CAJ | 학술논문

目的通过Microarray技术寻找环磷酸腺苷(cAMP)表达上调的机制.方法利用Microarray技术和生物信息分析方法,寻找与初级感觉神经元外周突预损伤促进中枢突损伤修复有关的关键微小RNA(miRNA,miR),并采用实时定量反转录聚合酶链反应(RT-qPCR)技术、Western blot技术、免疫组织化学染色、酶联免疫吸附试验(ELISA)、免疫荧光、反义miRNA寡核苷酸抑制剂和咯利普兰[磷酸二酯酶4A(PDE4A)抑制剂]等技术进行验证.结果与对照组比较,预损伤组背根神经节cAMP在各时间均有升高,而后索损伤组与对照组比较差异无统计学意义(P＞0.05).预损伤组NF-200累计吸光度值(79 473.86 ±2018.15)明显高于后索损伤组(89 623.43±1 984.69)且差异有统计学意义(P＜0.05).与对照组比较预损伤组和后索损伤组共有681个miRNA发生表达变化,预损伤组miR-139-5p在脊髓后索损伤后4h、3d和7d明显上调,而14 d开始下降.与对照组比较预损伤组各时间点PDE4A mRNA表达量差异无统计学意义(P＞0.05),而PDE4A蛋白含量在脊髓后索损伤后4h、3d和7d下降.培养基中加入AMO-139抑制背根神经节神经元的miR-139-5p后PDE4A蛋白含量较未加入AMO-139的背根神经节神经元高.加入咯利普兰的背根神经节神经元轴突较空白神经元明显延长,而在加入咯利普兰的同时加入AMO-139则轴突长度与空白神经元比较差异无统计学意义(P＞0.05).结论初级感觉神经元外周突预损伤使miR-139-5p上调,通过抑制PDE4蛋白的表达导致神经元内cAMP含量上升进而促进中枢突损伤修复.
목적 통과Microarray기술심조배린산선감(cAMP)표체상조적궤제.방법 이용Microarray기술화생물신식분석방법,심조여초급감각신경원외주돌예손상촉진중추돌손상수복유관적관건미소RNA(miRNA,miR),병채용실시정량반전록취합매련반응(RT-qPCR)기술、Western blot기술、면역조직화학염색、매련면역흡부시험(ELISA)、면역형광、반의miRNA과핵감산억제제화각리보란[린산이지매4A(PDE4A)억제제]등기술진행험증.결과 여대조조비교,예손상조배근신경절cAMP재각시간균유승고,이후색손상조여대조조비교차이무통계학의의(P＞0.05).예손상조NF-200루계흡광도치(79 473.86 ±2018.15)명현고우후색손상조(89 623.43±1 984.69)차차이유통계학의의(P＜0.05).여대조조비교예손상조화후색손상조공유681개miRNA발생표체변화,예손상조miR-139-5p재척수후색손상후4h、3d화7d명현상조,이14 d개시하강.여대조조비교예손상조각시간점PDE4A mRNA표체량차이무통계학의의(P＞0.05),이PDE4A단백함량재척수후색손상후4h、3d화7d하강.배양기중가입AMO-139억제배근신경절신경원적miR-139-5p후PDE4A단백함량교미가입AMO-139적배근신경절신경원고.가입각리보란적배근신경절신경원축돌교공백신경원명현연장,이재가입각리보란적동시가입AMO-139칙축돌장도여공백신경원비교차이무통계학의의(P＞0.05).결론 초급감각신경원외주돌예손상사miR-139-5p상조,통과억제PDE4단백적표체도치신경원내cAMP함량상승진이촉진중추돌손상수복.
Objective It has been proved that the repair of injured central branch of primary sensory neurons can be promoting by conditioning injury of the peripheral branch.Present study try to investigate the mechanism of upregulation of cyclic adenosine monophosphate (cAMP) levels via microarray and further interpret this phenomenon.Methods Microarray and bioinformatics were used to investigate the microRNA which is associated with the repair of dorsal column lesion that is promoted by sciatic nerve conditioning injury.To validate the result,real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR),Western blotting,immunohistochemistry,enzyme linked immunosorbent assay (ELISA),antisense oligonucleotide (AMO-139) and rolipram (PDE4 inhibitor) were applied.Results To compare with control group,cAMP levels increased in each check points of sciatic nerve conditioning injury group but has no statistical difference in each check points of simple dorsal column lesion group.The expression level of neurofilament protein (NF-200) in sciatic nerve conditioning injury group (79 473.86 ± 2 018.15) was higher than it in simple dorsal column lesion group (89 623.43± 1 984.69),and the difference was statistically significant.To compare with control group,totally 681 microRNAs' expression were altered in sciatic nerve conditioning injury group and simple dorsal column lesion group.Among them,miR-139-5p in sciatic nerve conditioning injury group was upregulated significantly at 4 h,3 d and 7 d post dorsal column lesion and started to decrease at 14 d post dorsal column lesion.After adding AMO-139 to inhibit miR-139-5p of dorsal root ganglion (DRG) neurons,the relative expression of PDE4A protein was increased,compared with the blank DRG neurons.The axonal length of DRG neurons with rolipram were significantly longer than blank DRG neurons.Whereas the axonal length has no statistical difference between DRG neurons with rolipram and AMP-139 and blank DRG neurons.Conclusion Injured peripheral axonal branch of primary sensory neurons induced the upregulation of miR-139-5p,which can increase cAMP levels through inhibiting the expression of PDE4 and further promote the repair of central axonal branch.