| Objectives1 To investigate the dedifferetiation process and principles of chondrocytes obtained from 4-month-old rabbit articular cartilage in vitro.2 To establish an immortalized rabbit articular chondrocyte line by transfecting the plasmid pSVSneo, which encoded the SV40LTAg gene and neo gene, into primary cultured chondrocytes obtained from mature rabbit articular cartilage using the lipofect methods.3 To investigate the biological characteristics of immortalized chondrocytes cultured in vitro.Methods1 Pieces of articular cartilage were cut from rabbit femoral articular surface in the femoropatellar area. Chondrocytes were isolated with trypsin and collagenase II. These cells were cultured in monolayer. The phenotypes of the cells were characterized by parameters such as cell morphology (convert microscopy), proliferative capacity (growth curve), GAG synthesis (toluidine blue staining and safranine O staining) and type- II collagen levels (immunohistochemistry), and so on.2 The 1st passage chondrocytes were transfected with the plasmid pSVSneo and cultured in selective medium containing 400 w g/ml G418 for 3 weeks. After several colonies of new cells were grown, they were transferred to 12 well plate to be expanded with small pieces of filter papers. The polygonal morphology cell clones within 12 well plate were isolated with small pieces of filter paper and limited dilution method. Some of them were expanded for further studies. In order to detect the expression of large Tantigen in transgenic cells, immunocytochemical staining of large T antigen was used.3 The immortalized chondrocytes were cultured in vitro. The variation of their morphology and the biological characteristics were observed by microscopy and growth curve. Type II collagen was detected by immunohistochemical methods. The synthasis of GAG were determined by toludine blue and safran O staining. The immortalized chondrocytes were compared with 3rd passage chondrocytes in cellular shape, proliferative capacity, transformation character, chondrocyte phenocyte, and so on.Results1 Convert microscopy showed that chondrocytes in primary culture underwent distinct morphological changes with respect to shape, size, and density of the cells. The majority of chondrocytes were in polygonal shape in the first 4 passages of culture, while more fusiform and spindle-shaped cells were found after 4-5 passages. After 10 passages the proliferating rate of chondrocytes slowed down, and finally stopped. GAG staining and type II collagen immunohistoric staining were positive within the first 4 passages and negative after the 5th passage.2 The majority of the transfected cells were dead after being selected by G418 for 3-4 weeks. There were 12 clone of new cells growing. Immunohistochemistry staining for large T antigen in the nuclear of transgenic cells was positive while that in the nuclear of untransgenic chondrocyte was negative.3 Five clones of transduced chondrocytes had been subcultured for 50 times in more than eight months, without any sign of senescence. Immortalized chondrocytes were polygonal-shaped, similar to primarily chondrocytes. Subculturing, freezing and recovering had no effect on cellular shape and proliferative capacity of immortalized chondrocytes. Analysis found that the size of immortalized chondrocytes was smaller than that ofchondrocytes (p<0.01). The population doubling time of immortalized rabbit articular chondrocytes and the 3rd chondrocytes were 75.7Ih and 54.66h resectively. Immortalized rabbit articular chondrocytes remained serum dependence and stopped proliferating in DMEM without serum. Proliferative rate of immortalized rabbit articular chondrocytes in 5% serum medium was obviously less than that in 10% and 20% serum medium. Both immortalized rabbit articular chondrocytes and normal chondrocytes cannotgrow in soft agar. When 2 X 106 immortalized rabbit articular chondrocyteswere injected intramuscularly in six-w...
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