Grigolo B, Roseti L, Neri S, Gobbi P, Jensen P, Major E O, Facchini A
Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA.
Osteoarthritis Cartilage. 2002 Nov;10(11):879-89. doi: 10.1053/joca.2002.0836.
To establish an immortalized normal human articular chondrocyte line which could be useful for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases.
Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 (HPV-16) early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium supplemented with an insulin-containing serum substitute and seeding on a hyaluronan-based non-woven structured biomaterial. The expression of markers characteristic of cartilage was shown in the mRNA by reverse transcriptase-polymerase chain reaction. Immunohistological staining and Western blotting analysis were performed to evaluate type II collagen synthesis. Proteoglycans deposition was detected by Alcian Blue staining. A Field Emission In Lens Scanning Microscopy was used to look at the morphology of the immortalized cells at very high magnification.
Normal human articular chondrocytes were efficiently transfected leading to the establishment of an immortalized cell line as confirmed by HPV-16 E7 mRNA and protein detection. These cells were able to re-express type II collagen both at mRNA and protein levels under the two defined cultured conditions we used, still maintaining type I collagen expression. Collagen IX mRNA was present only in early primary culture while collagen type X and aggrecan transcripts were always detected. Alcian Blue staining showed a proteoglycan-rich matrix production. The ultrastructural analysis of the immortalized cells revealed that their morphology strictly resembled that of normal chondrocytes.
The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation. Moreover, it appears to be a suitable model for pharmacological and toxicological studies related to rheumatic diseases relevant to humans.
建立永生化正常人关节软骨细胞系，以更好地理解与风湿性疾病新治疗方法开发相关的细胞分子机制。
采用高效阳离子脂质体介导（脂质转染）程序，将含有两个人乳头瘤病毒16型（HPV - 16）早期功能基因E6和E7的质粒转染至原代培养的成人健康关节软骨细胞。通过对E7 mRNA的逆转录 - 聚合酶链反应分析以及E7蛋白在细胞质中的免疫荧光定位来验证转染情况。在单层培养以及两种被描述为可重新诱导分化特征的培养条件下对建立的软骨细胞系进行检测：在添加含胰岛素血清替代物的无血清限定培养基中培养，以及接种于基于透明质酸的非织造结构化生物材料上。通过逆转录 - 聚合酶链反应在mRNA水平显示软骨特征性标志物的表达。进行免疫组织化学染色和蛋白质印迹分析以评估II型胶原蛋白的合成。通过阿尔辛蓝染色检测蛋白聚糖沉积。使用场发射透镜扫描显微镜在非常高的放大倍数下观察永生化细胞的形态。
通过HPV - 16 E7 mRNA和蛋白检测证实，正常人关节软骨细胞被高效转染，从而建立了永生化细胞系。在我们使用的两种限定培养条件下，这些细胞能够在mRNA和蛋白水平重新表达II型胶原蛋白，同时仍维持I型胶原蛋白的表达。IX型胶原蛋白mRNA仅在早期原代培养中存在，而X型胶原蛋白和聚集蛋白聚糖转录本始终可检测到。阿尔辛蓝染色显示产生了富含蛋白聚糖的基质。对永生化细胞的超微结构分析表明，它们的形态与正常软骨细胞极为相似。
我们获得的细胞系可能是一个有用的工具，可增加我们对软骨生长和分化过程中涉及的遗传和生化事件的了解。此外，它似乎是与人类风湿性疾病相关的药理和毒理学研究的合适模型。
