人脐血单个核细胞对大鼠腓肠肌失神经损伤的治疗作用
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摘要
目的制备大鼠坐骨神经离断后的腓肠肌萎缩模型,研究人脐血单个核细胞(CB-MNCs)肌肉多点注射对损伤的治疗效果。方法采集并制备新鲜人CB-MNCs;将30只4～6周龄Wistar大鼠随机分为正常对照组(Normal组,n=10)、CB-MNCs治疗组(Therapy组,n=10)和生理盐水治疗组(NS组,n=10),Therapy组和NS组大鼠离断坐骨神经,建立腓肠肌萎缩模型;在造模后第7天、第14天和第21天,分3次将CB-MNCs或等体积生理盐水多点注射到腓肠肌损伤局部,观察大鼠行动能力改变和足弓坏疽发展情况,并于第28天取样测定腓肠肌湿重比、肌肉横截面积,以及血液和腓肠肌中氧化应激相关指标、炎性因子水平和凋亡相关基因的表达,观察肌肉组织中血管内皮生长因子-α(VEGF-α)、α-肌动蛋白(α-actin)和肌营养不良蛋白(Dystrophin)的表达变化。结果 Therapy组大鼠足部坏疽面积和水肿程度明显比NS组减轻(P=0. 002),而且Therapy组中腓肠肌湿重比较NS组升高(P <0. 001);病理切片显示,Normal组、NS组和Therapy组肌肉横截面积分别为(12 452. 0±202. 8)、(6 287. 0±142. 2)和(8 193. 0±115. 5)μm2,差异有统计学意义(P <0. 001)。与NS组相比,Therapy组血清和肌肉肌酸激酶(CK)、丙二醛(MDA)含量明显降低,超氧化物歧化酶(SOD)和过氧化氢酶(CAT)含量明显增高(P <0. 001)。Therapy组较NS组半胱天冬酶-3(Caspase-3)、促凋亡基因Bax、肿瘤坏死因子-α(TNF-α)的mRNA表达量降低,抑凋亡基因Bcl-2、白细胞介素-10 (IL-10)、α-actin、Dystrophin和VEGF-α的mRNA表达量增高(P <0. 05)。与NS组相比,Therapy组α-actin、Dystrophin和VEGF-α蛋白表达均增高(P <0. 05)。免疫组化发现,NS组和Therapy组Dystro-phin表达均增加,但Therapy组明显高于NS组(P <0. 05)。结论肌肉注射CB-MNCs可有效促进腓肠肌失神经损伤的伤口愈合,缓解萎缩、促进肌肉修复,并降低凋亡细胞比例。
Objective To explore the effect of human cord blood mononuclear cells( CB-MNCs) on relieving the gastrocnemius muscle atrophy and promoting the damage repair in the isolated sciatic nerve rats after intramuscular administration.Methods CB-MNCs were isolated from healthy term infants and cultured in vitro. Thirty juvenile Wistar rats weighting about 200 g were randomized into three groups: CB-MNCs intramuscular administration group( Therapy group,n = 10),saline intramuscular administration group( NS group,n = 10) and healthy group( Normal group,n =10). The animal model of denervated gastrocnemius muscles were formed by cutting both sides of the sciatic nerve of ratswhich resulted in the nerve disconnection about 1 cm in the first two groups. One million of CB-MNCs were injected into the gastrocnemius muscles of rats in d7,d14,and d21 after despairing the nerves of Therapy group. The rats in Normal and NS groups were only injected saline with equal volume at the same time. Hind limb movements and gangrenous area ratios of rats were observed. The both sides of gastrocnemius muscles of each rat were weighed to measure wet weight ratios and HE staining was taken to determine cross-sectional area of muscle fiber at the end of the forth week.Peripheral blood serum and bilateral gastrocnemius muscles were taken to measure creatine kinase( CK),catalase( CAT),malondialdehyde( MDA),and superoxide dismutase( SOD) levels. The mRNA expressions of Bax,B-cell lymphoma-2( Bcl-2),Caspase-3,vascular endothelial growth factor-α( VEGF-α),tumor necrosis factor-α( TNF-α),interleukin-10( IL-10),α-actin and Dystrophin were tested by qRT-PCR. The expressions of α-actin,Dystrophin and VEGF-α protein were detected by Western blotting. The expression of Dystrophin in cells was also observed by immunohistochemistry.Results The gangrenous area ratio of the Therapy group was lower than that of the NS group( P =0. 002) and the wet weight of the gastrocnemius muscle in the Therapy group was higher than that in the NS group( P < 0. 001). The HE staining results showed that the muscle cross-sectional areas of the Normal group,NS group and Therapy group were respective( 12 452. 0 ± 202. 8),( 6 287. 0 ± 142. 2),and( 8 193. 0 ± 115. 5) μm2 with a statistical difference( P < 0. 001). Compared with the NS group,the CK and MDA contents of the serum and muscles in the Therapy group were significantly decreased,and the SOD and CAT contents were significantly increased( all P < 0.001). In contrast to the NS group,the mRNA levels of Caspase-3,Bax and TNF-α decreased while the mRNA levels of Bcl-2,IL-10,α-actin,Dystrophin and VEGF-α increased in the Therapy group( all P < 0. 05). Compared with the NS group,the expressions of α-actin,Dystrophin and VEGF-α protein in the Therapy group were increased( all P < 0.05). IHC analysis showed that the expression of Dystrophin was increased in both NS and Therapy groups,but the expression of Dystrophin in the Therapy group was significantly higher than that in the NS group( all P < 0. 05). Conclusion Injecting CB-MNCs into the paralyzed gastrocnemius can reduce the proportion of apoptotic cells,promote the wound healing,prevent muscle atrophy,and improve muscle functions.
