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首页> 外文期刊>Gene Therapy and Molecular Biology >Enhanced splenic protection and reduction of parasitaemia following transplantation of splenocytes overexpressing Bcl-2 in a murine malaria model
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Enhanced splenic protection and reduction of parasitaemia following transplantation of splenocytes overexpressing Bcl-2 in a murine malaria model

机译：小鼠疟疾模型中脾细胞过表达Bcl-2的脾细胞移植后增强的脾保护和减少寄生虫血症

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Malaria is one of the most important infectious diseases worldwide. During acute infection, the activation of the immune response and parasite clearance by the spleen are key host defence mechanisms. Apoptosis of lymphocytes is believed to compromise the immune response in malaria cases. As limited progress has been made using gene therapy against parasitic infections, we explored the potential beneficial effect of reconstituting Plasmodium chabaudi infected mice with transduced splenocytes overexpressing Bcl-2. An ecotropic retrovirus encoding GFP and Bcl-2 was packaged in 293T cells. A second retrovirus expressing only GFP was used as control. Initially, CBA/Ca mice were transplanted with 2 x 107 total splenocytes from syngenic donor mice transduced with retroviral particles. Two hours later recipient mice were infected with 5 x 104 P. chabaudi parasitised erythrocytes. Parasitaemia was evaluated every 24 h. Seven days post-infection, we evaluated splenic histology and Bcl-2 expression via RT-PCR and western blot. The protection against H2O2- induced apoptosis was tested on isolated total splenocytes. The efficiency of retroviral infection, expression of Bcl-2 and reporter gene was confirmed by Fluorescence microscopy, RT-PCR and western blotting of splenocytes from transplanted with cells transduced with GFP- or Bcl-2-expressing retroviruses. Parasitaemia in mice transplanted with GFP-transduced splenocytes transiently decreased 10-fold at day five after Plasmodium infection and was further decreased when mice were transplanted with Bcl-2-transduced splenocytes. The splenic architecture of mice that received transduced cells was preserved compared to control mice. Cytoprotection in vitro was enhanced with the Bcl-2 construct. The protective effect correlated with increased the cellularity in the spleen and overexpression Bcl-2. We tested the use of standard gene therapy strategies based on the reconstitution of irradiated mice with genetically modified heterologous cells in301Martínez-Flores et al: Enhanced splenic protection and reduction in a murine malaria modelan experimental murine model of malaria. We observed transient reduction of parasitaemia, preservation of splenic architecture and increased cell survival in vitro against oxidative stress. Although the experimental design aimed to test the effect of Bcl-2 overexpression, transplantation of cells transduced with the empty retroviral vector resulted in a significant protective effect accompanied by the increased expression of endogenous Bcl-2.
机译：疟疾是全世界最重要的传染病之一。在急性感染期间，脾脏对免疫反应的激活和寄生虫清除是关键的宿主防御机制。在疟疾病例中，淋巴细胞的凋亡被认为损害了免疫反应。由于使用针对寄生虫感染的基因疗法仅取得了有限的进展，因此我们探索了用过表达的Bcl-2转导的脾细胞重建chabaudi疟原虫感染小鼠的潜在有益作用。将编码GFP和Bcl-2的嗜性逆转录病毒包装在293T细胞中。使用仅表达GFP的第二逆转录病毒作为对照。最初，将CBA / Ca小鼠移植了2 x 107个脾细胞，这些细胞来自逆转录病毒颗粒转导的同基因供体小鼠。 2小时后，用5×104个沙巴氏假单胞菌寄生的红细胞感染受体小鼠。每24小时评估一次寄生虫血症。感染后7天，我们通过RT-PCR和Western blot评估了脾脏的组织学和Bcl-2表达。在分离的总脾细胞上测试了针对H2O2诱导的凋亡的保护作用。逆转录病毒感染，Bcl-2和报告基因的表达的效率已通过荧光显微镜，RT-PCR和Western印迹证实了脾脏细胞的脾细胞移植与GFP或Bcl-2表达的逆转录病毒转导的细胞。疟原虫感染后第5天，移植了GFP的脾细胞的小鼠体内的寄生虫血症瞬时降低了10倍，而移植了Bcl-2的脾细胞的小鼠中的寄生虫血症又进一步降低了。与对照小鼠相比，保留了转导细胞的小鼠的脾脏结构。 Bcl-2构建体可增强体外细胞保护作用。保护作用与脾脏中细胞增多和过表达Bcl-2有关。我们在301Martínez-Flores等人的试验中，基于经过基因修饰的异源细胞重建的辐射小鼠的重组，对标准基因治疗策略的使用进行了测试：增强的脾脏保护和减少鼠类疟疾模型和实验性疟疾鼠模型。我们观察到寄生虫血症的瞬时减少，脾结构的保存和体外抗氧化应激的细胞存活率的增加。尽管该实验设计旨在测试Bcl-2过表达的作用，但用空的逆转录病毒载体转导的细胞的移植导致了显着的保护作用，并伴随着内源性Bcl-2表达的增加。

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