Role of Nitric Oxide in the Terminal Differentiation of Rabbit Growth Plate Chondrocytes
|Course||Biochemistry and Molecular Biology|
|Keywords||growth plate chondrocytes nitric oxide terminal differentiation Endochondral ossification|
Objective: To explore the biological effect of nitric oxide on terminaldifferentiation of the rabbit growth plate chondrocytes cultured in monolayer andincubated in pellet culture, consequently, to analysis the role of nitric oxide on theterminal differentiation of rabbit growth plate chondrocytes. It would be beneficial todisclosing the mechanism of endochondral ossification and the etiopathogenesis ofosteocondlodysplasia.Methods: (1) Growth plate chondrocytes were isolated from rabbit costal cartilageand cultured in monolayer. The second passaged chondrocytes were plated in 24 wellplates and grew continuously in monolayer. After about 80% confluence:①the cellcultures were treated daily with AT-RA to a final concentration of 0、35、100、350、1000、3500nmol/L for 5 days, then measured the activity of ALP and NOS and theconcentration of NO.②the cell cultures were treated with AT-RA(1μmol/L)associated with L-NAME(0、1、2.5、5、10mmol/L), then detected the activity ofALP.③AT-RA(0、100、1000nmol/L) with or without SNOG(1mmol/L) were addedin medium of cell culture, then detected ALP activity.(2) Growth-plate chondrocyteswere incubated in pellet culture as three groups: the control group and the groupsAT-RA(1μmol/L) with or without L-NAME(10mmol/L). The expression of typeⅩ collagen and iNOS were detected by Immunohistochemical analysis and RT-PCRdetermination..Results: (1) On the condition of monolayer culture, ALP activity increasedsignificantly as the concentration of AT-RA increasing from 35nmol/L to1000nmol/L;The activity of NOS and the levels of NO were increased with the samepattern as ALP activity; As the concentration of L-NAME was increased from 1 to 10mmol/L, there was a dose-dependent decrease in ALP activity; SNOG causedsignificant increase in ALP activity compared with the control groups(servedAT-RA alone). (2) On the condition of pellet culture, the chondrocytes in the grouptreated with AT-RA had a relatively higher proportion of hypertrophy cells than theother groups. Immunohistochemical analysis and RT-PCR determination showedthat, the expression level of TypeⅩcollege was the highest in the AT-RA groupand raletively lower in AT-RA with L-NAME group and the lowest in control group;The expression of iNOS were higher in the AT-RA group and AT-RA with L-NAMEgroup than the control group.Conclusion: (1) Nitric oxide could promote the growth plate chongrocytes’development of ALP activity and expression of collagen typeⅩ, which indicatednitric oxide could promote the terminal differentiation of growth plate chongrocytes.(2) Nitric oxide is required for the process of growth plate chongrocytes’ differentiationof mature and hypertrophy chondrocytes phenotype.