Experimental Study On Influence Of YouGuiYin Decoction On Alkaline Phosphatase Expression Of Osteoblasts Under Stretch-stress Environment And Its Preliminary Mechanism

Objective Isolated Osteocytes and osteoblasts from the bone tissue of rats using sequential collagenase digestion, and then find the differences with osteoblasts’biological characteristic cell morphology, the first generation cells’alkaline phosphatase(ALP) and osteocalcin(BGP), and the second generation cells’secretion of ALP. In Stretch-stress environment, interfered the osteoblasts and osteocytes in neonatal SD rat with serum containing YouGuiYin soup, investigated the intervention function of osteoblasts proliferation differentiation and the influences on the expression of NO,PGE2in osteocytes. Explored the probably mechanism of YouGuiYin soup regulating and controlling the expression of osteoblasts ALP.Methods Under laboratory conditions, osteocytes and osteoblasts were isolated from the bone tissue of3-day-old rats using sequential collagenase digestion. The cells were identified through cell morphology after24hours later, the first generation cells’alkaline phosphatase(ALP) were stained by Kaplow-way, their osteocalcin(BGP) were stained by immunocytochemitry and the ALP were measured by AKP-test box. Cultured two group of cells in mechanical culture plate by the number of2mL×2×105, respectively culture cells with no serum, acting YouGuiYin Soup containing serum and control serum after culturing them without serum for24h.0.5Hz frequency, and the elongation rate of12%stretch plus mechanical stress was put on the experimental group cells, the control group cells were culture in the general condition. The content and activity of ALP were detected respectively after12h and24h. The number and distributuion of cAKP were detected by using diazotypeprocess and microscope after24h. The content and activity of NO and PGE2were detected by means of nitrate reductase and ELISA after24h. All date was analyzed with statistical software.Results Osteocytes and osteoblasts show obviously the differences in cell morphology, osteocytes were star-shaped or dendrite-shaped within more dendrites, while osteoblasts were spindle-shaped with short dendrites. Osteocytes are negative for ALP, but osteoblasts are positive for ALP. Osteocytes are more positive for BGP, and osteoblasts are less positive for BGP. The secretion of ALP is lower in osteocytes than in osteoblasts. The expression of ALP was inhibited in non-serum group in the condition of stretch compared with that in non-serum group in routine condition. In the condition of stretch stress YOUGUIYIN serum and NS serum could stimulate the expression of ALP compared with them in routine condition(P<0.05). ALP value was higher in YOUGUIYIN group than that in NS serum group in the stretch stress condition(P<0.05). NO value was higher in YOUGUIYIN group than that in NS serum group in the stretch stress condition(P<0.05). PGE2value has no obvious difference in each groups (P>0.05)Conclusion Osteocytes have obvious differences with osteoblasts in the expressions of ALP and BGP, YOUGUIYIN can stimulate the osteoblasts expression of ALP in the condition of stretch stress condition. This kind of stimulation has connection with the function of regulating and controlling the expression of NO in Osteocytes...