| Objective:1.In vitro culture obtained genetic stability,phenotypic consistent bone marrow mesenchymal stem cells(BMSCs),and had its' surface antigen biological identification.2.Intervene cells with Bakuchiol to observe their effect on cell proliferation,so as to obtain the safe concentration of Bakuchiol.3.To observe the effect of Bakuchiol on the migration of BMSCs in vitro,and to further explore the relationship between Bakuchiol and the non-classical pathway wnt5a/JNK of BMSCs during migration in vitro.Method:1.The whole bone marrow adherence method was used to train the BMSCs of Sprague Dawley(SD)rats,and the 3rd generation(Passage 3),which had good growth status,was detected by detection of cell surface antigen and three-line differentiation induction(osteogenesis differentiation,edipogenic differentiation,chondrogenic differentiation).2.Cell counting Kit-8(CCK8)method was used to screen the toxicity concentration of Bakuchiol.The next experiment was carried out by selecting concentrations that did not affect BMSCs proliferation.3.The effects of different concentrations of Bakuchiol on the migration of Bmscs cells were observed by cell migration experiments.4.Expression of Wnt5a protein in BMSCs after Bakuchiol intervention using Western-blot and cellular immunofluorescence method.5.Using sh-Wnt5a slow virus vector knockout Wnt5a gene in BMSCs,the expression of green fluorescent protein and polymerase chain locking reaction(PCR)were used to detect infection efficiency.Get sh-wnt5a Bmscs cells.6.The optimal migration concentration of Bakuchiol was selected to intervene cells.Transwell assay was used to compare the ability of migration in vitro.Subsequently,the expression of Wnt5a/JNK and its related protein gene was detected by PCR,and the reliability of the experimental results was verified by Western-Blot from protein level.Results:1.Flow cytometry detection of cell surface antigen markers showed positive expression of CD90,CD44 and CD29,the positive rate was 98.58%,95.50%,97.63%,CD11BC,CD34,CD45 expression negative,the positive rate was 0.47%,0.31%,0.24%,respectively.The results of three-line induction and differentiation showed that the cells used in the experiment had the potential of osteogenesis,adipogenic and chondrogenic differentiation,which further proved that the cells isolated and cultured in vitro were bmscs.2.Using different concentrations of gradient Bakuchiol to stimulate cells,it is concluded that high concentration psoralin(100?g/ml)inhibits BMSCS proliferation,the results are statistically significant,the concentration of less than or equal to 50?g/ml Bakuchiol does not affect BMSCS proliferation.The range of drug concentration without obvious inhibition of cell proliferation was obtained,and the concentration gradient was established to carry out cell migration experiment.3.The results of cell migration Transwell showed that compared witih the blank control group,25?g/ml was the best to promote the migration,the results were statistically significant(P<0.01),and 12.5?g/ml Bakuchiol also promoted BMSCs in vitro migration(P0.05).The rest of the concentration of Bakuchiol can also promote BMSCs in vitro migration,but the results are not statistically different.4.After using virus transfection cells,the positive expression of green fluorescent protein was observed under microscope,and the PCR results showed that Wnt5a expression decreased significantly in sh-Wnt5a BMSCs,and the results were statistically different(P<0.05).5.The intervention BMSCs with 25?mol/ml Bakuchiol and sh-Wnt5a BMSCS,PCR and WB showed that the expression of Wnt5a and JNK protein in the intervention group was higher than that of the blank complete medium group.Conclusion:Bakuchiol does not promote the proliferation of BMSCs,and the high concentration of Bakuchiol has cytotoxicity.Low concentrations of Bakuchiol can enhance the Bmscs migration capacity,possibly by activating the Wnt5a/JNK signaling pathway to promote cell displacement. |
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