| Antler may completely regenerate annually and the process is based on proliferation and differentiation of stem cells.Mesenchymalstemcells(MSCs)of antler play an important role in antler regeneration and rapid growth and development,while the mechanisms that affect the proliferation,migration and differentiation of antler MSCs have not been explored.MSCs can secrete a variety of cytokines,the most important of which is hepatocyte growth factor(HGF),which can combine with c-met to regulate cell proliferation and migration and promote tissue and organ regeneration.In this study,we used antler MSCs as a pilot material to clone the CDS region of HGF gene,construct a lentiviral overexpression HGF vector with HGF interferingRNA,upregulate and interfere with the expression of antler mscshgf gene,and examine the effects on the proliferation,migration and differentiation activities of antler MSCs,and the expression changes of downstream target genes of HGF/c-Met signalling.The results of the trial provide an experimental basis and rationale for uncovering the biological functions and mechanisms of action of HGF/c-Met signaling on antler MSCs.In the first experiment,using the full-length sequence of Tarim red deer HGF gene cloned by the research team,the CDS region specific primers of the gene were designed by Premier5.0 software,and the2276 bp sequence was cloned.The lentiviral vector of HGF overexpression was successfully constructed.After transfection of antler MSCs,the mRNA expression level of HGF gene was up-regulated by 23 times,and the protein expression abundance was increased by 1.5 times.At the same time,the siRNA sequence of HGF gene was designed,and the best siRNA was screened.After transfection of pilose antler MSCs,the mRNA expression level of HGF gene was down-regulated by 66%,and the protein expression abundance was decreased by 60%.The proliferation ability of cells in overexpression and interference group was detected by CCK-8 and Ed U.CCK-8 test showed that the OD value of pilose antler MSCs in overexpression HGF group increased significantly from 48 h to 96 h in vitro(P<0.05),while the cell OD interfering with HGF decreased significantly(P<0.05).The results of Ed U assay showed that the positive cell rate of overexpressed HGF group was significantly higher than that of empty vector group and control group(P < 0.05),but the opposite result was found after interfering with HGF(P < 0.05).The mRNA expression of cell proliferation related genes c-Met,RAS,ERK and MEK downstream of this signal pathway was similar to that of the above test.Therefore,HGF/c-Met signal can promote the proliferation of pilose antler MSCs.In the second experiment,MSCs migration of pilose antler was detected by scratch test and Transwell test.After overexpression of HGF gene,scratch test showed that the healing percentage of cultured24h-72 h cells at each time point was significantly higher than that of no load group and control group(P<0.0l),while interfering with HGF gene showed the opposite result(P<0.01).The results of Transwell test showed that the number of cells overexpressing HGF gene passing through membrane was significantly higher than that of no-load group and control group(P<0.0l),while interfering with HGF gene showed the opposite result(P<0.01).Furthermore,the mRNA expression of downstream cell migration and survival activity related genes Gab1,Grb2,AKT and PI3 K was detected,which was corresponding to the above experimental results.Therefore,it is considered that HGF/c-Met signal can promote the migration and survival of pilose antler MSCs.In the three experiment,during the cartilage induction of pilose antler MSCs in vitro,although the cells had the morphological characteristics of cartilage differentiation after 7 days,the results of toluidine blue staining showed that chondrocytes secreted proteoglycans both overexpressing HGF gene and interfering with HGF gene.However,the mRNA expression of Col Ⅱ,Sox9 and AGC in chondrocytes detected by RT-q PCR after overexpression of HGF gene was significantly lower than that in the control group(P<0.01),while the expression of HGF cartilage marker gene was significantly higher than that in the control group(P<0.01).The results showed that HGF/c-Met signal inhibited the cartilage differentiation of pilose antler MSCs to some extent.In summary,overexpression of HGF promotes the proliferation of deer antler MSCs through the HGF/c-Met signaling pathway to modulate c-Met,RAS,ERK,and MEK genes.Upregulating Gab1,Grb2,AKT and PI3 K genes promotes the migration of deer antler MSCs,while interfering with HGF downregulation of corresponding genes through the HGF/c-Met signaling pathway to inhibit the proliferation and migration of deer antler MSCs.HGF/c-Met signaling inhibits cartilage differentiation of deer antler MSCs to some extent. |
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