| Objective:This experiment is to observe the Yi Gu Tang granule drug-containing serum through classical Wnt/BMP-2 pairs of signaling pathways for in vitro culture of SD rat osteoblast proliferation,and on this basis to discuss its multiplication effect and related mechanism of the prevention and treatment of osteoporosis.Method:(1)the preparation of medicated serum.12 months of age 40 only female SD rats randomly divided into group A(Yi Gu Tang granule drug-containing serum group)and group B(Yi Gu Tang granule drug-containing serum bone soup+DKK1 group),group C(blank control group),group D(Yi Gu Tang granule drug-containing serum bone soup+Noggin group),A total of four groups,with Yi Gu Tang granule drug-containing serum and normal saline respectively to lavage of ABD and C group,the preparation of medicated serum group and blank control group serum.(2)the cultivation of newborn SD rats skull osteoblasts.The newborn SD rats skull by enzyme digestion method to produce the original generation of osteoblast,randomized to the ABCD four groups,take the third generation of osteoblasts after wedding to foster,after 24 hours of training,respectively,to join the concentration of 10%Yi Gu Tang granule drug-containing serum,10%of the profit of Yi Gu Tang granule containing medicine serum+0.2 ug/mL DKK 1,blank serum,and 10%of the profit Yi Gu Tang granule containing medicine serum+0.2 ug/mL Noggin,continue to cultivate 72 hours.Determined by MTT method,alkaline phosphatase(ALP)activity test,observation osteoblast proliferation of osteoblast mineralization condition,application westblot method and rt-pcr detection of double signaling pathways in Wnt,betacatenin,BMP-2,Runx2,Osx protein gene expression.Result:(1)by the enzyme digestion method of groups of osteoblast after 14 D train tested ALP activity to some extent,of which the most obvious group A,its alkaline phosphatase positive area reached 85%,more than,group B compared with group A and group D is low.Alizarin red mineralized nodules staining results also show that after staining positive nodes by up to A group.(2)through the determined by MTT method dynamic observations each group of osteoblasts in 24 h,48 h,72 h,96 h osteoblast proliferation regularity,48 and 96 h osteoblast a logarithmic growth,gradually after 96 h osteoblast proliferation is restrained.And in logarithmic growth in group A than in group C,the difference was statistically significant(P<0.05),group B and D group were lower than group A(good bone soup medicated serum group),the difference was statistically significant(P<0.05).(3)the Wnt/beta-catenin signaling pathway related to the influence of protein content:compared with group B and D groups,group A Wnt,beta-catenin,the expression of BMP-2,Runx2,Osx protein increases obviously,there are significant difference statistically significant(P<0.01),group C Wnt,beta-catenin,BMP-2,Runx2,Osx protein expression is lower than in group A,there are significant difference(P<0.01).(4)Wnt,beta-catenin,BMP-2,Runx2,Osx fluorescence quantitative PCR amplification curve analysis showed that the gene of fluorescent quantitative RT-PCT are specific amplification;At the same time,the fluorescence quantitative rt-pcr product curve analysis also showed that the dissolution of all the genes of fluorescence quantitative rt-pcr for the dissolution of a single peak.(5)each Wnt,beta-catenin,BMP-2,Runx2,Osx expression quantity:the influence of group C Wnt,beta-catenin,BMP-2,Runx2,Osx gene expression is significantly lowered,group A compared with group B and D groups,group A Wnt,beta-catenin,the expression of BMP-2,Runx2,Osx gene significantly raised,with significant difference statistically significant(P<0.01);Conclusions:Yi Gu Tang granule drug-containing serum can obviously promote the osteoblast proliferation and have a positive effect on the bone mineralization deposition and favorable for the formation of bone,so as to achieve the purpose of prevention and treatment of osteoporosis,and classic Wnt/BMP-2 pairs of signaling pathways is a main mechanism of the change. |
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