Study On Factors Related To EPCs Proliferation And Differentiation Affected By BMSC

OBJECTIVE: In this study,bone marrow-derived endothelial progenitor cells(EPCs)and bone marrow mesenchymal stem cells(BMSCs)were used as research cells,and the two cells were contactlessly co-cultured to verify that BMSCs have To promote the proliferation and tube formation of EPCs in vitro,and to study the expression of VEGFA,b-FGF and other cytokines and TRPC channels in EPCs,and further elucidate the role of BMSCs in promoting the proliferation and differentiation of EPCs and the related factors Expression provides a theoretical basis for follow-up experiments.Methods: 1.Isolation,culture and identification of canine bone marrow-derived EPCs and BMSCs: Healthy hybrid puppies were used for 1 to 2 months of age.After anesthesia,bone marrow puncture was performed on bilateral iliac bones and 3 ml bone marrow was collected.The canine bone marrow EPCs were isolated and cultured using canine lymphatic separation gradient centrifugation and conditional induction medium.BMSCs were isolated by differential adherence method.The morphology of the two cells was observed and recorded by inverted phase contrast microscopy,and EPCs were induced into tubes in vitro.BMSCs induced osteogenic experiments and flow cytometry to identify cell surface markers to identify cells.2.Proliferation and tube formation experiments of EPCs under co-culture system: Two experimental groups and one control group were set up.BMSCs and EPCs second generation cells were collected in a co-culture group.Transwell was used for non-contact co-culture.Induction group was used for induction medium.The group used control medium.Through the use of CCK-8 to detect the proliferation of three groups of EPCs and draw the growth curve,through the Matrigel tubing experiments to compare the ability of three groups of EPCs into the tube,verifying that BMSC has the biological functions to promote the proliferation and differentiation of EPCs.3.The expression of EPCs cytokines and TRPC channels in co-culture system: The co-culture group,the induction group and the control group were established,and three groups of VEGFA,b-FGF,CD133,CD34,TRPC1,and TRPC4 immunohistochemical staining were performed.The average optical density of each group was measured and compared.The total RNA extracted from EPCs from each group at 1d,3d,and 5d time points was collected and qRT-PCR was used to detect the expression level of the above target genes and compare them.Results: 1.In vitro isolation and culture of canine EPCs and BMSCs and their identification: The number of cells isolated from EPCs by density gradient centrifugation was small and the purity was high.After 5 days of primary culture,colony-like growth cells were seen under the EPCs microscope.The cell morphology was polygonal or long.Strips.After prolonged culture time,the cells showed a typical “paving stone” or “pebble” type;after 12 hours of tube-induction experiments,EPCs were observed to form a tube-like structure in a cord-like manner;the second-generation cells were detected by flow cytometry.CD133 and CD34 were positive.The BMSC primary cells collected by differential adherent method are more.After 4 hours of primary culture,BMSCs began to adhere to the wall,and after 5 days,the adherent cells were seen under the microscope and the cell morphology was seen as a strip or a horn.After twenty-one days of osteogenic differentiation test,calcium nodules were seen and alizarin red staining was positive.Flow cytometry showed CD29 positive and HAL-DR negative.2.Using CCK-8 to detect cell proliferation and draw the growth curve,the logarithmic phase of EPCs growth was earlier than that of the other two groups under the conditions of co-culture of BMSC and EPC.There was a statistically significant difference between the co-culture group and the other two groups at 4 days(P<0.05).<0.05),there was a significant difference between the co-culture group and the control group after 4 hours(P<0.05),and there was a statistically significant difference between the co-culture group and the other two groups after 8 hours(P<0.05).3.Immunohistochemistry results of VEGFA,b-FGF,and TRPC1 in the co-culture group were positive,and the difference between the two groups was statistically significant(P<0.05).The difference between the TRPC4 co-culture group and the control group was statistically significant(P<0.05).).After 1d of qRT-PCR,there was no significant difference in target genes between EPCs.After 3 days of culture,differences in target genes began to appear.After 5 days of culture,the VEGs of EPCs were co-cultured.Conclusion:1.BMSCs can promote the proliferation and tube formation of EPCs in vitro.2.EPCs are highly expressed by VEGF-BMSCs in vitro promote the expression of VEGFA,b-FGF,TRPC1,TRPC4,through the expression of these target genes can promote the biological function of EPCs.The co-culture system can promote the differentiation of EPCs,and the down-regulation of CD133 may be related to this.