| Seeded cells play an important role in tissue engineering. Chondrocytes undergo a limited number of division in culture and enter a non-dividing state called replicative senescence.In vitro mutiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generete neo-cartilage,but is known to induce cell dedifferentiation manifestes losing marker phenotype collagen type II after 5-6 passages,which poses a great difficult problem to cartilage tissue engineering. The attempt to immortalization of human articular chondrocytes shed interesting lights on the seeded cells of cartilage tissue engineering.Firstly, retrovirus vector pLXSN harbouring HPV16E7 gene segment was constructed through molecular cloning, followed by packaging cell line PA3 17 was electroporated with pLXSN-HPV16E7 after various parameters were optimized,then human articular chondrocytes were transfected with supernatant of viron containing HPV16E7. Results showedr(l) The HFV16E7 held a great homologous with the counterpart in the Genebank. (2)Optimized parameters of electroporation were as follows: recombinant plasmid 20μg, electric capacity 975 μ F, voltage 220v, time 15ms. PA317 packaging cell line of consistant producing viron was isolated with generating viral titer 5.0 × 106-6.8× 106CFU. (3) Immortalized human articular chondrocytes (IHAC) were established with HPV16E7 gene transfection which confirmed by immunohistochemistry, PCR and RT-PCRSecondly, biological characteristics between HAC and non-immortalizedhuman articular chondrocytes (NHAC) were compared, we found:(l) While NHAC appear typical trigonal or polygonal form, the size of MAC was slightly decreased and demonstrated spindle morphology. Maturation of NHAC and juvenile state of MAC were revealed by transmission elctronmicroscope. The small neclous plasma ratio as well as sparse microvilli were disclosed by transmission elctronmicroscope and scan elctronmicroscope respectively. (2)Compared with primary NHAC, more quick adhere and elongation rate,more viability and plating efficiency were in MAC. Growth curve of NHAC resembles that of MAC, but more suturation density was in MAC. More cells of MAC were in G0-M and S phase in cell circle than that of NHAC. (3)Judge tumorgenicity of MAC with below cretia: cell arrangement, growth anchorage dependence,neclous plasma ratio,microvilli,serum dependent test,soft agar cell clone forming test,karyotype analysis and nude mice tumor forming test. MAC arrayed orderly and had the characteristics of growth anchorage dependent, small neclous plasma ratio and sparce microvilli. MAC depended on serum to be confluence and had normal diploid karyotype, furthermore, MAC was not form any clones in soft agar and no tumor was observed in nude mice in one year duration. Therefore, IHAC was proved to be benign transformation. (4)Phenotype type II collagen of MAC was instability and convertd to type I collagen, as a result, type II collagen is needed to be inducted.Third, three different methods including Nutridoma-sp and ascorbate free serum medium(NA), centrifuge tube aggregate culture combinating with BMP-2,insulin,ascorbate and alginate three dimentional culture were performed to induct type II collagen in MAC. Results showed:(l)Fusiform MAC was changed into polygonal form and immunohistochemistry indicted that type II collagen was stained positive after 5d and can maintained as long as 14 d after NA inducted. (2)IHAC inducted with centrifuge tube aggregate culture demonstrated typical hyaline cartilage,round or oval cells inhabited in thelacuna and distributed evenly stained by HE; Alcian blue stain showed large of great amounts of extracellular matrix accumulation, immunohistochemistry revealed lots of type II collagen secretion.(3)IHAC cultured in three dimentional alginate system maintained a spherical shape,type II collagen was rich in round cells'plasma which evaluated with a confocal laser scanning microscope. Gene of type II collagen was coaxed to express in the above induction...
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