Objective To explore the effect of human cord blood mononuclear cells( CB-MNCs) on relieving the gastrocnemius muscle atrophy and promoting the damage repair in the isolated sciatic nerve rats after intramuscular administration.Methods CB-MNCs were isolated from healthy term infants and cultured in vitro. Thirty juvenile Wistar rats weighting about 200 g were randomized into three groups: CB-MNCs intramuscular administration group( Therapy group,n = 10),saline intramuscular administration group( NS group,n = 10) and healthy group( Normal group,n =10). The animal model of denervated gastrocnemius muscles were formed by cutting both sides of the sciatic nerve of ratswhich resulted in the nerve disconnection about 1 cm in the first two groups. One million of CB-MNCs were injected into the gastrocnemius muscles of rats in d7,d14,and d21 after despairing the nerves of Therapy group. The rats in Normal and NS groups were only injected saline with equal volume at the same time. Hind limb movements and gangrenous area ratios of rats were observed. The both sides of gastrocnemius muscles of each rat were weighed to measure wet weight ratios and HE staining was taken to determine cross-sectional area of muscle fiber at the end of the forth week.Peripheral blood serum and bilateral gastrocnemius muscles were taken to measure creatine kinase( CK),catalase( CAT),malondialdehyde( MDA),and superoxide dismutase( SOD) levels. The mRNA expressions of Bax,B-cell lymphoma-2( Bcl-2),Caspase-3,vascular endothelial growth factor-α( VEGF-α),tumor necrosis factor-α( TNF-α),interleukin-10( IL-10),α-actin and Dystrophin were tested by qRT-PCR. The expressions of α-actin,Dystrophin and VEGF-α protein were detected by Western blotting. The expression of Dystrophin in cells was also observed by immunohistochemistry.Results The gangrenous area ratio of the Therapy group was lower than that of the NS group( P =0. 002) and the wet weight of the gastrocnemius muscle in the Therapy group was higher than that in the NS group( P < 0. 001). The HE staining results showed that the muscle cross-sectional areas of the Normal group,NS group and Therapy group were respective( 12 452. 0 ± 202. 8),( 6 287. 0 ± 142. 2),and( 8 193. 0 ± 115. 5) μm2 with a statistical difference( P < 0. 001). Compared with the NS group,the CK and MDA contents of the serum and muscles in the Therapy group were significantly decreased,and the SOD and CAT contents were significantly increased( all P < 0.001). In contrast to the NS group,the mRNA levels of Caspase-3,Bax and TNF-α decreased while the mRNA levels of Bcl-2,IL-10,α-actin,Dystrophin and VEGF-α increased in the Therapy group( all P < 0. 05). Compared with the NS group,the expressions of α-actin,Dystrophin and VEGF-α protein in the Therapy group were increased( all P < 0.05). IHC analysis showed that the expression of Dystrophin was increased in both NS and Therapy groups,but the expression of Dystrophin in the Therapy group was significantly higher than that in the NS group( all P < 0. 05). Conclusion Injecting CB-MNCs into the paralyzed gastrocnemius can reduce the proportion of apoptotic cells,promote the wound healing,prevent muscle atrophy,and improve muscle functions.
引文
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[16]Correa A,Ottoboni GS,Senegaglia AC,et al. Expand-ed CD133+cells from human umbilical cord blood im-proved heart function in rats after severe myocardial in-farction[J]. Stem Cells Int,2018,2018:5412478.doi:10. 1155/2018/5412478.
[17]Bertaggia E,Scabia G,Dalise S,et al. Haptoglobin isrequired to prevent oxidative stress and muscle atrophy[J]. PLoS One,2014,9(6):e100745. doi:10. 1371/journal. pone. 0100745.
[18] Reid MB,Moylan JS. Beyond atrophy:redox mecha-nisms of muscle dysfunction in chronic inflammatory dis-ease[J]. J Physiol(Lond),2011,589(Pt 9):2171-2179.
