The Effect Of Prolactin On Cultured Osteoblast Of Cow In Vitro

Objective:To observe the effect of prolactin to proliferation,differentiation and related proteins secreted protein function of gene expression,of cultured osteoblast obtained from the rib of newborn cow in vitro.Methods:To obtain osteoblasts from neonatal cow's rib adopting the method of tissue culture,conduct primary culture and sub-culturing,take the second generation cells as experimental model after having been identified by means of morphological observation and drum dyeing of ALK etc,and then co-culture these cells with different concentrations of the prolactin.The effect of prolactin on cell proliferation was measured by MTT method(OD value at wave-length 570nm),on cell differentiation was measured by Golden's method(ALP activity),and on cell secretion bone gla protein and TNF-αwere observed by means of radicalization immunity(RI method,expressed by ng/ml). The effect of prolactin on the expressions of BGP mRNA and TNF-αmRNA were dectected by semin quantitative reverse transeription polymerase chain reaction (RT-PCR.).Results:Relatively rarefied osteoblasts could be obtained by means of cell culture in vitro using the method of tissue culture.Seven days after communal cultivation,certain concentrations of prolactin could inhibit the proliferation of the osteoblasts.Contrast with the control group,prolactin groups could inhibit the proliferation of the osteoblasts in the concentration of 0.1μg/mL,1.0μg/mL and 10.0μg/mL whereas that had insignificant difference with control group(P＞0.05),0.01μg/mL group had significant difference (P＜0.01),but there had no dose-dependent.The activity of alkaline- phosphotase was decreased in experimental groups which were compared with control group respectively, 0.1μg/mL,1.0μg/mL and 10.0μg/mL group had insignificant difference(P＞0.05), 0.01μg/mL group had significant difference(P＜0.01).Seven days later,the quantity of BGP was decreased in 0.1μg/mL,1.0μg/mL and 10.0μg/mL groups which were compared with control group respectively,there had insignificant difference(P＞0.05);the quantity of BGP was increased in 0.01μg/mL group which were compared with control group,and it had significant difference(P＜0.01).Seven days later,the quantity of TNF-αwas increased in experimental groups which were compared with control group respectively,1.0μg/mL and 10.0μg/mL group had insignificant difference(P＞0.05),but 0.01μg/mL and 0.1μg/mL group had significant difference(P＜0.01).After having been affected by prolactin for 10 days,the cells were used for RNA extraction,then the RNA was carried on RT-PCR to test the mRNA of the BGP and TNF-α,the result indicated that:0.1μg/mL, 1.0μg/mL and 10.0μg/mL group could inhibit the BGP to express,but the 0.01μg/mL group could promote the BGP to express;all the concentration of prolactin could promote the TNF-αto express.Conclusions:prolactin can inhibit bone formation by decrease the proliferation and the differentiation of osteoblasts in vitro.In a certain concentration of prolactin,low concentration to promote the secretion of BGP,high concentration promote the secretion of BGP.The quantity of TNF-αwas increased in all experimental groups.The effect of prolactin on the expressions of BGP mRNA and TNF-αmRNA were the same with cell secretion.