Study On The Method Of Determining The Biological Activity Of The Stock Solution Of Osteopeptide

Objective:In this study,multi-component biochemical bone peptide was used as the research object.Based on the pharmacodynamic study of bone peptide stock solution,the suitable method was selected as the biological activity evaluation method of bone peptide,and then Methodological verification of established bioactivity assay methods was carried out,and determination of the limit dose and quality criteria,so that the quality standards of existing bone peptide drugs are more complete.Methods:Part ? Pharmacodynamic study of bone peptide stock solution I.In vivo pharmacodynamics study(?)Improve osteoporosis in ovariectomized rats The bilateral ovarian rats were used as a model of osteoporosis,non-extracted ovarian rats were set as sham operation group.The model rats were randomly divided into 12 groups according to their body weight: model group,positive control group(estradiol)and test groups(1~10 manufacturers of bone peptides stock solution),8 rats per group.The drug administration period was 12 weeks.The femur was collected for bone mineral density,bone calcium,bone phosphorus and bone pathology.Serum was collected for alkaline phosphatase(ALP)and tartrate-resistant acid phosphatase(TRACP)activity.The mice were randomly divided into 6 groups according to their body weight: a blank group,a positive control group,and a test sample group(one batch of each of the four manufacturers of bone peptide stock solution),and 10 mice per group.After10 minutes of administration,the model of auricle swelling caused by croton oil was applied to the left ear of the mouse,and the right ear was an autologous control.After4 hours,the mice were sacrificed by cervical vertebrae,and the ears were cut.The discs were placed at the same position on the left and right ears with a puncher.The left and right ear differences of each group of mice were weighed and the anti-inflammatory inhibition rate of the test group was calculated.?.In vitro pharmacodynamics and mechanism of action(?)Promoting the proliferation of rat osteosarcoma cells(UMR106)UMR106 cells were selected as the research object,and the bone peptide stock solution was diluted to a concentration of 0.06 ~ 2.0 mg / m L.After 48 h and 72 h,the proliferation of UMR106 cells was determined by CCK-8 method,and the cell proliferation rate was calculated.(?)Preliminary study on the osteogenic differentiation and mechanism of fetal bone marrow mesenchymal stem cells(F-BMSCs)F-BMSCs were selected as the research object to induce osteogenic differentiation..A negative control group,an induction group,and a test group were set.The early osteogenesis index ALP was detected on the 7th day of F-BMSCs induction.On the 14 th day,the advanced osteogenic index was stained with alizarin red mineralized nodules for osteogenic differentiation.After induction of F-BMSCs,m RNA expression levels of osteogenic differentiation-related genes COl2a1,Sox9,ISBP,OPN,OCN,Runx2,ALP at 6th and8 th day were detected by RT-PCR;The expression levels of Runx2 and OCN on the6 th,8th and 10 th day were detected by Western blotting,and the potential mechanism of osteogenic differentiation was analyzed.(?)Anti-inflammatory effects on lipopolysaccharide(LPS)-induced mouse inflammatory model of mononuclear macrophage cell line(RAW264.7)RAW264.7 was selected as the research object,and LPS was used to cause a cellular inflammation model.A blank group,a model group,a positive control group,and a test group were set.After 24 hours of action,the cell supernatant was taken and the secretion of cytokine IL-6 was detected by ELISA.?.Screening of determination methods for bioactivity According to the above-mentioned pharmacodynamic evaluation index and experimental results,according to the requirements of reasonable design,simple operation,low cost,clear detection index,high sensitivity and good reproducibility,the suitable method was selected as the biological activity evaluation method of bone peptide stock solution.Part ? Establishment and validation of a method for determining the biological activity of proliferating UMR106 cells by bone peptide stock solution I.Optimization and determination of experimental conditions for promoting UMR106 cell proliferation The orthogonal experimental design was used to optimize the experimental conditions of UMR106 cell proliferation.According to the experimental results,determine the maximum influencing factors of the experiment,further optimize the experimental conditions,and determine the experimental plan.?.Methodological verification(?)Repeatability : Take the same batch of test samples(manufacturer B,batch:1806004),and four independent tests were repeated,and the results were evaluated.