| Bone defects and nonunion have always been important problems for clinicians suffering from orthopedics.With the incidence of severe fractures increasing significantly,the problems of bone defects and nonunion caused by it are becoming more and more serious.Therefore,it is particularly necessary to study the mechanism of promoting bone repair and shortening the time of fracture healing.The method of activating blood circulation to remove blood stasis in Chinese medicine has a significant positive effect in the treatment of fracture healing,and a large number of experiments in the early stage have confirmed that the blood circulation and phlegm-removing safflower can promote the migration of bone marrow mesenchymal stem cells,and thus has a significant effect on the treatment of fracture healing.In order to explore the germination of bone marrow mesenchymal stem cells to promote the molecular biological mechanism,this experiment through a series of screening found that safflower effective monomer palmitic acid has a significant role in promoting the migration of bone marrow mesenchymal stem cells,select safflower The effective monomer palmitic acid was further studied to investigate the effect of palmitic acid on the migration of bone marrow mesenchymal stem cells.At the same time,relevant experimental studies were carried out to confirm whether palmitic acid promoted osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells.Purpose:To observe the effects of safflower effective monomeric palmitic acid on migration,osteogenic differentiation and chondrogenic differentiation of bone marrow mesenchynal stem cells,and to explore the molecular biological mechanism of palmitic acid on bone marrow mesenchymal stem cells migration and differentiation.Therefore,it provides basic experimental research basis for palmitic acid to promote fracture healing,lays a foundation for the development of new drug products,and declares patents.Method:1.The whole bone marrow mesenchymal stem cells of SD rats were cultured by whole bone marrow perfusion method.Due to the characteristics of cell adherent growth,the purity of the cells was continuously improved by the adherence screening method,and the third generation cells with high purity and good state were taken.Cell surface antigen flow detection and induction of osteogenic,adipogenic and chondrogenic triad differentiation.2.Thiazole blue(MTT)colorimetric assay for the effect of palmitic acid on the proliferation of bone marrow mesenchymal stem cells.3.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the expression of CXCR4 gene in the migration of bone marrow mesenchymal stem cells by palmitic acid and the expression of CXCR4 gene in palmitic acid after CXCR4 silencing.4.In vitro migration assay(Transwell)was used to detect the effect of palmitic acid on the migration of bone marrow mesenchymal stem cells and cell migration under the intervention of palmitic acid after CXCR4 silencing.5.Western blot(Western Blot)was used to detect the expression of CXCR4 protein in the migration of bone marrow mesenchymal stem cells by palmitic acid and the expression of CXCR4 protein in palmitic acid after CXCR4 silencing.6.Alizarin red staining was used to detect the effect of palmitic acid on osteogenic differentiation of bone marrow mesenchymal stem cells.7.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the effects of palmitic acid on the expression of osteogenic and chondrogenic differentiation genes(ALP,OPN,RUNX2,SOX9,COL2,ACAN)in bone marrow nesenchymal stem cells.Result:1.Cell identification results:The results of detecting cell surface antigen markers showed that the positive rates of CD105,CD29,CD44,CD73 and CD90 were 98.62%,99.20%,98.82%and 98.57%,respectively,and the positive rates were all greater than 95%.The positive rates of CD34 and CD45 for expressing hematopoietic cells were 0.40%and 0.10%,respectively,and the positive rates were less than 5%.At the same time,the results of three-dimensional differentiation of cells:under the intervention of osteogenic,adipogenic and chondrogenic induction fluids,cells can achieve osteogenicdifferentiation,adipogenic differentiation and chondrogenic differentiation.It was confirmed that the cells used in this experiment were all bone marrow mesenchymal stem cells.2.MTT colorimetric cell proliferation results:under the intervention.of palmitic acid gradient concentration,each group of bone marrow mesenchymal stem cells showed different degrees of cell proliferation,and under different intervention time(24h,48h,72h)Maintain different degrees of proliferation.The optimal intervention time was 48h,the optimal concentration was 10?g/mL,and the growth rate was 29.5%.Compared with the blank group,cell proliferation