| Objective 1. To establish and culture the osteoblast(OB) in vitro. 2. Tostudy the effects and mechanisms of soybean isofla- vone(SI) on cultured ratfirst generation OB in vitro, and provide scientific evidence for the preventionand control for the post- menopausal osteoporosis(PMO).Methods 1. The isolated, cultured and identification rat OB in vitro:Newborn rat calvarid OB were isolated by the Trypsin and collagenase typeâ…¡digesting several times alternatively and cul- tured in vitro. OB were identifiedby Wright's staining and ALP dyeing after 5,7,10 days. 2. The study of SIon the proliferation and differentiation of cultured rat OB in vitro: (1)Theexperiment was divided into seven groups: They were Normal Control group,different dose of S1 group (10-9M,10-8M 10-7M, 10-6M, 10-5M, respectively) andE2 group (10-10M 17-βestradiol). (2) The effect of SI on OB in proliferation anddifferentiation: After cultured with SI 1～7 days, the MTT(OD) of OB weremeasured. After 2d and 4d, the contents of extracellular ALP and BGP weredetected. 3. The influence of SI on the expression of Collagenâ… genes inosteoblast: After cultured with 4h, total RNA was extracted from the cells.Collagenâ… cDNA were amplified by RT-PCR.β-actin cDNA was also amplifiedat the same time and used as control. The pictures of PCR production werescanned. The specific values of relative abundance of Collagenâ… /β-actin werecalculated, and the expression levels of Collagenâ… genes were determined.Result 1. The isolated, cultured and identification rat OB in vitro: Thecells have the characteristics of OB typical morphology. ALP dyeing positiverate exceeds 95%. 2. The effect of SI on the proliferation and differentiation ofcultured rat OB in vitro: (1) From the 1st day to the 4th day, the MTT(OD) of10-8M～10-5M SI groups were higher than that in normal control group(P＜0.05), especially in the 2nd and 3rd days;after the 5th day, proliferation of 10-8M～10-5M SI group were slower than that in normal control group. Sincethe 3rd day, the MTT(OD) of 10-9M SI group were lower than that in normalcontrol group (P＜0.05). The growth curve of the normal control group showthat latent period was from the 1st day to the 4th day,the exponential growthperiod was from the 4th day to the 7th day.Compare to the normal controlgroup, the growth curves of SI groups and E2 was early for the exponentialgrowth period. The growth curves of E2,10-8M,10-6M and 10-5M SI wereobviously like ladder. (2) In the 2nd day, the ALP activity of E2 and 10-6M SIgroups were higher than that in normal control group (P＜0.05); in the 4thday,the ALP activity of E2,10-6M and 10-5M SI group were higher than that innormal control group (P＜0.05). The ALP of 10-5M and 10-9M～10-7M SI groupswere lower than that in E2 group (P＜0.05). (3) In the 2nd day,the BGP activityof E2 and 10-7M SI group was higher than that in normal control group (P＜0.05);in the 4th day,the BGP activity of E2,10-8Mand10-7M SI groups were higherthan that in normal control group (P＜0.05). The BGP of 10-9M 10-8M 10-6Mand 10-5M SI groups were lower than that in E2 group (P＜0.05). (4) The effectof SI on the expression of the collagenâ… : The collagenâ… of 10-7M and 10-5M SIgroups and E2 group were higher than normal control group (P＜0.05).Conclusion 1. We can get OB isolated from newborn rat calvarid by themethod of Trypsin and collagenase typeâ…¡digesting several timesalternatively,and OB that we got were pure and active. 2. The SI has effect onOB, dependently on time and dose. The effective concentration of SI on theproliferation of OB was from 10-8M to 10-5M, especially in the 2nd and 3rddays. Since the 3rd day, the MTT(OD) of 10-9M SI group were lower than thatin normal control group. The growth curves of every groups were different andeach period was diversity. Possibly, OB in different growth period havedifferent reaction to effective components on SI. 3. 10-6M, 10-5M SI canenhance ALP activity of OB; 10-8M, 10-7M SI can enhance BGP activity of OB.4. 10-7M and 10-5M SI increased the expressions of Collagenâ… genes and enhanced ability of bone formation 5. SI was identical with E2 on theproliferation and differentiation of cultured rat OB.
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