Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9
Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9
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DOI：
10.1089/107632704323061933
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发表时间：
2004-03-01
影响因子：
--
通讯作者：
Hawkins, RE
中科院分区：
文献类型：
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作者：
Li, Y;Tew, SR;Hawkins, RE
软骨细胞形成并维持软骨的细胞外基质。这些细胞可以从软骨中分离出来用于组织工程等应用，但它们在单层培养中的扩增会导致软骨形成表型的逐渐丧失。在这项工作中，我们研究了在关节置换时从骨关节炎（OA）软骨中分离人关节软骨细胞，在单层培养中扩增，以及使用编码绿色荧光蛋白的基因作为标记基因，用腺病毒、逆转录病毒和慢病毒载体转导它们。研究发现，在细胞培养过程中添加生长因子（转化生长因子β（1）、成纤维细胞生长因子2和血小板衍生生长因子BB）可大大增加细胞增殖，从而选择性地提高逆转录病毒的转导效率。腺病毒和慢病毒载体的转导效率分别为 95% 和 85%。使用添加生长因子的培养基和逆转录病毒载体，效率超过 80%。通过荧光激活细胞分选流分析和免疫印迹进行分析时，逆转录病毒和慢病毒的表达稳定数月。研究了 SOX9 转导作为在传代的人 OA 软骨细胞中重新启动软骨基质基因表达的方法。使用腺病毒和逆转录病毒载体的单层培养物中内源性 II 型胶原表达（mRNA 和蛋白质）均增加。此外，藻酸盐珠培养物刺激了用 SOX9 逆转录病毒转导的软骨细胞中的胶原蛋白 II 基因表达，而在对照软骨细胞中则不然。这些结果证明了快速扩增和高效转导人 OA 软骨细胞的方法，以及通过 SOX9 转导恢复软骨细胞表型关键特征的潜力。
Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.