(?)Intermediate precision:Three experimental personnel took the same batch of test samples(manufacturer B,batch: 1806004),and conducted independent tests,and the results were evaluated.(?)Durability:Take the same batch of test samples(manufacturer B,batch:1806004),respectively,to examine some small changes in the experimental conditions,including: different cell plating density: 4500,5000,5500 cells / well;The drug acts on different sites;the CCK-8 reagent is added for different times: 1.5,2,2.5 h;the drug action time: 68,72,76 h.The results of the measurements are not affected to ensure the reliability of the method.?.Determination of UMR106 cell proliferation limit dose and judgment criteria According to the final experimental protocol,13 manufacturers of bone peptide stock solution were determined.According to the experimental results,the limits and criteria for the proliferation of UMR106 cells were determined.The biological activity of the bone peptides was determined.Data statistics: The experimental data were expressed as mean ± standard deviation(x ± s),and statistical analysis was performed using t-test(p < 0.05,the difference was statistically significant),and mapping was performed using GRAPHPAD software.Result:Part ? Pharmacodynamic study of bone peptide stock solution I.In vivo pharmacodynamics study(?)Improve osteoporosis in ovariectomized rats1.Test results of femoral bone density,bone calcium,bone phosphorus Compared with the model group,the bone density of the six manufacturers of J,I,E,D,B and C were increased,with significant difference(p<0.05),four manufacturers of F,A,G and H were increased bone density to some extent,but without significant difference(p>0.05).The bone calcium content of the three manufacturers of I,J,C were increased,with significant differences(p<0.05),seven manufacturers of G,B,E,A,D,H and F increased bone calcium content to some extent,but without significant difference(p>0.05);The bone phosphorus of the manufacturers C were increased,with significant difference(p<0.05).Nine manufacturers of H,D,I,F,J,E,G,B and A increased bone phosphorus to some extent,but without significant difference(p>0.05).2.Test results of bone pathology Compared with the model group,the area,number,and area percentage of the trabecular bone of the manufacturer of D were increased,and the trabecular bone separation were decreased,with significant difference(p<0.05);Nine manufacturers of A,E,C,G,J,H,I,B and F increased the area,number and area of trabecular bone to a certain extent,and reduced the trabecular bone separation degree,but without significant difference(p>0.05).The results of HE staining showed that the trabecular bone status of test groups was better than the model group,and the effect of the manufacturer of D was the most significant.3.Test results of ALP,TRACP activity Compared with the model group,the ALP activity of the six manufacturers of D,A,B,C,I and F were increased,with significant difference(p<0.05),and the four manufacturers of H,E,G and J were increased ALP activity to some extent,but without significant difference(p>0.05);The TRACP activity of the seven manufacturers of H,B,D,G,J,C and A E were decreased,with significant difference(p<0.05).The three manufacturers of E,F and I were reduced TRACP activity to some extent.,but without significant difference(p>0.05).(?)inhibiting the swelling and anti-inflammatory effect of mouse ear caused by croton oil There were not anti-inflammatory effects on the bone peptide stock solution of the four manufacturers of A,B,C and L.?.In vitro pharmacodynamics and mechanism of action(?)Promoting the proliferation of rat osteosarcoma cells(UMR106)The manufacturer of A did not promote proliferation of UMR106 cells;the manufacturers of B,C,L promoted the proliferation of UMR106 cells,and the proliferation of UMR106 cells had a dose-dependent manner in the range of0.06~1.0mg/m L.Therefore,the drug-acting cells were selected for 72 hours as the detection time.