Investigating The Effects Of Gu Ben Zeng Gu Capsule Contained With Medicated Serum On Rat’s Bmscs Proliferation And Osteogenic Differentiation By Combining With BMPs Signaling Pathway

1 Purpose1.1 To observe the effects of different concentrations Guben Zenggu capsule drug-containing serum on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs),intracellular distribution and cell biological behavior;detection of Guben Zenggu capsule drug-containing serum osteogenic related protein The expression of genes associated with BMPs signaling pathways;explore the mechanism of Guben Zenggu capsule in the treatment of OP.1.2 To provide a theoretical reference for the clinical application of Guben Zenggu Capsule.2 Method2.1 Rat BMSCs were cultured and passaged to prepare different concentrations of Guben Zenggu capsule drug-containing serum(5%,10%,20%),set the control group,and measured the proliferation rate of rat BMSCs by MTT after administration;Alkaline phosphatase staining was used to observe the osteogenic differentiation of BMSCs.2.2 Using ELISA technology to detect the concentration of osteogenic related proteins:type I collagen,alkaline phosphatase,osteocalcin,osteopontin.To investigate the effect of serum containing Guben Zenggu capsule on osteogenic differentiation of rat BMSCs.2.3 Real-time PCR technique was used to detect the gene expression levels of BMP2,BMP4,Smad1,and RUNX2 in BMPs related to osteoblasts,and the activation of BMPs signal was investigated to investigate the effect of Guben Zenggu capsule drug-containing serum on osteogenic differentiation.Mechanism.3 Results3.1 Successful passage of Wistar rat BMSCs.3.2MTT assay cell proliferation rate: There was a statistically significant difference in MTT between groups at different time points(P<0.05).From the time point perspective,there was a statistically significant difference in MTT between 96 h and 120 h and 24 h,48h,and 72h(P<0.05),and the former were all higher than the latter;from the perspective of intervention measures,the MTT of the 10%,20%,and 30% drug-containing serum groups was statistically different from the other groups(P<0.05).MTT was higher than the latter,and the 20%drug-containing serum group was the highest.There was no significant difference in the MTT between the other groups(P>0.05).The other groups had statistical significance(P<0.05).Tip 20% drug-containing serum group has the best effect.The detailed results are shown in Table 1 and Figure 2.3.3 Alkaline phosphatase staining: After staining with alkaline phosphatase,cells of different Guben Zenggu capsule containing serum and traditional induction group showed blue-violet cell aggregation area.In the drug-containing serum group(5%,10%,and 20%),the blue-violet region gradually became denser as the concentration increased,and when the concentration reached 20%,the staining area was the most dense and significantly higher than the blank group and the traditional osteogenic induction group.When the concentration increases to 30%,the staining area decreases.With the increase of drug concentration,osteogenic differentiation of BMSCs was increased,and osteogenic differentiation was best at20% concentration.Detailed results are shown in Figure 3.3.4 Osteogenesis-associated protein detection: The 5%,10%,and 20% drug-containing serum groups had significant differences in osteocalcin,osteopontin,alkaline phosphatase,type I collagen,and other groups.(P<0.05),and 5%,10%,20% drug-containing serum histone concentrations were higher than other groups,while 30% drug-containing serum group,blank group,and traditional induction of histone protein concentration had no significant difference.Among them,the concentration of histones in the 20% drug-containing serum was the highest,suggesting that the 20% drug-containing serum group had the best results.The detailed results are shown in Table 2-5 and Figure 4-7.3.5 Detection of BMP Signal Pathway Related Genes: Real-time PCR results showed that the expression levels of BMP2,BMP4,Smad1,and RUNX2 genes in the 10%,20%,and30% drug-containing serum groups were statistically significant compared with other groups(P<0.05),and the levels of BMP2,BMP4,Smad1,and RUNX2 in the 10%,20%,and 30%drug-containing serum groups were higher than those in other groups,while the expression levels of related genes in the 30% drug-containing serum groups,blank groups,and traditional induction groups.No significant difference.The 20% drug-containing serum group had the highest gene expression level,suggesting that the 20% drug-containing serum group had the best results.The detailed results are shown in Table 6-9 and Figure 8-11.4 Conclusion4.1 Guben Zenggu Capsule drug-containing serum can significantly promote the osteogenic differentiation of rat BMSCs.4.2 Guben Zenggu capsule containing serum can increase the concentration of osteocalcin,osteopontin,alkaline phosphatase,type I collagen;4.3 Guben Zenggu capsule drug-containing serum can significantly up-regulate the expression of BMP2,BMP4,Smad1,and RUNX2 mRNA and promote bone formation.