高压氧联合NgR基因沉默骨髓间充质干细胞移植治疗大鼠脊髓损伤  

Hyperbaric oxygen combined with NgR gene silencing bone marrow mesenchymal stem cells transplantation for spinal cord injury in rats

作　　者：梅运运 张建军[2] 王东[2] Mei Yunyun;Zhang Jianjun;Wang Dong(Fourth Clinical College of Tianjin Medical University,Tianjin 300070,China;Tianjin Fourth Central Hospital,Tianjin 300140,China)

机构地区：[1]天津医科大学第四中心临床学院,天津市300070 [2]天津市第四中心医院,天津市300140

出　　处：《中国组织工程研究》2022年第1期12-19,共8页Chinese Journal of Tissue Engineering Research

基　　金：天津市自然科学基金项目(17JCYBJC27300),项目负责人:王东。

摘　　要：背景:高压氧治疗和Nogo-A/NgR-RhoA/ROCK信号通路的抑制有助于移植细胞的增殖、分化。目的:探讨高压氧联合携带miRNA沉默NgR基因的大鼠骨髓间充质干细胞移植对大鼠脊髓损伤修复的影响。方法:靶向NgR的miRNA重组质粒转染大鼠骨髓间充质干细胞,RT-qPCR和Western blot检测转染后NgR的表达。80只SD大鼠,随机选取15只大鼠作为假手术组,其余65只大鼠构建脊髓损伤模型,将造模成功的60只大鼠随机分为脊髓损伤组、高压氧组、骨髓间充质干细胞组和高压氧+骨髓间充质干细胞组。骨髓间充质干细胞组和高压氧+骨髓间充质干细胞组于造模24 h后注射5×106个转染重组质粒的大鼠骨髓间充质干细胞(5μL),脊髓损伤组和高压氧组注射等体积的PBS,随后高压氧组和高压氧+骨髓间充质干细胞组接受连续7 d高压氧治疗。细胞移植后采用BBB评分评估大鼠后肢运动功能,每周1次,连续4周;造模后第4周,检测感觉诱发电位评估神经传导恢复情况,苏木精-伊红染色、TUNEL染色和荧光金逆行追踪观察脊髓组织损伤、细胞凋亡以及轴突再生情况。高压氧治疗后随即对损伤区脊髓组织中Nogo-A、NgR、RhoA、ROCK-1、Lingo-1、p75NTRmRNA相对表达进行RT-qPCR检测。结果与结论:①骨髓间充质干细胞转染后NgR表达明显下降;②造模后2-4周BBB评分:高压氧+骨髓间充质干细胞组>骨髓间充质干细胞组、高压氧组>脊髓损伤组(P<0.05);③与其他治疗组相比,高压氧+骨髓间充质干细胞组感觉诱发电位的潜伏期缩短,波幅升高(P<0.05);骨髓间充质干细胞组与高压氧组差异无显著性意义(P>0.05);④与脊髓损伤组比较,高压氧组和骨髓间充质干细胞组的神经元数量有所增加,具有轴浆运输能力的轴突数量也明显增多,神经元再生效果明显,凋亡细胞减少,而高压氧+骨髓间充质干细胞组的修复效果优于其他2个治疗组;⑤与脊髓损伤组相比,3个治�BACKGROUND:Hyperbaric oxygen therapy and inhibition of Nogo-A/NgR-RhoA/ROCK signaling pathways are conducive to the proliferation and differentiation of transplanted cells.OBJECTIVE:To explore the effects of hyperbaric oxygen plus rat bone marrow mesenchymal stem cells transplantation carrying miRNA silencing NgR gene on repair of rat spinal cord injury.METHODS:Rat bone marrow mesenchymal stem cells were transfected using constructed miRNA recombinant plasmid targeting NgR.The expression levels of NgR were detected by real-time quantitative PCR and western blot assay after transfection.The 15 of 80 Sprague-Dawley rats were randomly selected as sham group and the remaining 65 rats were used to construct models of spinal cord injury;and 60 successful models were randomly divided into spinal cord injury group,hyperbaric oxygen group,rat bone marrow mesenchymal stem cells group and rat bone marrow mesenchymal stem cells plus hyperbaric oxygen group.The rat bone marrow mesenchymal stem cells group and hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group were injected with 5μL rat bone marrow mesenchymal stem cells suspension(5×106 cells)after 24 hours,respectively,and the spinal cord injury and hyperbaric oxygen groups were injected with PBS of the same volume.The hyperbaric oxygen and hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group received continuous treatment for 7 days.The hind limb motor function was evaluated by Basso Beattie Bresnahan score before and after the modeling,once a week,for 4 consecutive weeks.After 4 weeks of modeling,somatosensory evoked potential testing was performed to detect the recovery of neural conduction.The histopathological damage,apoptosis of cells and axon regeneration in spinal cord tissues was observed by hematoxylin-eosin staining,TUNEL staining and fluorescent gold retrograde tracing,respectively.After hyperbaric oxygen therapy,the mRNA relative expression levels of Nogo-A,NgR,RhoA,ROCK-1,Lingo-1 and p75NTR were detected by real-time quantitativ

关 键 词：干细胞 骨髓间充质干细胞 miRNA干扰 NGR 高压氧 脊髓损伤 电生理 运动功能 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]