| Objective1.Observe the effect of Guben Zenggu Capsule drug-containing serum on the osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs),and detect the expression of related proteins,and preliminary explore the effect of drug-containing serum on BMSCs.2.To detect the expression of genes related to Wnt/?-catenin signaling pathway and to explore the mechanism of Guben Zenggu capsule drug-containing serum treatment of osteoporosis based on Wnt/?-catenin signaling pathway.The target provides experimental evidence.Method1.Prepare Guben Zenggu capsule drug-containing serum;Set control experiment,MTT method to measure the proliferation rate of rat BMSCs cells cultured with different concentrations of drug-containing serum;Alizarin red staining to observe the degree of mineralization;Western-blot technology to detect type I The expression of collagen-I,bone-specific transcription factor(Runx2)and osteocalcin(BGP)were investigated to investigate the effects of different concentrations of Guben Zenggu capsule drug-containing serum on osteogenic differentiation of rat BMSCs.2.Real-time PCR was used to detect the mRNA expression ofLRP-5,?-catenin,LEF-1,and Runx2 genes related to Wnt/?-catenin signaling pathway and to explore the Wnt/?-catenin signal of Guben Zenggu capsule drug-containing serum.Pathway treatment of osteoporosis mechanism of action;through the above-mentioned experimental study for Guben Zenggu capsule clinical treatment of OP provides a theoretical basis.Result1.Subculture of Wistar rat BMSCs:After the resuscitation of P1 Wistar rat BMSCs,the cells showed a short spindle-shaped growth.After 5 days of culture,the cells gradually became dispersed colony and grew to form small clones;BMSCs were subcultured for 12 hours.After24 hours of culture,the cells were needle-like spindle-shaped and homogeneously distributed.After 4 to 5 days of culture,the degree of cell fusion reached more than 80%,and they grew in groups.2.The MTT method was used to measure the cell proliferation rate:There was a statistically significant difference in OD values between different time points(P<0.05).From the time point perspective,72 h,96h,and 120 h OD values were different from 24 h and 48 h OD values.Significance(P<0.05),and the former was higher than the latter;from the perspective of intervention measures,the OD values of 10%,20%,and30% drug-containing serum groups were different from those of other groups.Both were statistically significant(P<0.05).The OD values of the former were higher than the latter and were highest in the 20% drug-containing serum group.There was no significant difference in OD values between the other groups(P>0.05).The other groups were statistically significant(P<0.05),suggesting that the 20%drug-containing serum group had the best effect,and after 96 hours of action,the cell proliferation was the strongest.3.Observation of alizarin red staining:After 28 days of osteogenic induction culture,the serum containing drug-containing serum(5%,10%,20%)increased with increasing concentration,and the number of red calcium nodules also gradually increased,especially at a concentration of 20 At %,the number of red calcium nodules was the most significant and significantly higher than that of other groups.When the concentration continued to increase to30%,the number of calcium nodules showed a downward trend.4.Detection of Osteogenesis-Associated Protein:Western-blot showed that the expression of collagen-I,BGP,and Runx2 was positively correlated with the concentration when the concentration of drug-containing serum was 5%,10%,and 20%,especially when the concentration was 20%.The protein expression level showed a peak value,which was significantly higher than that of the blank group and the traditional induction group,and the difference was statistically significant(P<0.05).When the serum concentration of the drug-containing serum continued to increase to 30%,the expression of osteogenic related proteins declined.5.Osteogenesis-related gene detection:Real-time PCR results showed that Wnt/?-catenin signaling pathways LRP-5,?-catenin,and the concentrations of serogroups containing sera were 5%,10%,and 20%.The expression of LEF-1 and Runx2 mRNA showed a significant increase trend,of which the 20% drug-containing serum had the best effect and the mRNA expression was highest,which was statistically significant compared with other groups(P<0.05);when the serum concentration continued to increase to 30%,the mRNA expression decreased.Conclusion1.The serum containing Guben Zenggu Capsule can obviously promote the osteogenic differentiation and mineralization ability of rat BMSCs and promote the formation of calcium nodules;and the concentration of 20% of drug-containing serum has the best effect,and the cell proliferation is the strongest after 96 hours.2.Guben Zenggu capsule drug-containing serum can promote the expression of osteogenic-related proteins collagen-I,BGP,Runx2 and significantly up-regulate Wnt/?-catenin signaling pathway LRP-5,?-catenin,LEF-1,Runx2 mRNA Expression ultimately promotes bone formation. |
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