Preliminary Study On The Mechanism Of Gefitinib Inhibiting EGFR Signaling Pathway Affecting Long Bone Fracture Healing In Rats

ObjectiveTo compare the differentiation,proliferation and apoptosis of periosteal mesenchymal stem cells(PSCs),endometrial mesenchymal stem cells(ESCs)and bone marrow mesenchymal stem cells(BMSCs)in rats during long bone fracture healing,and to explore the effects of gefitinib on the biological function of PSCs,ESCs and BMSCs in rats by inhibiting EGFR signaling pathway.MethodsHealthy male 4-week-old SD rats were randomly divided into experimental groups and control groups after successful establishment of femoral fracture model of right lower extremity.The experimental group was given gefitinib 100mg/Kg/D and the control group was given the same dose of methylcellulose.Bilateral femur and tibia were taken one week after operation,and the PSCs,ESCs and BMSCs were isolated and extracted,respectively.The ability of cloning by CFU detection,expression of cell surface antigens CD29,CD90,CD34,CD45 and ability of multidirectional differentiation(osteogenesis,chondrogenesis and adipogenesis)were used to identify the stem cell attributes.PCR was used to detect osteogenic differentiation related genes(osteocalcin,bsp,osterix,RUNX2),chondrogenic differentiation related genes(aggrecan,Col2al,Sox9),adipogenic differentiation related genes(1p1,pparγ,cebpα),cell cycle inhibitor-related genes(p15,p16,p21,p27)and apoptosis related genes(bcl2,bax,caspase 3)were detected.BrdU incorporation assay was used to detect cell proliferation.Detection of apoptosis by flow cytometry to detected the percentage of living cells in each sample.ResultsCFU test showed that the number of ALP + and CFU + cells and cell colony diameter of PSCs were the largest and BMSCs the smallest,and the experimental group was significantly larger than the control group(P<0.05).Expressions of cell surface markers in each group,CD29 and CD90 were overexpressed,while CD34 and CD45 were underexpressed.Von Kossa staining showed obvious calcium nodule formation after osteogenic differentiation.After chondrogenesis induction differentiation,there were obvious blue-stained granular nodules in Alsine blue staining.Oil red O staining showed obvious red-stained lipid droplets after adipogenic differentiation.The value of 2-△△Ct of osteogenic differentiation related genes(osteocalcin、bsp、osterix、RUNX2),chondrogenic differentiation related genes(aggrecan,Col2a1,Sox9)and adipogenic differentiation related genes(lpl,ppary,cebpa)expressed,PSCs were largest,BMSCs were smallest,experimental group was significantly smaller than that in control group(P<0.05).The value of 2-△△Ct of cell cycle inhibitor-related genes(p15,p16,p21,p27)expressed,PSCs were the smallest,BMSCs were the largest,and the experimental group was significantly larger than the control group(P<0.05).There was no significant difference between PSCs,ESCs and BMSCs in the expression of anti-apoptotic genes(bcl2)(P<0.05).The expression of apoptotic genes(Bax and Caspase-3)in PSCs was the smallest and BMSCs was the largest and experimental group was significantly larger than the control group(P<0.05).BrdU incorporation assay showed that the BrdU value of PSCs was the largest and BMSCs was the smallest,and the BrdU value of experimental group was significantly smaller than that of control group and the inhibition rate of BrdU value of PSCs was the smallest and BMSCs was the largest(P<0.05).Detection of apoptosis by flow cytometry showed that the percentage of living cells was the highest in PSCs and the smallest in BMSCs and experimental group xwas significantly larger than that in control group(P<0.05),but there was no significant statistical difference in apoptotic promotion rate.ConclusionThe biological functions of mesenchymal stem cells from different regions of long bone are different in rats.The differentiation,proliferation and anti-apoptotic ability of PSCs is stronger than those of ESCs and BMSCs,and ESCs is stronger than those of BMSCs,indicating that the closer to the source of bone surface structure,the stronger the differentiation,proliferation and anti-apoptotic ability of mesenchymal stem cells.Gefitinib inhibits EGFR signaling pathway and inhibits proliferation,promotes differentiation and promotes apoptosis of PSCs,ESCs and BMSCs,but has the weakest inhibitory effect on proliferation of PSCs,and the strongest inhibitory effect on proliferation of BMSCs.The mechanism of EGFR signaling pathway inhibitor gefitinib promoting fracture healing in rats may be as follows:Firstly,through promoting the differentiation of PSCs in extramedullary space into osteoblasts and chondroblasts to form intrachondral ossification centers,thus forming strong external callus to make fracture fast and stable.Secondly,by promoting osteogenic differentiation of BMSCs and ESCs at cortical fracture sites to form intramembranous ossification centers to accelerate fracture healing.However,the specific impact mechanism remains to be further studied in the follow-up related experiments.