慢病毒載體感染成年食蟹猴骨髓間充質干細胞

任振華，王淑艷，張穎，鄒春林，張愚

1 首都醫科大學 宣武醫院細胞治療中心，北京 100053

2 安徽醫科大學 人體解剖學教研室，合肥 230032

組織工程與細胞培養

慢病毒載體感染成年食蟹猴骨髓間充質干細胞

任振華1,2，王淑艷1，張穎1，鄒春林1，張愚1

1 首都醫科大學 宣武醫院細胞治療中心，北京 100053

2 安徽醫科大學 人體解剖學教研室，合肥 230032

骨髓間充質干細胞 (Mesenchymal stem cells，MSCs) 具有增殖和多向分化潛能，臨床應用廣泛，近年來備受關注。另一方面，MSCs易于轉導和表達外源基因，是理想的基因工程細胞。非人靈長類 (NHPs) 和人類具有非常相近的遺傳背景，NHPs模型在評價藥物療效和移植治療等方面具有不可替代的價值。本研究采用密度梯度離心法分離成年食蟹猴骨髓單核細胞 (Marrow mononuclear cells，MNCs)，貼壁培養 MSCs。同時構建表達綠色熒光蛋白 (Green fluorescent protein，GFP) 的慢病毒載體，感染成年食蟹猴MSCs。結果顯示，體外培養的成年食蟹猴MSCs均感染猴泡沫病毒 (Simian foamy virus，SFV)，體外培養成年食蟹猴MSCs必須添加抗病毒藥物Tenofovir。但由于食蟹猴MSCs感染SFV，以及培養中添加了抗病毒藥物Tenofovir，慢病毒載體的感染效率明顯降低 (＜10%)。本研究通過停用抗病毒藥，在細胞復蘇后6 d轉染慢病毒，可大幅提高慢病毒的感染效率 (＞50%)。為成年食蟹猴MSCs作為基因工程細胞應用于實驗和臨床研究提供了技術保證。

食蟹猴骨髓間充質干細胞，綠色熒光蛋白，慢病毒載體，猴泡沫病毒，轉導

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.

Keywords: cynomolgus monkey mesenchymal stem cells, green fluorescent protein, lentiviral vectors, simian foamy viruses,transduction

骨髓間充質干細胞 (MSCs) 是來自于骨髓的成體干細胞，因其具有增殖和多向分化潛能、造血支持、免疫調控等特點而備受關注[1-2]。MSCs在體外可分化成為脂肪、成骨、軟骨等中胚層的組織細胞。……