摘要
目的玻璃体腔注射重组ND4基因的腺相关病毒血清型2(Adenovirus associated virus 2-NADH dehydro-genase subunit 4,AAV2-ND4)是Leber遗传性视神经病变(Leber hereditary optic neuropathy,LHON)的基因治疗方法,观察基因治疗后不同免疫抑制方案组全身免疫抑制效果及ND4基因表达情况。方法各实验组(A～D组)均单次玻璃体腔注射4μL AAV2-ND4,注射后灌胃予以口服药物28d,A组不予以免疫抑制治疗,B组口服环孢霉素A[起始剂量10mg/(kg.d),14d后减半],C组口服强的松[起始剂量5mg/(kg.d),14d后减半],D组玻璃体腔同时注射曲安奈德,并口服强的松(剂量同C组),对照组(E组)单次玻璃体腔注射4μL磷酸缓冲盐溶液。分别于注射AAV2-ND4后3、7、28、56、84d后检测血清中AAV2和ND4的抗体含量,同时于第84d观察鼠视网膜上ND4的表达。结果 A～D各组抗ND4基因抗体浓度在第56天时均明显增加,以A、C组为显著;第84天时,除D组外各组抗体浓度均出现不同程度下降。A～D各组抗AAV2抗体浓度在第28天时明显增加,以A、C组为显著;第56天时除B组外各组抗体浓度均出现下降。实验各组的视网膜神经节细胞层在注射第84天仍有明显ND4表达。结论免疫反应主要集中在玻璃体腔注射AAV2-ND4后56d内;各个方案的免疫抑制效果均不理想;不同免疫抑制治疗方案对ND4的表达效率无明显影响。
Objective To examine the systemic immunosuppressive effect and the expression of adenovirus associated virus 2-NADH dehydrogenase subunit 4 gene（AAV2-ND4） in different immunosuppressive therapeutic strategies following intravit- real injection of AAV2-ND4 for treatment of leber hereditary optic neuropathy（LHON）. Methods Twenty SD rats were alloca- ted to 5 groups（groups A,B,C,D and E） with 4 in each group. Single introvitreal administration of 4 μL AAV2-ND4 was given to rats in groups A to D. Rats in groups B,C and D were later intragastrically administered with immunosuppressive agents for 28 days. In group B,cyclosporin A was given at 10 mg/（kg · d） for 14 days and at 5 mg/（kg · d） for another 14 days. In group C,prednisone at 5 mg/（kg ·d） was given for consecutive 14 days and the dose was reduced by half in the following 14 days. In group D,the drug treatment was the same as that in group C except that triamcinolone acetonide was introvitreally administra- ted simultaneously. Animals in the group E were introvitreally given phosphate buffered saline once and served as blank con- trol. Serum levels of anti-AAV2 and anti-ND4 were measured by enzyme-linked immunosorbent assay（ELISA） on day 3,day 7, day 28, day 56 and day 84 after intravitreal injection. The expression of ND4 in retinal ganglion cell layer was detected by immu- nofluorescence staining on day 84. Results The serum anti-ND4 antibody concentration was obviously increased in groups A to D on day 56,especially in groups A and C. On day 84, the antibody levels in groups A,B and C were decreased to varying de- gree. The serum anti-AAV2 antibody concentration in groups A to D was significantly increased on day 28,especially in groups A and C. On day 56, there was a decrease in the antibody levels of serum anti-AAV2 in groups A to D except group B. ND4 was found to be expressed in the retinal ganglion cells layer（RGCs layer） in groups A to D on day 84. Conclusion The immune re- sponses mainly occurred in the period of 56 days after intravitreal injection of AAV2-ND4. The immunosuppressive effect was unsatisfactory in all the immunosuppressive strategies. However, the different immunosuppressive strategies had no significant influence on the expression of ND4.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2013年第2期187-191,共5页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.81271015)
同济医院新技术新业务基金资助项目(No.2010021)
关键词
LEBER遗传性视神经病变
ND4基因
基因转染
免疫抑制
leber hereditary optic neuropathy
NADH dehydrogenase subunit 4 gene
gene transfer
immunosuppres-sion
