CAJ | 학술논문

目的：观察改良Tet-On系统修饰慢病毒( Lv-TH-GD-NF)目的基因的表达调控及纹状体内直接转移对帕金森病(PD)大鼠的作用。方法①用 Lv-TH-GDNF 与 rtTA2s-M2病毒感染 HeLa 细胞,免疫印迹法检测强力霉素( Dox )对TH、GDNF基因表达的调控。②用 Lv-TH-GDNF 与 rtTA2s-M2病毒共同注射到PD大鼠患侧纹状体,Dox诱导目的基因表达。通过观察阿扑吗啡( APO)诱导旋转行为、黑质多巴胺能神经元数量、患侧纹状体 DA、DOPAC 含量评估 Lv-TH-GDNF治疗效应；通过移植侧纹状体内TH与GDNF蛋白量评估外源基因在体内的表达。结果①在体外HeLa细胞实验,仅Dox阳性组见TH、GDNF蛋白条带。②在动物体内实验,病毒移植4周后,与PBS对照组相比,仅病毒+Dox组大鼠旋转行为明显改善( P<0．01),损伤侧黑质致密部TH阳性细胞数、纹状体DA、DOPAC含量及TH和GDNF蛋白量明显增高( P<0．01)。结论新型Tet-On系统修饰的Lv-TH-GDNF目的基因表达受四环素类抗生素调控,在体外实验未见基础活动,且纹状体内直接转移对PD大鼠有一定治疗作用。
목적：관찰개량Tet-On계통수식만병독( Lv-TH-GD-NF)목적기인적표체조공급문상체내직접전이대파금삼병(PD)대서적작용。방법①용 Lv-TH-GDNF 여 rtTA2s-M2병독감염 HeLa 세포,면역인적법검측강력매소( Dox )대TH、GDNF기인표체적조공。②용 Lv-TH-GDNF 여 rtTA2s-M2병독공동주사도PD대서환측문상체,Dox유도목적기인표체。통과관찰아복마배( APO)유도선전행위、흑질다파알능신경원수량、환측문상체 DA、DOPAC 함량평고 Lv-TH-GDNF치료효응；통과이식측문상체내TH여GDNF단백량평고외원기인재체내적표체。결과①재체외HeLa세포실험,부Dox양성조견TH、GDNF단백조대。②재동물체내실험,병독이식4주후,여PBS대조조상비,부병독+Dox조대서선전행위명현개선( P<0．01),손상측흑질치밀부TH양성세포수、문상체DA、DOPAC함량급TH화GDNF단백량명현증고( P<0．01)。결론신형Tet-On계통수식적Lv-TH-GDNF목적기인표체수사배소류항생소조공,재체외실험미견기출활동,차문상체내직접전이대PD대서유일정치료작용。
Aim To investigate TH and GDNF genes expression and regulation of lentivirus ( Lv-TH-GDNF ) based on improved Tet-On system and the effect of the Lv-TH-GDNF intrastriatal transfer on a rat Parkinson’ s disease( PD) model. Methods 1. HeLa cells were infected by obtained Lv-TH-GDNF and rtTA2 s-M2 vi-rus. The expression of tyrosine hydroxylase ( TH ) and glial cell line-derived neurotrophic factor( GDNF) genes was induced by doxycycline( Dox) which was examined by Western blot. 2. The Lv-TH-GDNF together with rtTA2 s-M2 viruses were injected into lesion-side stria-tum of a rat PD model, and the expression of GDNF and TH genes was induced by Dox. Then, the effects of Lv-TH-GDNF were evaluated by the apomorphine-induced rotational behavior, the number of dopaminer-gic neurons in substantia nigra,DA and DOPAC levels in the lesion-side striatum. In addition, Western blot was performed to check the expression of TH and GD-NF genes in the transplanted striatum. Results 1. In vitro studies on HeLa cells, Western blot showed clear protein bands of TH and GDNF in the Dox-positive group, but not in the Dox-negative group. 2. In vivo experiments in animals, the results showed that, 4 weeks after transplantation, the apomorphine-induced turning effect was significantly improved ( P<0 . 01 ) , the number of TH-positive cells in the lesion-side sub-stantia nigra pars compacta as well as the content of DA and DOPAC, the protein level of GDNF and TH genes in the lesion-side striatum was significantly in-creased ( P<0 . 01 ) , each of which was only in Lv-TH-GDNF+rtTA2 s-M2+Dox-treated rats as compared with PBS-treated rats. Conclusion The expression of TH and GDNF genes in Lv-TH-GDNF based on im-proved Tet-On system is effectively regulated by tetra-cycline antibiotics without basal activity in vitro, and the intrastriatal transfer of which has certain therapeutic effect on PD rats.