under the intervention of palmitic acid was statistically significant(P<0.01).It can be seen that palmitic acid can promote the proliferation of bone marrow mesenchymal stem cells.3.Transwell cell migration test results:Under the intervention of 10h palmitic acid gradient concentration,each group of bone marrow mesenchymal stem cells showed different degrees of cell migration.Among thenb the amount of submedullary cells of bone marrow mesenchymal stem cells increased significantly under the intervention of 10?g/mL palmitic acid.Compared with the blank group,the difference was highly statistically significant(P<0.01).Under the intervention of 5?g/mL and 20?g/mL palmitic acid,the number of submucosal cells of bone marrow mesenchymal stem cells decreased significantly,but still more than the blank group.Compared with the blank group,the difference was highly statistically significant(P<0.01).).It can be seen that palmitic acid has a positive effect on the migration of bone marrow mesenchymal stem cells.4.RT-PCR gene expression results:Under the different concentration gradients of palmitic acid,the expression of CXCR4 messenger ribonucleic acid(mRNA)can be increased to different extents,especially the concentration of 10?g/mL is the best,the difference is highly statistical.Academic significance(P<0.01).It can be seen that under the intervention of palmitic acid,the expression of CXCR4 gene in bone marrow mesenchymal stem cells is significantly improved.5.The expression of RT-PCR gene after CXCR4 silencing results:10?g/mL palmitic acid significantly promoted the expression of CXCR4 gene in silencing cells,which was statistically significant(P<0.05).It can be seen that under the intervention of palmitic acid,bone marrow mesenchymal stem cells partially restore CXCR4 gene expression.6.Transwell cell migration assay after CXCR4 silencing:10?g/mL palmitic acid significantly promoted the migration of BMSCs after silencing of CXCR4.Compared with the blank control group,the palmitic acid+slow group was not statistically significant(P>0.05).Compared with the silent group,the palmitic acid+slow group had a statistical significance of P<0.01.It can be seen that under the intervention of palmitic acid,the bone marrow mesenchymal stem cells partially restore the migration ability of the cells.7.Western blot immunoblot results:10?g/mL palmitic acid significantly promoted the expression of CXCR4 protein in bone marrow mesenchymal stem cells after CXCR4 silencing.Compared with the blank control group,the palmitic acid+slow group was not statistically significant(P>0.05).The palmitic acid+silencing group was highly statistically significant compared with the silent group(P<0.01).It can be seen that under the intervention of palmitic acid,bone marrow mesenchymal stem cells partially restore CXCR4 protein expression.8.Palmitate+osteogenic induction,alizarin red staining and reverse transcription-polymerase chain reaction(RT-PCR)results:compared with the induction group,palmitic acid+induction group on the expression of RUNX2 gene in bone marrow mesenchymal stem cells It has a significant promoting effect,especially at the concentration of 20?g/mL,which is statistically significant(P<0.05).It can be seen that under the intervention of palmitic acid,bone marrow mesenchymal stem cells have a tendency to differentiate into osteoblasts.9.Reverse transcription-polymerase chain reaction(RT-PCR)results of palmitic acid+chondrogenic induction:Compared with the induction group,the palmitate+induction group significantly promoted the expression of SOX9 gene in bone marrow mesenchymal stem cells.Especially at the optimal concentration of 20?g/mL,and compared with the induction group,it was statistically significant(P<0.05).It can be seen that under the intervention of palmitic acid,bone marrow mesenchymal stem cells have a tendency to differentiate into chondrocytes.Conclusion:The effective monomeric palmitic acid of activating blood and removing peony safflower has positive effects on the proliferation,migration and osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells,which further confirms the significant effect of promoting blood circulation and removing blood stasis on fracture healing.Safflower provides molecular biological basis in the treatment of fracture healing,which lays a foundation for palmitic acid to promote fracture healing.At the same time,the optimization of traditional Chinese medicine design based on the theory of traditional Chinese medicine and blood circulation opens up new therapeutic ideas for accelerating fracture healing,.and provides a research basis for the third-phase differentiation and treatment principle of fractures. |
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