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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。

Inhibition of retinoblastoma in vitro and in vivo with conditionally replicating oncolytic adenovirus H101.

机构信息

Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P R China.

出版信息

Invest Ophthalmol Vis Sci. 2010 May;51(5):2626-35. doi: 10.1167/iovs.09-3516. Epub 2009 Dec 10.

Abstract

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-RB(44) 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-RB(44) 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-RB(44) 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-RB(44) cells expressed CAR and were sensitive to adenoviral infection. HXO-RB(44) cells treated with H101 had reduced cell viability compared with AdGFP-treated cells (P < 0.01). Abundant replication of H101 in HXO-RB(44) 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.

摘要

目的

研究溶瘤腺病毒 H101 对体外及体内人视网膜母细胞瘤的治疗效果。

方法

通过 RT-PCR、Western blot、免疫荧光和免疫细胞化学染色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。用携带绿色荧光蛋白的腺病毒(AdGFP)通过流式细胞术确定合适的感染复数。采用细胞计数试剂盒-8 法测定 HXO-RB(44)细胞经 H101 或 AdGFP 处理后的活力。通过终点稀释滴定和实时 PCR 测定病毒在体外的增殖情况。通过流式细胞术分析 HXO-RB(44)的细胞周期和凋亡活性。荷人视网膜母细胞瘤异种移植瘤的 NOD-SCID 小鼠经瘤内注射 H101、AdGFP 或 PBS 治疗。记录肿瘤体积和存活时间。通过免疫组化检测腺病毒纤维蛋白和 Western blot 检测腺病毒六邻体蛋白评估 H101 病毒在体内的复制情况。

结果

HXO-RB(44)细胞表达 CAR,对腺病毒感染敏感。与 AdGFP 处理的细胞相比,用 H101 处理的 HXO-RB(44)细胞的细胞活力降低(P<0.01)。HXO-RB(44)细胞中 H101 的大量复制导致 G2/M 期阻滞,最终肿瘤细胞裂解,但未激活凋亡途径。与 PBS 和 AdGFP 对照组相比,用 H101 治疗的荷瘤小鼠的肿瘤负荷降低,存活时间延长(均 P<0.01)。免疫组化和 Western blot 检测显示 H101 在肿瘤内广泛复制。

结论

这些结果表明,H101 可有效抑制体外和体内人视网膜母细胞瘤细胞的生长,可能成为治疗视网膜母细胞瘤的一种新方法。

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