Highly efficient baculovirus-mediated gene transfer into rat chondrocytes
Highly efficient baculovirus-mediated gene transfer into rat chondrocytes
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DOI：
10.1002/bit.20239
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发表时间：
2004-12-05
影响因子：
3.8
通讯作者：
Hu, YC
中科院分区：
文献类型：
--
作者：
Ho, YC;Chen, HC;Hu, YC
为探讨杆状病毒作为基因载体在关节软骨组织工程中的应用潜力，对杆状病毒介导的基因转导大鼠原代软骨细胞的效率进行了评价，发现通常采用的转导方案(以多重感染的浓缩病毒200作用1h)是无效的(1%)。因此，采用了一种改进的方案，显著提高了效率(68%)。对转导参数的优化，如孵育时间(8h)、温度(25℃)和周围溶液(PBS)，进一步将转导效率提高到88%，并将表达持续时间延长到21天，这表明以前被认为不允许杆状病毒转导的细胞可以使用替代的转导方案重新检查它们的通透性。定量实时聚合酶链式反应(Q-PCR)表明，效率的提高与延长孵育时间后病毒摄取量的增加有很好的相关性。Q-PCR还显示了病毒DNA随着培养时间的延长而降解。病毒转导虽然在一定程度上阻碍了细胞的增殖，但在长期培养中可以恢复生长速度。更重要的是，转导细胞可以分泌关节软骨特异性的11型胶原和糖胺多糖，以及模拟转导细胞，证实杆状病毒转导后大鼠软骨细胞保持了正常的分化状态。综上所述，这些数据表明杆状病毒是一种安全高效的大鼠软骨细胞基因载体。(C)2004年威利期刊公司。
To explore the potential of baculovirus serving as a gene delivery vector in tissue engineering of articular cartilage, the efficiencies of baculovirus-mediated gene delivery into primary rat chondrocytes were evaluated and the transduction protocol commonly employed by others (using concentrated virus at multiplicity of infection [MOI] 200 for 1 h) was found to be ineffective (< 1%). Therefore, a modified protocol was adopted, which markedly enhanced the efficiency (68%). Optimization of the transduction parameters, such as incubation time (8 h), temperature (25degreesC), and surrounding solutions (PBS), further increased the efficiency to 88% and prolonged the duration of expression to 21 days, suggesting that the cells previously considered nonpermissive to baculovirus transduction may be reexamined for their permissiveness using alternative transduction protocols. The elevated efficiency correlated well with increased virus uptake upon extended incubation time, as demonstrated by quantitative real-time polymerase chain reaction (Q-PCR). The Q-PCR also revealed the degradation of viral DNA over culture time. Although the virus transduction somewhat hindered the cell proliferation, growth rate could be restored in the long-term culture. More importantly, transduced cells could secrete articular cartilage-specific type 11 collagen and glycosaminoglycan as well as mock-transduced cells, confirming that normal differentiation state of rat chondrocytes is retained upon baculovirus transduction. Taken together, these data indicate that baculovirus is a safe and highly efficient gene delivery vehicle into rat chondrocytes. (C) 2004 Wiley Periodicals, Inc.