腺伴随病毒介导的脑源性神经营养因子对体外培养的鼠视网膜神经节细胞转染及生长特性的影响

Transfection of brain-derived neurotrophic factor gene by recombinant adeno-associated virus vector in retinal ganglion cells in vitro

目的探讨重组腺伴随病毒载体介导的脑源性神经营养因子（rAAV-BDNF）对体外培养的鼠视网膜神经节细胞（RGCs）转染及其生长活性的影响。方法实验研究。（1）应用rAAV-BDNF对体外培养2d的RGCs进行转染；（2）应用逆转录聚合酶链反应（RT-PCR）技术，检测外源性BDNF基因在RGCs细胞mRNA水平的表达情况；（3）应用酶联免疫吸附测定（ELISA）法，对细胞培养液中BDNF含量进行检测；（4）对rAAV—BDNF转染细胞、未转染细胞及加入BDNF的培养细胞进行MTT比色分析；（5）应用Annexin V-FITC凋亡检测试剂盒和流式细胞仪，检测rAAV-BDNF转染细胞、未转染细胞及加入BDNF培养细胞的凋亡比率。结果（1）RT-PCR检测结果：转染细胞表达外源性BDNF基因，而未转染细胞不表达BDNF基因。（2）ELISA法检测结果：rAAV．BDNF转染细胞的培养液中BDNF含量-3染7d后为（616．1±40．0）ng／L，转染14d后为（1075．1±48．7）ng／L。（3）MTT比色结果-3染3和6d后，rAAV-BDNF转染细胞与未转染细胞间的吸光度（A）值差异无统计学意义（t=1．084，1．582；P=0．284，0．120）；转染9d后，转染细胞的A值高于未转染细胞（t=4．854，P=0．000）。（4）流式细胞仪检测结果：rAAV-BDNF转染细胞和加入BDNF培养细胞的凋亡率明显低于未转染细胞的凋亡率，差异有统计学意义（P=0．015，0．017）。结论rAAV-BDNF可有效转染体外培养的鼠RGCs，转染细胞可在转录水平和翻译水平表达外源性BDNF基因，且生长活性改善，凋亡细胞减少。这为青光眼视神经保护的基因治疗提供了理论和技术支持。

Objective To determine whether rat retinal ganglion cells （RGCs） could be infected by rAAV-BDNF in vitro and to evaluate the influence of rAAV-BDNF transfection on the survival and apoptosis of rat RGCs. Methods It was a experimental study. （ 1 ） RGCs were isolated from neonatal Sprague-Dawley rats （postnatal within 24 h）. （2） Two days after the cultivation, the RGCs were transfected with rAAV- BDNF at a dosage of MOI = 105 and then incubated for 7 days. Total RNA were extracted from rAAV-BDNF transfected cells using Trizol reagent. The gene expression of BDNF gene in RGCs was analyzed by reverse transcription polymerase-chain reaction （RT-PCR）. （ 3 ） Supernatant of the rAAV-BDNF transfected cells was collected at 7 days and 14 days after transfection. The protein expression of BDNF in the cell supernatant was examined with ELISA assay. （4） The survival and apoptosis of rAAV-BDNF transfected cells, untransfected cells and the cells with addition of BDNF in culture medium were evaluated by MTT colorimetric assay and flow cytometry with Annexin V-FITC staining, respectively. Results （ 1 ） RT-PCR analysis showed that mRNA expression of BDNF gene could be detected in transfected cells but not in untransfected cells. （2）The concentrations of BDNF protein in the conditioned medium of the rAAV-BDNF transfected cells were （616. 1 ± 40. 0） ng/L and （ 1075.1 ± 48. 7 ） ng/L 7 days and 14 days after the transfection, respectively. （3） MTT colorimetric assay showed that the OD values of rAAV-BDNF transfected cells and untransfected cells were similar at the time of 3 and 6 days after transfection（t = 1. 084 and 1. 582, P =0. 284 and 0. 120）. The OD value of transfected cells was higher than that of untransfected cells 9 days after the transfection （ t = 4. 854, P = 0. 000）. （ 4 ） The apoptosis rate in rAAV-BDNF transfected cells and the cells with BDNF exposure was lower than that of the untransfected cells （ P = 0. 015, 0. 017 ） . Conclusions Rat RGCs are able to be transfected by rAAV-BDNF in vitro. The transfected cells can express BDNF gene at the level of both mRNA and protein. Apoptosis rate is low in the transfected cells. This study indicates that rAAV-BDNF transfection can be used for the potential gene therapy in glaucoma neurolarotection.