The Effect Of Chondrocyte Desdifferentiation On Hypoxia And Relationship Between Collagen Prolyl 4-hydroxylase

Objective: To observe the effect of hypoxia on the dedifferentiation of chondrocytes in vitro,and to explore the role of collagen prolyl 4-hydroxylase(C-P4Hs)and hypoxia-inducible factor-1 a(HIF-1α).Methods: After selecting the optimal Cocl2 concentration for hypoxia induction,the mouse costal chondrocytes were divided into the normal oxygen group and the hypoxia group,and the indexes of the 3rd generation 0.5-72 h and the 1st,3rd,5th and 7th generation at 72 h were detected respectively.The proliferation rate was determined by CCK8 method and cell count,and the dynamic changes of HIF-1α,P4Hα1,P4Hα2 and Col-II in each group were detected by RT-q PCR,IF and Western blot.Results: 1.Costal chondrocytes were cultured under different concentration of Cocl2 for 48 h.When the concentration of Cocl2 >150μM/L,the proliferation rate(P<0.05)was significantly decreased.2.Normal oxygen and hypoxia induced rib chondrocytes for 0-72 h,and RT-q PCR showed significant increases in P4 H α 2 and Col-II m RNA in hypoxia group(P <0.05).Immunofluorescence showed that HIF-1α and P4Hα2 accumulated in the nucleus under hypoxia,and P4Hα2 gradually entered the cytoplasm from the nucleus.Western blot analysis showed that HIF-1αand P4Hα2 protein expressions were significantly increased in hypoxia group(P<0.05).3.The expression of Col-II protein in hypoxia group(P<0.05)increased at the induction stage.CCK8 and cell count results showed that the proliferation rate and cell number of each generation in the hypoxic group were significantly increased(P<0.01),and there was still potential for proliferation when the cells were transferred to the 6-7 generation.RT-q PCR showed that hypoxia group each generation cells P4Hα2,Col-II m RNΑ were significantly increased(P<0.01).Western blot results showed that HIF-1α,P4Hα2 and Col-II protein expressions were increased in each generation of hypoxia group(P<0.05).Conclusion: 1.When the Cocl2 concentration of chondrocytes was 100 microns in vitro,the proliferation of chondrocytes was promoted and the process of cell dedifferentiation was slowed down.When Cocl2 concentration is greater than 150 microns,it can produce obvious drug toxicity on cells.Therefore,the optimal concentration of cobalt chloride for cultured chondrocytes in vitro is 100 microns.2.Increased expression of P4Hα2 through hypoxia induced HIF-1α can accelerate posttranslational modification of Col-II in chondrocytes and increase synthesis and accumulation of Col-II.3.P4Hα2 is the main component of C-P4 Hs,while P4Hα1 is the secondary component,which is regulated by HIF-1α in chondrocytes.4.P4Hα2 may be responsible for increased proliferation rate and delayed dedifferentiation of chondrocytes cultured in vitro under hypoxia condition.