慢病毒载体感染成年食蟹猴骨髓间充质干细胞

Improving infection efficiency of adult cynomolgus monkey mesenchymal stem cells with lentiviral vectors

摘要:

骨髓间充质干细胞 (Mesenchymal stem cells，MSCs) 具有增殖和多向分化潜能，临床应用广泛，近年来备受关注。另一方面，MSCs易于转导和表达外源基因，是理想的基因工程细胞。非人灵长类 (NHPs) 和人类具有非常相近的遗传背景，NHPs模型在评价药物疗效和移植治疗等方面具有不可替代的价值。本研究采用密度梯度离心法分离成年食蟹猴骨髓单核细胞 (Marrow mononuclear cells，MNCs)，贴壁培养MSCs。同时构建表达绿色荧光蛋白 (Green fluorescent protein，GFP) 的慢病毒载体，感染成年食蟹猴MSCs。结果显示，体外培养的成年食蟹猴MSCs均感染猴泡沫病毒 (Simian foamy virus，SFV)，体外培养成年食蟹猴MSCs必须添加抗病毒药物Tenofovir。但由于食蟹猴MSCs感染SFV，以及培养中添加了抗病毒药物Tenofovir，慢病毒载体的感染效率明显降低 (＜10％)。本研究通过停用抗病毒药，在细胞复苏后6 d转染慢病毒，可大幅提高慢病毒的感染效率 (＞50％)。为成年食蟹猴MSCs作为基因工程细胞应用于实验和临床研究提供了技术保证。

Abstract:

Mesenchymal stem cells (MSCs) have received considerable attention for various therapeutic approaches in recent years. MSCs are also easy to genetically modify to express therapeutic genes by using lentiviral vectors. Because of the similarities in genetics, physiology and metabolism between non-human primates (NHPs) and humans, NHPs models are invaluable for researching human disorders and for developing therapeutic strategies. Therefore, MSCs derived from NHPs could be a powerful tool for cell therapy and genetic engineering. Studies from captive and free-ranging adult NHPs show that up to 100% were infected with simian foamy virus (SFV). In this study, we found that all cultured MSCs derived from adult cynomolgus monkey were infected with SFV by RT-PCR. Therefore, antiviral drugs must be added in MSCs culture. However, because of SFV infection and additive antiviral drugs, the infection efficiency of the lentiviral vectors reduced significantly. In this study, we improved the infection efficiency by disabled antiviral drugs before lentiviral infection. It might be provide technical assistance for the culture of adult cynomolgus monkey MSCs as genetically engineered cells applied to clinical and experimental research.