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首页> 外文期刊>Osteoarthritis and cartilage >Vital marking of articular chondrocytes by retroviral infection using green fluorescence protein.
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Vital marking of articular chondrocytes by retroviral infection using green fluorescence protein.

机译：使用绿色荧光蛋白通过逆转录病毒感染对关节软骨细胞进行重要标记。

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OBJECTIVE: One of the main open questions in chondrocyte transplantation is the fate of the implanted cells in vivo. We intended to establish prerequisites for such studies in animal models and to show the feasibility of this approach in rabbits. Isolated articular chondrocytes were retrovirally marked using green fluorescence protein (GFP) as a cell-specific marker in order to allow an in vivo follow-up of these cells. METHODS: Chondrocytes from rabbits, sheep, cattle and humans were isolated and infected with murine leukemia virus-derived retroviruses carrying the GFP gene. The influence of the host range of three packaging cell lines (PA317, PT67, PG13), start cell concentrations, number of cell passages and number of infection cycles on the efficiency of infection was investigated. Stability of GFP expression was followed by FACS analysis, confocal imaging and fluorescence microscopy. For in vivo follow-up of GFP expression we used marked allogeneic chondrocyte populations grown on scaffold material and implanted them into full-thickness defects in knee joints of rabbits. RESULTS: Retroviruses from all three packaging cell lines were able to infect rabbit and human chondrocytes, whereas only retroviruses released from PG13 cells were able to infect sheep and bovine chondrocytes efficiently. Optimization of the infection with these viruses resulted in efficiencies of 60-90% GFP-expressing chondrocytes. Populations of 100% marked chondrocytes were obtained by cell sorting. GFP expression stability of such marked chondrocyte populations was followed in monolayer culture and in 3-D culture on different scaffold materials. The expression of GFP was stable on all tested materials for at least 4 weeks. In monolayer culture GFP expression was stable for more than 8 months. In vivo, we observed stable GFP expression in the transplants during a four-week time course. CONCLUSION: Retroviral GFP gene transfer led to long-term expression in chondrocytes from rabbits, sheep, cattle and humans. Transgene expression and the number of implanted chondrocytes remain stable for at least 4 weeks in vivo. This method permits a rapid monitoring of chondrocytes and provides a basis for following the fate of these cells in vivo. Copyright 2002 OsteoArthritis Research Society International.
机译：目的：软骨细胞移植的主要开放性问题之一是体内植入细胞的命运。我们打算为在动物模型中进行此类研究建立先决条件，并证明这种方法在兔中的可行性。使用绿色荧光蛋白（GFP）作为细胞特异性标记物对分离的关节软骨细胞进行逆转录病毒标记，以便对这些细胞进行体内随访。方法：分离兔，绵羊，牛和人的软骨细胞，并用鼠白血病病毒衍生的携带GFP基因的逆转录病毒感染。研究了三种包装细胞系（PA317，PT67，PG13）的宿主范围，起始细胞浓度，细胞传代数和感染周期数对感染效率的影响。通过FACS分析，共聚焦成像和荧光显微镜观察GFP表达的稳定性。对于GFP表达的体内随访，我们使用在支架材料上生长的显着的同种异体软骨细胞群，并将其植入兔膝关节的全厚度缺损中。结果：来自所有三个包装细胞系的逆转录病毒均能感染兔和人的软骨细胞，而只有从PG13细胞释放的逆转录病毒才能有效地感染绵羊和牛的软骨细胞。对这些病毒的感染的优化导致表达GFP的软骨细胞的效率为60-90％。通过细胞分选获得100％标记软骨细胞群。在不同的支架材料上进行单层培养和3-D培养后，跟踪这些标记软骨细胞群的GFP表达稳定性。 GFP的表达在所有测试材料上稳定至少4周。在单层培养中，GFP表达稳定超过8个月。在体内，我们在四个星期的时间内观察到了移植物中稳定的GFP表达。结论：逆转录病毒GFP基因转移导致兔，绵羊，牛和人的软骨细胞中长期表达。转基因表达和植入的软骨细胞数量在体内至少持续4周保持稳定。这种方法可以快速监测软骨细胞，并为在体内追踪这些细胞的命运提供基础。版权所有2002 OsteoArthritis Research Society International。

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