6许雯,张翼鷟,赵丹丹,赵智刚,于泳,赵伟鹏.微孔膜法比较Stro-1~＋和Stro-1^-间充质干细胞的免疫调节作用[J].中国组织工程研究与临床康复,2012,16(1):111-115. 被引量：2
7鲁俊山,王建伟,马勇.人脐血间充质干细胞定向成骨分化的研究进展[J].现代中西医结合杂志,2012,21(13):1475-1477. 被引量：2
8何晓婷,李怀芳,王建军,孙向华.人脐血间充质干细胞的改良分离培养[J].同济大学学报（医学版）,2012,33(2):33-37.
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10魏晓巍,李玉云,张强.脐带血间充质干细胞的分离培养及诱导表达IDO对免疫介导再生障碍性贫血小鼠的治疗研究[J].蚌埠医学院学报,2012,37(10):1147-1150.

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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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