| ObjectiveTo observate the effect of Jiangu Granula in osteocalcin(OCN),Osterix(OSX),core binding factor aphal(Cbfal),alkaline phoSphatase(AKP),Collagen Typeâ… (ColI),hydroxyproline(HYP) and Mineralization tubercle on Rat Osteoblast and research the effects of Jiangu granula (JGG) on OB in differentiation,so that to probe into the detailed mechanism of prevention and cure of postmenopause osteoporosis with traditional Chinese medicine.Methods(1) Establishing the culture in vitro system of OB:Primary cells were isolated from calvaria of newborn SD rats by enzymatic digestion.OB characteristics were identified by AKP stain of azo dye method and calcification scobination of alizarin bordeaux dyeing. Cells at the third passage were selected for experiment.(2) Preparation for JGG rat serum:20 male SD rats(3-month-old) were randomly divided into 2 groups:JGG-group and normal saline(NS)group,10 rats each group.The rats of JGG-group perfused with JGG 2g·kg-1·d-1,and NS-group with normal saline 2ml·d-1.The serum containing JGG or NS were extracted after 7 days.(3) Detective index:Cell proliferation of OB cultured in differenent concentration serum was measured by MTT Method,and the optimization concentration of the serum containing JGG would be obtained.Then the serum containing JGG or NS were respectively added and cultured the cells belong to JGG-group or NS-group for 7 day.The expression of OCN was measured by ELISA.The expression of AKP,HYP was measured by color method.The expression of Cbfal,OSX,ColI was measured by SYBR GREEN.Takes the 4th generation of cell according to 6×105/ml's density vaccination in 12 orifices,observes 15 days to use the sodium alizarinsulfonate dyeing dyeing mineralization tubercle continuously.Results(1) It showed that cell proliferation rate of OB was the fastest when the concentra- tion of the serum containing JGG was 20%,which was added to the OB of JGG-group.And it was significantly faster than that of NS-group(P＜0.05).(2) It showed that the calcification tubercle was presented in the group including the medicine JGG 20%,then was in the groups including the medicine JGG 30%and 15%.Compares including the medicine group and the physiological saline blood serum group,the former the latter has tubercle quantity many tendencies.(3) ELISA showed that JGG-group increases the OCN content along with the number of days to increase gradually;is highest 4th day of OCN content,then increases along with the number of days drops gradually,and is quite even with the same density NS-group has the significance difference(P＜0.05).(4) Color method showed that the NS-group and the JGG-group increases the AKP and HYP content along with the number of days to increase gradually,and the JGG-group is higher than the NS-group,two groups quite even have the significance difference.(P＜0.05).(5)The results measured by SYBR GREEN were mainly coincident.Cbfal in OB of JGG-group was significantly higher than that in NS-group(P＜0.05) at 1 day,2 day and 3 day(P(0.05).Two group were non-significance difference at 7 day.Col I and OSX in OB of JGG-group was significantly higher than that in NS-group(P＜0.05) at 1 day,2 day,3 day and 7day.(P(0.05).Conclusion1,JGG can increase the expression of OCN,OSX,Cbfal,AKP,ColI,HYP and Mineralization tubercle,so that to multiply OB,to speed up the cell differentiation.2,It is perhaps one of mechanisms that JGG can prevention and cure postmenopausal osteoporosis:JGG→surrenders to the state the most essential specificity duplication activation factor Cbfal→active OSX→active OB protein to duplicate and synthesis→multiply OB.
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