| Objective To investigate the effects and mechanisms of SI on cultured rat first generation OB in vitro, and compared with E2 Provide scientific evidence for the prevention and control for the POP.Methods 1. The isolated and cultured rat OB in vitro: Newborn rat calvarid OB were isolated by the enzyme digesting several times and cultured in vitro. The experiment lasted 24 days. The cells were observed by inverted phase contrast microscope. OB were identified by ALP dyeing and bone nodules dyeing. Cell growth was measured by MTT assay and the growth curves were drawn. 2. The study of SI on OB in metabolize: (l)The cells divide into six groups : They were Normal Control group, different dose of SI (10-8, 10-7, 10-6, 10-5M,respectively) group and E2 group ( 10-10M 17- P estradiol).(2) The effect of SI on OB in proliferation and differentiation: After cultured with SI 48h and 72 h, the MTT(OD) of OB were measured. After 72h, the contents of cell protein, ALP and BGP were detected. (3) The effect of SI on OB in bone formation: After 72h,Collagen I in cellular were determined. After 18 days, alizarin red stains dying was used to count bone nodules. (4)The effect of SI on TGF-Pi: After 72h, the content of TGF-Pi were detected. (5)The effect of SI on OB in cellular Ca2+: The experiment lasted 72h. The content of cellular Ca2+ of Normal Control group, 10-5M SI group and E2 group were measured. (6) The effect of SI on OB in ultrastructure: After 72h, the cells of Normal Control group, 10-5M SI group and E2 group were observed by transmission electron microscope.Results 1. The isolated and cultured rat OB in vitro: The cell possessed the characteristics of OB typical morphology. ALP dyeing positive rate exceeds 95%. The cell can form bone nodules in vitro. The growth curve was made up of incubation period, Growth period of logarithm .Platform period and Decline phase. 2. The study of SI on OB in metabolize: (1) The effect of SI on OB in proliferation and differentiation: After 48h and 72h,the MTT(OD) of all SIgroup and E2 group were significantly higher than that in normal control group (P<0.01) , 10-8 M-10-6M SI group lower than E2 group (P<0.01) . The MTT(OD) of Normal Control group and 10-8M-10-6M SI group in 72h were significantly higher than that in 48h (P<0.01) . The protein of 10-6M -10-5M SI group and E2 group were significantly higher than that of normal control group (P<0.01) . The ALP activity and BGP of all SI groups and E2 group were significantly higher than that in normal control group (P<0.01) , the BGP of SI groups was significantly lower than that in E2 group (P<0.01) .The level of above index were correlated with the dose of SI. (2) The effect of SI on OB in bone formation: 10-5M SI group and E2 group could enhance the OB ability of forming bone nodules, the difference was significance (P<0.01) . The ability of forming bone nodules of 10-8M-10-6M SI group were significantly lower than E2 group (P>0.01) . 10-5M SI group were significantly higher than 10-8M~10-6M SI group (P>0.01) .The collagen I of 10-7M-10-5M SI group and E2 group was higher than normal control group (P<0.01) , but that of 10-8M-10-7M SI group were significantly lower than that of E2group (P<0.01) . 10-7M-10-5M SI group were significantly higher than 10-8M-10-6M SI group (P>0.01) . (3)The effect of SI on TGF-B1: The content of TGF-B1 of 10-6M-10-5M SI group and E2group were significantly higher than that in normal control group(P0.01 ),and 10-8M-10-7M SI group were significantly lower than that in E2 group (P<0.01). 10-6M-10-5 SI group were significantly higher than 10-8M-10-7M SI group (P<0.01). (4)The effect of SI on OB in cellular Ca2+: Compared with normal control group, the Ca2+ in OB of 10-5M SI group was no significant difference (P>0.05) , but the difference of E2 group was significant (P<0.01) . (5) The effect of SI on OB in ultrastructure: By transmission electron microscope, the organelle of supplying energy and synthesize of 10-5M SI group and E2 group were increase, the function was more active than normal control... |
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