Objective To observe the replicative senescence of rat articular chondrocyte cultured in vitro so as to provide reference for the succeeding experiment of using medicine interfere and reverse the cataplasia of tissue engineering cartilage or probing cataplasia mechanism．Methods Different generations(P1, P2, P3 and P4) of the chondrocytes were detected with the methods of histochemistry for β-galactosidase (β-gal)， electronmicroscope for ultromicrostructure， immunocytochemistry for proliferating cell nuclear antigen (PCNA)，alcian blue stain for content and structure of sulfatglycosaminoglycan (GAG) of extracellular matrix (ECM)，reverse transcriptionpolymerase chain reaction (RTPCR) for content of collagen Ⅱ，flow cytometry for cell life cycle and proliferative index(PI) to observe senescence of chondrocytes．Results In the 4th passage，the chondrocytes emerging quantitively positive express of β-gal，cyto-architecture cataplasia such as caryoplasm ratio increasing and karyopycnosis emerging under electronmicroscope ，cell life cycle being detented on G1 phase(83．8％)，while in P1, P2, P3 the content of G1 phase was 79.1%, 79.2%, 80.8% respectively. In the 4th passage, PI decreased(16．2％)，while in P1, P2, P3, it was 20.9%, 20.8%, 19.2%. The positive percentage of PCNA，the content of GAG(long chain molecule) and the positive expression of collagen Ⅱ diminished,all detections above were significantly different (Plt;0.01) when compared the 4th passage with the preceding passages．Conclusion Chondrocytes show the onset of senescence in the 4th passage．
Objective To construct a replicationdefective recombinant adinovirus including the target gene human bone morphogenetic protein 4(fragment hBMP-4). Methods The hBMP-4 gene fragment was cut down from pCS2(+)/hBMP-4, cloned into the eukaryotic expressive vector pcDNA 3.1(+), then subcloned into pShuttle-CMV and transformed into the competent E. coli BJ5183/p by electroporation. The resulting recombinant plasmid pAdE/hBMP-4 was transformed into the packaging of thecell lines HEK293 to produce the replication-defective recombinant adenovirusescontaining the hBMP-4 gene. These replication-defective recombinant adinoviruses were transfected into HEK293 and HeLa cells. Then, total RNA and total protein were detected by RT-PCR and the Western-blot assay. Results The pAdE/hBMP-4 was confirmed by the restrictional endonuclease digestion. In HEK293 and HeLa cells, the specific transcription of the hBMP-4 gene was confirmed by RT-PCR, and the expression of the hBMP-4 protein was confirmed by theWestern-blot assay. Conclusion The replication-defective recombinant adinovirus expression vector containing the hBMP-4 gene can be constructed and expressed successfully, which has laid a foundation for the further research on the genetherapy of hBMP-4.
OBJECTIVE: To explore the SV40-mediated immortalization, the related factors and their roles in cell immortalization. METHODS: The original articles about cell immortalization and replicative senescence in recent decade were reviewed. RESULTS: Cell immortalization was a multifaceted phenomenon, it was involved in viral DNA integration, activation of telomerase, inactivation of growth suppressors, and so on, and their roles were closely related. CONCLUSION: The research on cell immortalization may be expected to provide important insights into a broad range of cellular biological phenomenon, and the immortalized cells can play important roles in the research of cell engineering and tissue engineering as standard cells.