[19]Marzetti E,Privitera G,Simili V,et al. Multiple path-w ays to the same end:mechanisms of myonuclear apop-tosis in sarcopenia of aging[J]. Scientific World Jour-nal,2010,10:340-349. doi:10. 1100/tsw. 2010. 27.
[20]Iwakura T,Fujimoto S,Kagimoto S,et al. Sustainedenhancement of Ca(2+)influx by glibenclamide in-duces apoptosis in RINm5F cells[J]. Biochem BiophysRes Commun,2000,271(2):422-428.
[21] Shehata AS,Al-Ghonemy NM,Ahmed SM,et al.Effect of mesenchymal stem cells on induced skeletalmuscle chemodenervation atrophy in adult male albinorats[J]. Int J Biochem Cell Biol,2017,85:135-148.doi:10. 1016/j. biocel. 2017. 01. 016.
[22]Kuchroo P,Dave V,Vijayan A,et al. Paracrine factorssecreted by umbilical cord-derived mesenchymal stemcells induce angiogenesis in vitro by a VEGF-independ-ent pathw ay[J]. Stem Cells Dev,2015,24(4):437-450.
[23]Sun HL,Liu J,Ding F,et al. Investigation of differen-tially expressed proteins in rat gastrocnemius muscle dur-ing denervation-reinnervation[J]. J M uscle Res CellM otil,2006,27(3/4):241-250.
[24]Pallafacchina G,Blaauw B,Schiaffino S. Role of satel-lite cells in muscle grow th and maintenance of musclemass[J]. Nutr M etab Cardiovasc Dis,2013,23(Suppl1):S12-S18.
[25]刘晓蓉,张成,张为西,等.骨髓移植治疗Duchenne型肌营养不良模型鼠的实验研究[J].中国病理生理杂志,2005,21(8):1462-1466.LIU Xiaorong,ZHANG Cheng,ZHANG Weixi,et al.Experimental treatment of the model mice of Duchennemuscular dystrophy by bone marrow transplantation[J].Chinese Journal of Pathophysiology,2005,21(8):1462-1466.
[26] Witt R,Weigand A,Boos AM,et al. Mesenchymalstem cells and myoblast differentiation under HGF andIGF-1 stimulation for 3D skeletal muscle tissue engineer-ing[J]. BM C Cell Biol,2017,18(1):15. doi:10.1186/s12860-017-0131-2.
[2] Kern H,Carraro U. Home-based functional electricalstimulation for long-term denervated human muscle:his-tory,basics,results and perspectives of the vienna reha-bilitation strategy[J]. Eur J Transl M yol,2014,24(1):3296. doi:10. 4081/ejtm. 2014. 3296.
[3]Biazar E. Use of umbilical cord and cord blood-derivedstem cells for tissue repair and regeneration[J]. ExpertOpin Biol Ther,2014,14(3):301-310.
[4] Tang HB,Inoki K,Lee M,et al. mTORC1 promotesdenervation-induced muscle atrophy through a mechanisminvolving the activation of FoxO and E3 ubiquitin ligases[J]. Sci Signal,2014,7(314):ra18. doi:10. 1126/sci-signal. 2004809.
[5] Rudolf R,Deschenes MR,Sandri M. Neuromuscularjunction degeneration in muscle w asting[J]. Curr OpinClin Nutr M etab Care,2016,19(3):177-181.
[6]赵志强,刘强,李钢.骨骼肌卫星细胞移植对延缓失神经肌肉萎缩的作用[J].中华骨科杂志,2006,26(1):51-55.ZHAO Zhiqiang,LIU Qiang,LI Gang. The effect oftransplantation of skeletal muscle satellite cell on retard thedenervated muscles atrophy[J]. Chinese Journal of Or-thopaedics,2006,26(1):51-55.
[7] Kim S,Kim K. The effects of exercise and conjugatedlinoleic acid intake on IGF-1 and pro-inflammatory cyto-kines in atrophied skeletal muscle of rats[J]. Integr M edRes,2013,2(4):166-173.
[8]Arakawa T,Katada A,Shigyo H,et al. Electrical stimu-lation prevents apoptosis in denervated skeletal muscle[J]. NeuroRehabilitation,2010,27(2):147-154.
[9]裴艳宏,刘坤祥.黄芪丹参联合应用可延缓大鼠失神经骨骼肌萎缩[J].解剖学报,2014,45(2):278-282.PEI Yanhong,LIU Kunxiang. Role of Huangqi combinedw ith Danshen in the denervated skeletal muscle atrophy[J]. Acta Anatomica Sinica,2014,45(2):278-282.
