| Objective: The effect of antler polypeptide on the activity of rat bone marrow mesenchymal stem cells was studied by enzymatic ultrafiltration.So as to lay the experimental foundation for the construction of cell scaffold by using antler polypeptide.Method: ① Four kinds of enzymes were selected for enzymatic hydrolysis,qualitative analysis and amino acid analysis.② Preparation of antler polypeptide by star point effect surface design method,The amount of enzyme,substrate concentration,temperature as independent variables,Polypeptide content area as the dependent variable.Respectively,multiple linear regression calculation and binomial equation fitting calculation,combined with the best mathematical model to describe the effect surface.Determine the best technology,and the use of the verification test.③The molecular weight of Mr（800-1500）was extracted by ultrafiltration.The subculture of BMSCs was carried out under sterile conditions.The optimum concentration was selected to investigate the effect of the best concentration of antler polypeptide on the growth,growth cycle and ALP of BMSCs.Result: ① TLC detection of deerhorn glue,the test results meet the Pharmacopoeia standards;by pepsin deerhorn glue,can be obtained with molecular weight of 800-1500 amino acid concentration of the deer antler polypeptide;peptide solution for 21.025mol/m L.② The optimum process conditions were as follows: 4% of the enzyme dosage,substrate concentration of 6%,and temperature of 40℃.The deviation is 1.41%.of the best process is scientific and normative in the best process validation test results,and the predicted values of the binomial fitting equation.③ Ultrafiltration extracted from antler polypeptide fragments with molecular weight of 800-1500;BMSCs grew well;when the antler polypeptide fragments（molecular weight 800-1500）concentration of1.261×10-1g/m L,the cell proliferation rate was 45.84%,promote cell proliferation was better than the rest of the concentration of the sample;antler polypeptide fragments （molecular weight 800-1500）significantly increased the rat bone marrow.Mesenchymal stem cells ALP,compared with the control group with a very significant difference（P<0.001）;flow cytometry showed that the antler polypeptide fragment group（molecular weight 800-1500）on osteoblast proliferation index was 38.68%,significantly better than the other groups.Conclusion: The pepsin antler glue is better than other three kinds of enzymes;the ccd-rsm optimization of antler polypeptide preparation,the result is relatively stable,predictable strong,simple and rational;antler polypeptide fragments(molecular weight800-1500,the concentration of 1.261×10-1g/m L)has contributed to the ossification effect on BMSCs. |
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