(?)Preliminary study on the osteogenic differentiation and mechanism of fetal bone marrow mesenchymal stem cells(F-BMSCs)1.Osteoblast identification1.1 ALP activity On the 7th day after induction of F-BMSCs,compared with the negative group,the ALP activity of the induction group was significantly increased(p<0.05).compared with the induction group,the ALP activity of the manufacturer of C was significantly increased(p<0.05);the manufacturers of B,L was higher than the induction group,but without significant difference(p>0.05).1.2 Alizarin red staining On the 14 th day of F-BMSCs induction,compared with the negative group,the induction group showed positive staining for alizarin red,indicating that the induction was successful;compared with the induction group,the red color of the manufacturers B,C,L was significantly enhanced;Under the microscope,the alizarin red staining positive nodular coverage area of B,C,L was significantly higher than that of the induction group.2.Preliminary study on the mechanism of osteogenic differentiation of F-BMSCs2.1 Test results of RT-PCR On the 8th day after the induction of F-BMSCs,the m RNA expression of the osteogenic differentiation marker gene in the manufacturer of B was increased,and was significantly higher than that in the induction group.2.2 Test results of western blotting On the 10 th day after the induction of F-BMSCs,the expression of the manufacturer of B Runx2 and OCN protein was significantly higher than that in the induction group.(?)Anti-inflammatory effects on LPS-induced mouse inflammatory model of RAW264.7There were not anti-inflammatory effects on the bone peptide stock solution of the four manufacturers of A,B,C and L.?.Screening of determination methods for bioactivity The results of pharmacodynamic experiments showed that the bone peptide stock solution had no anti-inflammatory effect.The advantages and disadvantages of the experimental methods for the pharmacodynamics of bone peptides stock solution in bone and body were compared.In vivo pharmacodynamic experiments required many experimental animals,and the experimental process was cumbersome and did not meet the "3R" principle.In vitro pharmacodynamic experiments,the cost of inducing osteogenic differentiation of F-BMSCs was high,and had many the influencing factors;the cell strains UMR106 cells were easy to obtain,the parallelism was good,the operation was simple,and the cost was low.Since the internal and external pharmacodynamic experiments have good consistency,we selected the UMR106 cell proliferation experiment in vitro to establish a biological activity assay method for bone peptide.Part ? Establishment and validation of a method for determining the biological activity of proliferating UMR106 cells by bone peptide stock solution I.Optimization and determination of experimental conditions for promoting UMR106 cell proliferation According to the experimental results,the determination method of the biological activity of UMR106 cell proliferation experiment is as follows: the concentration of bone peptide stock solution is 1.0 mg/ml,the serum content of the medium is 2.5%FBS,and the number of plated cells is 5000 cells/well.?.Methodological verification(?)Repeatability:Independent experiment four times,the average proliferation rate was 247%,RSD=13%.(?)Intermediate precision:The average proliferation rate of the independent experiment of the three experimental staff was 261%,RSD =16%.(?)Durability:1.The different of cell plating density The average proliferation rate was224%,RSD=2.3%.2.The different of locations The average proliferation rate was214%,RSD=5.3%.3.The different of times(drug)The average proliferation rate was208%,RSD=7.5%.?.Determination of UMR106 cell proliferation limit dose and judgment criteria According to the experimental results of the proliferation rate of UMR106 cells by a total of 39 batches from 13 manufacturers,the drug concentration of 1.0 mg/m L in the experiment of proliferating UMR106 cells was determined as the limit dose,and the proliferation rate was not less than 150%,as a criterion for compliance.The manufacturer pass rate is greater than 75%.Conclusion:In this experiment,the experiment of promoting the proliferation of UMR106 cells was established as a method for determining the biological activity of bone peptide stock solution.The drug concentration of the bone peptide stock solution is 1.0 mg/m L as the limit dose,and the proliferation rate is not less than 150% as the test product meets the prescribed determination standard.The optimal experimental conditions were determined that the serum content of the medium was 2.5% FBS,and the number of plated cells was 5000 cells/ well.