Study On Tonifying Kidney And Promoting Blood Circulation To Prevent Traumatic Femoral Head Necrosis By CircRNA-25487

Objective: To study the relationship between kidney-invigorating and blood-activating drugs and CircRNA-25487 and the mechanism of synergistic regulation of h BMSCs.Observe whether it is possible to promote the osteogenic differentiation of h BMSCs by regulating the expression level of CircRNA-25487.Treatment TIONFH provides new and effective methods.Method:1.When performing artificial total hip arthroplasty in TIONFH patients who signed an informed consent form and 6 patients with no femoral neck fracture,necrosis was performed,and 10 ml of autologous bone marrow fluid was extracted respectively,and h BMSCs were extracted,separated,and separated by density gradient centrifugation.Purified and subcultured at a ratio of 1: 3,and observed the cell growth status and morphological changes on the 3rd and 12 th days after the cultivation with an optical microscope.2.Select the third-generation h BMSCs and inoculate cells in 12-well or 96-well plates.Divided into two groups: experimental group and control group.The experimental group was h BMSCs of TIONFH patients,and the control group was h BMSCs of patients with no fracture and necrosis.They were cultured with PRMI1640 complete medium,and the alizarin red staining was used to detect the osteogenic differentiation ability.The osteogenic activity of ALP stained cells was detected by QPCR on the 3,6,and 8 days of osteogenic induction.The c RNA expression levels of osteogenic genes BMP2,Runx2,Osterix,ALP,and OPN in the two groups were detected.Western blotting detected the two groups after 8days of osteogenic induction Group of related osteogenic genes: BMP2,Runx2,Osterix,ALP,OPN,GAPDH(as reference protein).According to the results of pathological staining and detection of osteogenic genes,the relationship and mechanism of CircRNA-25487 expression level and h BMSCs osteogenic differentiation were analyzed.3.Select the third-generation h BMSCs cells of well-grown TIONFH patients for testing,inoculate the cells in 12-well or 96-well cell culture plates,and culture them with PRMI1640 complete medium.Divided into inducer group and inducer + FHNHC group: add osteoblast inducer(2m L per well),β-glycerol sodium phosphate,dexamethasone and ascorbic acid;inducer + kidney activating blood Chinese medicine: add FHNHC(400ug / m L)on the basis of the inducer group.ALP staining was performed on the 3rd,6th and 8th days after osteogenic induction to test the osteogenic activity.Alizarin red staining was used to observe the osteogenic differentiation of the cells,and the inverted microscope was used to observe the staining of the two groups of cells.Western blotting was used to detect the expression levels of related osteogenic genes BMP2,Runx2,Osterix,ALP,OPN,and GAPDH(as reference proteins)after 8 days of osteogenic induction intervention;QPCR was performed on days 3,6,and 8 after osteogenic induction The expression levels of CircRNA-25487 in two groups were detected.Result:1.The cell morphological characteristics of h BMSCs extracted in this experiment at various stages such as isolation,culture and passage are consistent with the bone cell map.The extracted h BMSCs gradually adhered after culturing for 24 h,and almost adhered after 3 days,and gradually fused to about 80%-90% after 12 days,with typical "paving stone"-like changes.The adherent cells grow closely,such as long,short fusiform,oval,etc.,with a certain direction,the whole is arranged in a colony-like,swirling arrangement,which is consistent with the characteristics of h BMSCs cell morphology.2.The relationship between the expression level of CircRNA-25487 and h BMSCs osteogenic proliferation and differentiation;(1)Alizarin red staining results: after osteogenic induction,the number of calcified nodules in the Normal group is more obvious than that in the ONFH group;(2)ALP staining results showed that the ONFH group had higher ALP activity than the Normal group;(3)Denatured agarose gel electrophoresis to detect RNA:electrophoresis results showed good RNA integrity;(4)Fluorescence quantitative PCR detection to detect gene expression: amplification-related Bone gene primers such as BMP2,Runx2,Osterix,ALP,OPN,GAPDH;(5)QPCR detection of c RNA osteogenic genes: 3,6,and 8 days after osteogenic induction,BMP2,Runx2,Osterix,ALP,OPN,etc.of Normal group The expression of c RNA related to osteogenesis was higher than that in the ONFH group,and the difference was statistically significant(P <0.05);(6)Western blotting measured the protein expression of each group: BMP2,Runx2,Osterix,ALP,OPN measured by the ONFH group The expression of other related osteogenic genes was lower than that of Normal group.3.The effect of Bushen Huoxue drugs on the expression level of CircRNA-25487 signal and osteogenic differentiation in h BMSCs:(1)Alizarin red staining results: the number of calcified nodules in the inducer + FHNHC(400ug / m L)group was significantly higher than that of the inducer Group;(2)ALP staining results showed that after 3,6,and 8 days of osteogenic induction intervention,the ALP activity of the inducer group was lower than that of the inducer + FHNHC(400ug / m L)group;(3)QPCR detection of CircRNA-25487 Expression level: After 3,6,and 8 days of osteogenic induction intervention,the expression level of CircRNA-25487 in the inducer + FHNHC(400ug / m L)group was significantly lower than that in the inducer group,and the difference was statistically significant(P <0.05);(4)Western blotting to measure the protein expression of each bone gene: after 8 days of osteogenic induction intervention,the expression levels of BMP2,Runx2,Osterix,ALP,OPN and other osteogenic genes in the inducer + FHNHC(400ug / m L)group were significantly Higher than the inducer group.Conclusion:1.The osteogenic proliferation and differentiation ability of h BMSCs in patients with TIONFH and patients with non-necrosis of femoral neck fracture were negatively correlated with the level of CircRNA-25487 expression.2.Observed by alizarin red staining and ALP staining,it can be concluded that the traditional Chinese medicine for tonifying the kidney and promoting blood circulation can improve the osteogenic differentiation and osteogenic activity of h BMSCs.3.Bushen Huoxue Chinese medicine can regulate the proliferation and differentiation ability of h BMSCs osteoblasts by regulating the expression level of CircRNA-25487 in h BMSCs.