微囊泡包裹溶瘤病毒的制备与鉴定及对Hepa 1-6细胞体外杀伤作用的探究

Microvesicle-encapsulated oncolytic virus: preparation, identification, and killing effect on Hepa 1-6 cells in vitro

摘要:

【背景】 溶瘤病毒生物治疗是肿瘤治疗发展的新方向，其中新城疫病毒(Newcastle disease virus, NDV)因具有生物安全及靶向性强等优势备受关注。然而，中和抗体的病毒清除将削弱溶瘤病毒的持续有效发挥的作用。基于肿瘤细胞源性的微囊泡递送体系有望解决上述瓶颈，因此微囊泡包裹溶瘤病毒的制备具有非常重要的现实意义。【目的】 探究建立基于肿瘤细胞源性的新城疫病毒微囊泡递送体系和评估其体外杀瘤效应。【方法】 采用差速离心的方法，提取出微囊泡包裹的新城疫病毒(MV@NDV)；粒度仪检测MV、NDV、MV@NDV的粒径大小；透射电子显微镜观察MV@NDV中MV和NDV的相对位置；免疫印迹技术(Western blotting, WB)检测MV@NDV的Integrin β-1、Gpc 3和Flotillin-1蛋白表达；激光共聚焦显微镜观察MV@NDV感染Hepa 1-6细胞24 h，细胞NDV-HN蛋白表达；倒置显微镜和CCK-8法分别检测感染Hepa 1-6细胞24、48、72 h的细胞形态以及活力变化；实时荧光定量PCR (quantitative real-time PCR, qPCR)和免疫印迹技术(WB)分别检测MV@NDV感染Hepa 1-6细胞24 h凋亡相关基因Caspase-3和Caspase-9表达以及凋亡相关蛋白Caspase-3表达。【结果】 粒径和透射电子显微镜检测结果表明MV粒径范围为141−342 nm，NDV粒径范围为91−825 nm，MV@NDV粒径范围为164−712 nm。WB结果表明MV@NDV能够表达MV所表达的Integrinβ-1、Gpc 3、Flotillin-1蛋白。激光共聚焦显微镜观察结果表明MV@NDV、NDV感染Hepa 1-6细胞，出现红色荧光，均表达NDV-HN蛋白。CCK-8结果表明MV@NDV组与NC组相比，Hepa 1-6细胞活力下降(P<0.05)。qPCR结果表明MV@NDV与NC组相比凋亡相关基因Caspase-3和Caspase-9均上调(P<0.05)。WB结果表明MV@NDV与NC组相比凋亡相关蛋白Caspase-3上调(P<0.05)。【结论】 成功构建NDV微囊泡递送体系MV@NDV，有望解决治疗型溶瘤新城疫病毒在临床转化上存在的瓶颈，为临床新城疫病毒抗肿瘤方面提供新的思路及依据。

关键词:溶瘤病毒;新城疫病毒;微囊泡;抗肿瘤

Abstract:

[Background] Oncolytic virus therapy is a new direction in the research on tumor treatment, and Newcastle disease virus (NDV) has attracted much attention because of its high biosafety and accurate targeting. However, viral clearance by neutralizing antibodies will weaken the sustained effect of oncolytic virus. The tumor cell-derived microvesicle delivery system is expected to break the above bottleneck. [Objective] To establish the tumor cell-derived microvesicle delivery system of oncolytic NDV and evaluate its antitumor effect in vitro. [Methods] Microvesicle-encapsulated NDV (MV@NDV) was extracted by differential centrifugation. The particle sizes of MV, NDV, and MV@NDV were measured by a particle size meter. The relative positions of MV and NDV in MV@NDV were observed by transmission electron microscopy. Western blotting (WB) was employed to determine the protein levels of Integrin β-1, Gpc 3, and Flotillin-1 of MV@NDV. Laser confocal microscopy was employed to observe MV@NDV 24 h post infection in Hepa 1-6 cells and the expression of NDV-HN protein in cells. An inverted microscope and the cell counting kit-8 (CCK-8) were used to examine the changes the morphology and viability of infected Hepa 1-6 cells 24, 48, and 72 h post infection, respectively. Quantitative real-time polymerase chain reaction (qPCR) and WB were employed to determine the mRNA levels of apoptosis-associated genes Caspase-3 and Caspase-9 and the protein level of Caspase-3, respectively, in the MV@NDV-infected Hepa 1-6 cells 24 h post infection. [Results] The particle sizes of MV, NDV, and MV@NDV were within the ranges of 141−342 nm, 91−825 nm, and 164−712 nm, respectively. WB results indicated that Integrin β-1, Gpc 3, and Flotillin-1 were expressed in MV@NDV. The Hepa 1-6 cells infected with MV@NDV and NDV showed red fluorescence and expressed NDV-HN protein. In addition, the viability of Hepa 1-6 cells decreased in the MV@NDV group compared with that in the NC group (P<0.05). The Hepa 1-6 cells in the MV@NDV group showed up-regulated mRNA levels of Caspase-3 and Caspase-9 and up-regulated protein level of Caspase-3 compared with the NC group (P<0.05). [Conclusion] The successful construction of NDV microvesicle delivery system MV@NDV is expected to solve the bottleneck of therapeutic oncolytic Newcastle disease virus (NDV) in the clinical translation, and provide new ideas and rationale for clinical NDV anti-tumor aspects.

Key words:oncolytic virus;Newcastle disease virus;microvesicles;anti-tumor