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Objective To find the seed cells for replacing bone marrow mesenchymal stem cells(BMSCs) to treat bone defects by compared the capacity of osteogenesis from the BMSCs, human umbilical cord mesenchymal stem cells(UC-MSCs) and human placental mesenchymal stem cells(P-MSCs). Methods Three MSCs were cultured in DMEM/Ham's F-12 medium containing 10% fetal bovine serum,cell proliferation curve was drawn by CCK8 detec-ted,and the cell surface antigens were measured using flow cytometry. Osteogenic ability was confirmed by the al-kaline phosphatase(ALP) staining,alizarin red staining. To further explore the difference in organic components, their underlying genotypes and proteins, including RUNX2, ALP and osteocalcin(OCN), were analyzed by RT-qPCR and Western blot. Results Cell growth curve analysis indicated that three MSCs were in the exponential stage at 3 days following incubation. Flow cytometry analysis showed that more than 98% cells were positive for CD44, CD90 and CD105. ALP and Alizarin red staining displayed that three MSCs presented good mineralizationg ability by osteogenesis induced medium. RT-qPCR and Western blot showed that the three MSCs experiment group higher expression levels of osteogenic markers including RUNX2, ALP and OCN during the initial 9 and 18 days when compared with control(P<0.05). Conclusions UC-MSCs and P-MSCs have good osteogenic ability, which may function as a potential bone tissue engineering seed cells for the treatment of bone defects.
RESUMO
Objective To find the seed cells for replacing bone marrow mesenchymal stem cells(BMSCs) to treat bone defects by compared the capacity of osteogenesis from the BMSCs, human umbilical cord mesenchymal stem cells(UC-MSCs) and human placental mesenchymal stem cells(P-MSCs). Methods Three MSCs were cultured in DMEM/Ham's F-12 medium containing 10% fetal bovine serum,cell proliferation curve was drawn by CCK8 detec-ted,and the cell surface antigens were measured using flow cytometry. Osteogenic ability was confirmed by the al-kaline phosphatase(ALP) staining,alizarin red staining. To further explore the difference in organic components, their underlying genotypes and proteins, including RUNX2, ALP and osteocalcin(OCN), were analyzed by RT-qPCR and Western blot. Results Cell growth curve analysis indicated that three MSCs were in the exponential stage at 3 days following incubation. Flow cytometry analysis showed that more than 98% cells were positive for CD44, CD90 and CD105. ALP and Alizarin red staining displayed that three MSCs presented good mineralizationg ability by osteogenesis induced medium. RT-qPCR and Western blot showed that the three MSCs experiment group higher expression levels of osteogenic markers including RUNX2, ALP and OCN during the initial 9 and 18 days when compared with control(P<0.05). Conclusions UC-MSCs and P-MSCs have good osteogenic ability, which may function as a potential bone tissue engineering seed cells for the treatment of bone defects.
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Objective:To construct a eukaryotic expression vector of leptin gene,and to transfect it into human placental mesenchy-mal stem cells(HPMSCs)and appraise its expression.Methods:Primers were designed and the leptin gene was obtained by RT-PCR from human adipose tissue.The aimed segments were inserted into the eukaryotic expression vector pIRES2-EGFP,plasmid pIRES2-EGFP-LEP was constructed and identified by restricted enzymatic resection,and then transfected it into HPMSCs by liposome.The ex-pression of leptin in the transfected cells was detected by RT-PCR and Western blotting,the multi-differentiation potential of the cells was indentified.Results:The length of specific fragment was 500 bp,the recombinant plasmid pIRES2-EGFP-LEP presented 5.3 kb and 500 bp bands by restriction enzyme digestion.Leptin gene was expressed in transfected HPMSCs and the transfected HPMSCs maintained multi-directional differentiation potential.Conclusion:The eukaryotic expression vector of leptin can be transfected and ex-pressed in HPMSCs.