[10]Reza MM,Subramaniyam N,Sim CM,et al. Irisin is apro-myogenic factor that induces skeletal muscle hyper-trophy and rescues denervation-induced atrophy[J]. NatCommun,2017,8(1):1104. doi:10. 1038/s41467-017-01131-0.
[11]Farjah GH,Fazli F,Karimipour M,et al. The effect ofbone marrow mesenchymal stem cells on recovery ofskeletal muscle after neurotization surgery in rat[J]. IranJ Basic M ed Sci,2018,21(3):236-243.
[12]Pimentel-Coelho PM,Rosado-de-Castro PH,da FonsecaLM,et al. Umbilical cord blood mononuclear cell trans-plantation for neonatal hypoxic-ischemic encephalopathy[J]. Pediatr Res,2012,71(4 Pt 2):464-473.
[13]练诗梅,王晓波,薛祖光,等.脐血与外周血树突状细胞及细胞组成的比较[J].临床和实验医学杂志,2005,4(2):71-73.LIAN Shimei,WANG Xiaobo,XUE Zuguang,et al.Analysis of dendritic cells and cellular component of um-bilical cord blood and peripheral blood[J]. Journal ofClinical and Experimental M edicine,2005,4(2):71-73.
[14]Galieva LR,Mukhamedshina YO,Arkhipova SS,et al.Human umbilical cord blood cell transplantation in neuro-regenerative strategies[J]. Front Pharmacol,2017,8:628. doi:10. 3389/fphar. 2017. 00628.
[15]Hosseini SR,Kaka G,Joghataei MT,et al. Assessmentof neuroprotective properties of melissa officinalis incombination w ith human umbilical cord blood stem cellsafter spinal cord injury[J]. ASN Neuro,2016,8(6):1759091416674833. doi:10. 1177/1759091416674833.
[16]Correa A,Ottoboni GS,Senegaglia AC,et al. Expand-ed CD133+cells from human umbilical cord blood im-proved heart function in rats after severe myocardial in-farction[J]. Stem Cells Int,2018,2018:5412478.doi:10. 1155/2018/5412478.
[17]Bertaggia E,Scabia G,Dalise S,et al. Haptoglobin isrequired to prevent oxidative stress and muscle atrophy[J]. PLoS One,2014,9(6):e100745. doi:10. 1371/journal. pone. 0100745.
[18] Reid MB,Moylan JS. Beyond atrophy:redox mecha-nisms of muscle dysfunction in chronic inflammatory dis-ease[J]. J Physiol(Lond),2011,589(Pt 9):2171-2179.
[19]Marzetti E,Privitera G,Simili V,et al. Multiple path-w ays to the same end:mechanisms of myonuclear apop-tosis in sarcopenia of aging[J]. Scientific World Jour-nal,2010,10:340-349. doi:10. 1100/tsw. 2010. 27.
[20]Iwakura T,Fujimoto S,Kagimoto S,et al. Sustainedenhancement of Ca(2+)influx by glibenclamide in-duces apoptosis in RINm5F cells[J]. Biochem BiophysRes Commun,2000,271(2):422-428.
[21] Shehata AS,Al-Ghonemy NM,Ahmed SM,et al.Effect of mesenchymal stem cells on induced skeletalmuscle chemodenervation atrophy in adult male albinorats[J]. Int J Biochem Cell Biol,2017,85:135-148.doi:10. 1016/j. biocel. 2017. 01. 016.
[22]Kuchroo P,Dave V,Vijayan A,et al. Paracrine factorssecreted by umbilical cord-derived mesenchymal stemcells induce angiogenesis in vitro by a VEGF-independ-ent pathw ay[J]. Stem Cells Dev,2015,24(4):437-450.
[23]Sun HL,Liu J,Ding F,et al. Investigation of differen-tially expressed proteins in rat gastrocnemius muscle dur-ing denervation-reinnervation[J]. J M uscle Res CellM otil,2006,27(3/4):241-250.
[24]Pallafacchina G,Blaauw B,Schiaffino S. Role of satel-lite cells in muscle grow th and maintenance of musclemass[J]. Nutr M etab Cardiovasc Dis,2013,23(Suppl1):S12-S18.
[25]刘晓蓉,张成,张为西,等.骨髓移植治疗Duchenne型肌营养不良模型鼠的实验研究[J].中国病理生理杂志,2005,21(8):1462-1466.LIU Xiaorong,ZHANG Cheng,ZHANG Weixi,et al.Experimental treatment of the model mice of Duchennemuscular dystrophy by bone marrow transplantation[J].Chinese Journal of Pathophysiology,2005,21(8):1462-1466.
[26] Witt R,Weigand A,Boos AM,et al. Mesenchymalstem cells and myoblast differentiation under HGF andIGF-1 stimulation for 3D skeletal muscle tissue engineer-ing[J]. BM C Cell Biol,2017,18(1):15. doi:10.1186/s12860-017-0131-2.