The Effect Of Gradiont Glucose And Different Treatment Of Qianggubao Fang On Cultured Osteoblast In Vitro

Objective: To observe the effect of high glucose and different treatment of qianggubao decoction on cultured osteoblast in vitro.Methods:OB was isolated from the skull of 1-2 day newly born SD rats by means of Trypsin--collagenase digestion and identified by image analysis observed by inverted microscope, collagen I detected by V-G collagen staining, ALP staining used by adopting ameliorative Gomori' s calcium-cobalt, calcification nod staining used by alizarin red staining method etc. To take the second -fifth generation cells as experimental model. The first step, the proliferation of osteoblasts was monitored by MTT analysis : â‘  not-containing and containing different concentrations of glucose culture medium were incubuted; â‘¡ not-containing and containing different dose of qianggubao decoction culture medium were respectively added to the OB cultured by normal and high glucose(300mg/dl) and incubuted. â‘¢not-containing and containing serm from diabetic rat-model fed with Qiang-gu-bao decoction were respectively added to the OB and incubated, which was obtained depended on time(perfus stomach three days or five days, last perfus stomach one hour or three hours )and dose(5%, 10% 20%). The next, on the base of first step all group but the group of containing different dose of qianggubao decoction culture medium by high glucose were monitored by Alpactivity analysis measured by Golden' s method, Mineralization of Formation used by alizarin red staining method .The last, the cytokines were assayed by radio-immunity and AGEs were assayed by spectrofluorometer on the liquid cultured the group of serm from diabetic rat-model fed with Qiang-gu-bao decoction for 48 or 72 h and 7 or 10 days respectively. The effect of iang-gu-bao decoction on theformation of AGEs and secretion of cytokines were obtained.Result: (1)relatively rarefied osteoblasts can be obtained by means of cell culture in vitro using the method of collagenase-pancreatic enzyme digestion. (2)different concentrations of glucose promoted for osteoblastic cell proliferation for 48 hour , but inhibited for 72 hour beyond 15mmol/L among of these concentrations, no more than 15mmol/L promoted for osteoblastic cell proliferation on the earlier period of cultivation (P<0. 01, P<0.05) and was not suppression on the relative long time, and no less than 30mmol/L inhibited secretion of ALP for 7 day (P<0. 05) , and beyond lOmmol/L delaying formation of cell mineralzation nodes for 18 days, below lOmmol/L glucoses didnot inhibit the secretion of ALP and formation of mineralization. (3) 100u g/ml > 50ug/m^ 10p g/ml ^ 5 p g/ml of qianggubao decoction promoted for osteoblastic cell proliferation on the early stage ( P<0.05 or P<0.01 ), 50ug/ml was the best. Moreover, 50P g/ml > 10u g/ml of qianggubao decoction also promoted for osteoblastic cell secretion of ALP for seven days ( P<0.05 or P<0.01 ) and mineralization of formation for eighteen days (P<0. 05 or P<0. 01) . (4) 100 P g/ml> 50 p g/ml of qianggubao decoction added to the 0B cultured by high glucose promoted for osteoblastic cell proliferation on the early stage (P<0. 05) . (5)The serm from diabetic rat-model fed with Qiang-gu-bao decoction promoted for osteoblastic cell proliferation on the early stage. The best effectest of serm from diabetic rat-model fed with Qiang-gu-bao decoction was serm of perfus stomach three days^ last perfus stomach one hou^ 20% density and not-Inactivation. All serms of diabetic rat-model fed with Qiang-gu-bao decoction promoted the secretion of ALP and the formatiom of mineralzation nodes (P<0. 05 or P<0. 01) . There was stepped-up trend on the secretion of Interleukin-6 , but weaken trend on the secretion of Tumo necrosis factor-a depended on time , There was insignificant difference among the experimental groups and control group for 48h. However, both of serum obviously inhibited the secretion of Tumo necrosis factor-a for 72 hours(P<0. 01). both of the serums inhibited the formation of AGEs in the supernatant fluid for 7 days and 10 days(P<0. 05 or P<0. 01).Conclusion:(l)High glucose had an influence on the proliferation,differentition and mineralization of osteoblastic cell.The tolerance concentrations were below 15mmol/L on the proliferation of OB and were below lOmmol/L on the differentiation and mineralization of OB. (2) Its one of cytology mechanisms of Qiang-gu-bao decoction on preventing and curing diabetes osteoporosis were related with promoting for osteoblastic cell proliferation tlilTurunliLion and mineralization of formation, moreover inhibiting nonenzymic glycation reaction and reducing the formation of AGEs on the early stage, inhibiting the secretion of osseous absorptive cytokine, Consequently, Qiang-gu-bao decoction could inhibited bone resorption and promote for bone formation as well as regulated the bone metabolization. (3)osteoblastic cultivate system on the stage of high glucose and diabetes mellitus body fluid founded by this research could regard as one of cell model for the inordinate bone metabolization of diabetes mellitus, and were used with the mechanism of diabetes osteoporosis as well as prevention and cure decoction.