Inhibition of Retinoblastoma In Vitro and In Vivo with Conditionally Replicating Oncolytic Adenovirus H101
Inhibition of Retinoblastoma In Vitro and In Vivo with Conditionally Replicating Oncolytic Adenovirus H101
复制标题
使用条件复制溶瘤腺病毒 H101 在体外和体内抑制视网膜母细胞瘤。
DOI：
10.1167/iovs.09-3516
复制
发表时间：
2010-05-01
影响因子：
4.4
通讯作者：
Fan, Xianqun
中科院分区：
文献类型：
--
作者：
Song, Xin;Zhou, Yixiong;Fan, Xianqun
目的.观察溶瘤腺病毒H101对视网膜母细胞瘤的治疗作用。采用RT-PCR、Western blot、免疫荧光和免疫细胞化学染色等方法检测人视网膜母细胞瘤细胞系HXO-RB 44中柯萨奇病毒-腺病毒受体（CAR）的表达。在视网膜母细胞瘤细胞中使用流式细胞术用表达绿色荧光蛋白的腺病毒（AdGFP）确定适当的感染复数。使用基于细胞计数试剂盒-8的程序测量用H101或AdGFP处理的HXO-RB 44细胞的活力。通过终点稀释滴定法和实时PCR法测定病毒体外增殖。流式细胞仪检测细胞周期和凋亡活性。携带视网膜母细胞瘤异种移植物的NOD-SCID小鼠用瘤内注射H101、AdGFP或PBS处理。记录肿瘤体积和生存时间。对视网膜母细胞瘤异种移植物进行腺病毒纤维蛋白的免疫组织化学和腺病毒六邻体蛋白的Western印迹，以评估H101病毒在体内的复制。HXO-RB 44细胞表达CAR，对腺病毒感染敏感。H101处理的HXO-RB 44细胞的存活率较AdGFP处理的HXO-RB 44细胞明显降低（P < 0.01）。H101在HXO-RB 44细胞中的大量复制导致G2/M期阻滞，最终导致肿瘤细胞溶解，但凋亡途径未被激活。与PBS和AdGFP对照组相比，用H101治疗的荷瘤小鼠具有降低的肿瘤负荷和延长的存活时间（均P < 0.01）。免疫组化和Western blot检测显示H101在肿瘤内广泛复制。这些结果表明，H101在体外和小鼠中有效地抑制视网膜母细胞瘤细胞的生长，并可能作为一种新的治疗视网膜母细胞瘤。（Invest Ophthalmol维斯科学。2010;51：2626-2635）DOI：10.1167/iovs.09-3516
PURPOSE. To determine the therapeutic effect of oncolytic adenovirus H101 on retinoblastoma in vitro and in vivo.METHODS. The expression of coxsackievirus-adenovirus receptor (CAR) in human retinoblastoma cell line HXO-RB44 was determined by RT-PCR, Western blot, immunofluorescence, and immunocytochemistry staining. Appropriate multiplicity of infection was determined using flow cytometry in retinoblastoma cells with green fluorescent protein-expressing adenovirus (AdGFP). The viability of HXO-RB44 cells treated with H101 or AdGFP was measured using a cell counting kit-8-based procedure. Viral proliferation in vitro was measured by end point dilution titration and real-time PCR. Cell cycle and apoptotic activity of HXO-RB44 were analyzed by flow cytometry. NOD-SCID mice bearing retinoblastoma xenografts were treated with intratumoral injection of H101, AdGFP, or PBS. Tumor volume and survival time were recorded. Immunohistochemistry for adenoviral fiber protein and Western blot for adenoviral Hexon protein of retinoblastoma xenografts were performed to evaluate H101 virus replication in vivo.RESULTS. HXO-RB44 cells expressed CAR and were sensitive to adenoviral infection. HXO-RB44 cells treated with H101 had reduced cell viability compared with AdGFP-treated cells (P < 0.01). Abundant replication of H101 in HXO-RB44 cells resulted in G(2)/M-phase arrest and finally tumor cell lysis, but the apoptosis pathway was not activated. Tumor-bearing mice treated with H101 had reduced tumor burdens and prolonged survival times compared with PBS and AdGFP controls (both P < 0.01). Immunohistochemical and Western blot examination revealed widespread replication of H101 within the tumor.CONCLUSIONS. These results suggest that H101 effectively inhibits the growth of retinoblastoma cells in vitro and in mice and may serve as a novel therapy for retinoblastoma. (Invest Ophthalmol Vis Sci. 2010;51:2626-2635) DOI:10.1167/iovs.09-3516