Establishment of effective methods for transducing genes into iris pigment epithelial cells by using adeno-associated virus type 2
Establishment of effective methods for transducing genes into iris pigment epithelial cells by using adeno-associated virus type 2
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DOI：
10.1167/iovs.04-1351
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发表时间：
2005-09-01
影响因子：
4.4
通讯作者：
Tamai, M
中科院分区：
文献类型：
--
作者：
Sugano, E;Tomita, H;Tamai, M
目的.目的建立一种利用重组腺相关病毒2型（rAAV 2）将人脑源性神经营养因子（hBDNF）基因转染人虹膜色素上皮细胞（hIPE）的方法。用羟基脲-丁酸钠（HUSB; DNA合成抑制剂）或酪氨酸磷酸化抑制剂-1（Tyr;表皮生长因子受体[EGFR]酪氨酸激酶抑制剂）或HUSB和Tyr的组合（HUSB-Tyr）处理培养的hIPE细胞。每次处理后，将细胞暴露于rAAV2（rAAV-LacZ或rAAV-hBDNF）。BDNF的水平通过ELISA测定，也通过Western印迹分析测定。对每种类型的处理细胞进行Southern印迹分析。用rAAV-hBDNF-hIPE与视网膜神经节细胞（RGCs）共培养，定量评价BDNF对RGCs的神经保护作用。hIPE细胞的感染显著低于ARPE和HT1080细胞，ARPE和HT1080细胞是rAAV2的高度容许细胞。在rAAV-hIPE细胞中，HUSB-Tyr的处理比用这些试剂之一处理后更能增强转基因表达。Southern杂交结果显示，Tyr处理后病毒复制型单体（RFm）的数量明显少于HUSB或HUSB-Tyr处理后病毒复制型单体（RFm）的数量，且HUSB和HUSB-Tyr处理后病毒基因组向双链形式的转化没有差异。然而，加入Tyr处理刺激JNK1/2和p38途径并改变靶转基因表达。与未感染hIPE细胞相比，HUSB-Tyr处理的rAAV-hBDNF-hIPE细胞对RGCs的拯救作用显著增强（P <0. 01），而HUSB-Tyr处理的rAAV-hBDNF-hIPE细胞对RGCs的拯救作用显著增强（P> 0. 05）。HUSB-Try的联合处理是利用AAV介导的基因转移增加转基因表达的有效方法。HUSB和Tyr在基因表达增加中的作用可能不同，分别与病毒转化到宿主基因组和转录增强有关。
PURPOSE. To establish an efficient method of transferring the human brain-derived neurotrophic-factor (hBDNF) gene into human iris pigment epithelial (hIPE) cells by using recombinant adeno-associated virus type 2 (rAAV2).METHODS. Cultured hIPE cells were treated with either hydroxyurea-sodium butyrate (HUSB; DNA synthesis inhibitor), or tyrphostin-1 (Tyr; epidermal growth factor receptor [ EGFR] tyrosine kinase inhibitor), or a combination of HUSB and Tyr (HUSB-Tyr). After each treatment, cells were exposed to rAAV2 (rAAV-LacZ or rAAV-hBDNF). The levels of BDNF were measured by ELISA and also determined by Western blot analysis. Southern blot analysis was performed on each type of treated cell. The neuroprotective effect of BDNF on the retinal ganglion cells (RGCs) was quantitatively assessed by culturing rAAV-hBDNF-hIPE with RGCs.RESULTS. The infection of hIPE cells was significantly lower than ARPE and HT1080 cells, which are highly permissive cells for rAAV2. The treatment of HUSB-Tyr enhanced the transgene expression more than that after treatment with one of these agents in rAAV-hIPE cells. Southern hybridization revealed that the amount of replicative form monomer (RFm) was less in Tyr than in HUSB or HUSB-Tyr treatment and there was no difference in conversion of virus genome to double stranded form after HUSB and HUSB-Tyr treatment. However, adding Tyr treatment stimulated the JNK1/2 and p38 pathways and modified the target transgene expression. BDNF had a significantly greater rescue effect of RGCs with the HUSB-Tyr-treated rAAV-hBDNF-hIPE cells ( P < 0.01) than that with the HUSB-treated rAAV-hBDNF-hIPE cells ( P > 0.05) compared with noninfected hIPE cells.CONCLUSIONS. The combined treatment of HUSB-Try is an effective method of increasing transgene expression with the AAV-mediated gene transfer. The role of HUSB and Tyr in the increase of gene expression may be different and related to the conversion of virus into the host genome and the enhancement of the transcription, respectively.