Effect Of Insulin-like Growth Factor-1 (IGF-1) On Proliferation And Apoptosis Of Bmscs

Objective: In this study,BMSCs were stimulated by adding Insulin-Like Growth Factor-1(IGF-1)into Bone Mesenchymal Stem Cells(BMSCs)culture medium to stimulate BMSCs and investigate the effect of insulin-like growth factor-1(IGF-1)on proliferation and apoptosis of BMSCs,and verify the specific mechanisms of IGF-1-induced changes in biological characteristics of BMSCs through the addition of PI3 K inhibitors LY294002 and Akt phosphorylation inhibitors MK2206,which can provide experimental basis for the safe application of BMSCs in targeted therapy of tumors.Materials and Methods: 1.Experimental Animals: SPF grade male SD rats,3-4 weeks old,weighing about 50 g,were purchased from Experimental Animal Center of Chongqing Medical University.Experimental Cells: SD rat BMSCs were isolated by adherent method.Experimental groups: The study is divided into two parts and The first part is divided into the following 4 groups: Blank control group: BMSCs group;2.Experimental group 1: BMSCs+ IGF-1 50 ng/mL;Experimental group 2: BMSCs+ IGF-1 150ng/mL;Experimental group 3: BMSCs+IGF-1 300ng/mL.The second part is divided into the following 4 groups: Blank control group: BMSCs group;Experimental group 1: BMSCs+IGF-1;Experimental group 2: BMSCs+IGF-1+LY294002 inhibitors;Experimental group 3: BMSCs+IGF-1+MK2206 inhibitors.Experimental methods: 1.BMSCs were separated and screened by adherent method,The morphology of the cells was observed by inverted microscope;2.BMSCs were detected by Flow cytometry and indirect immunofluorescence;3.Cell proliferation ability in each group was assayed by CCK-8;4.The phase distribution of the cell cycle in each group was detected by flow cytometry;5.Changes in cell mitochondrial membrane potential was analyzed by JC-1;6.The cell migration ability of each group was examined by cell scratch test;7.Cell senescence was detected by ?-galactosidase staining;8.protein expression levels analysis of p-Akt in BMSCs cells stimulated with different concentrations of IGF-1 by Western Blot;9.The optimal working concentration of inhibitors was determined CCK-8 Cytotoxicity Test;10.Hoechst 33342 live cell staining was used to observe the nuclear morphology and apoptosis ratio;11.Annexin V-FITC/PI double staining method was used to detect the apoptosis ability of each group;12.The expression of Akt,Bad,Bcl-xl,c-Myc,STAT3,P53 mRNA in each group was detected by RT-PCR;13.The protein expression levels of Akt,p-Akt,p-Bad,Bad,Bcl-xl,c-Myc,p-STAT3,STAT3 and P53 were detected by Western Blot in each group.Results: 1.The BMSCs extracted from SD rats were identified by flow cytometry.The positive rate of CD29 in bone marrow mesenchymal stem cells(P3)was 99.9%,the positive rate of CD90 was 98.8%,and CD34 and CD45 were not expressed in the third generation of bone marrow mesenchymal stem cells(P3).The IF method detected the expression of CD90 in BMSCs mainly in the nucleus,and CD105 did not express CD45 in both cytoplasm and nucleus.It shows that the purity of primary BMSCs extracted from rats is higher,which meets the basic requirements for follow-up experiments.2.BMSCs cultured in BMSCs cultured in BMSCs cultured on an inverted microscope for 2 weeks showed adherent growth,spindle shape,swirling arrangement,and poor refractive index.After 2 weeks of IGF-1 stimulation,the cells became thinner and sharper,the ratio of nucleus to cytoplasm increased,disorder was arranged,intracellular particles increased,and the refractive index increased.The morphology of cells treated with LY294002 and MK2206 was more regular than that of cells stimulated with IGF-1.3.CCK-8 proliferation experiments found that the proliferation of IGF-1 stimulation group cells compared with blank control cells,the proliferation of cells was significantly increased,the difference was statistically significant(P<0.05): plus blocker treatment group of two Compared with the IGF stimulation group,the proliferation ability of the group cells was decreased,and the difference was statistically significant(P<0.05).4.Flow cytometry analysis of cell cycle results showed that the proportion of cells in the G2+S phase of the IGF-1 stimulation group was significantly higher than that of the BMSCs alone culture group,and the experimental group 3> experimental group 2> experimental group 1.This result shows that IGF-1 can increase the proportion of cells in the G2+S phase of BMSCS in a concentration-dependent manner.5.Detection of mitochondrial membrane potential changes by flow cytometry revealed that the percentage of cells with decreased mitochondrial membrane potential was significantly lower in IGF-1-stimulated BMSCs than in BMSCs alone,indicating that early apoptosis was inhibited.6.Through the scratch test,it was found that the three different concentrations of IGF-1 can promote the healing ability of bone marrow mesenchymal stem cells(P<0.05).It shows that IGF-1 can improve the migration ability of BMSCs.7.The result of cell aging staining suggested that the proportion of BMSCs positive cells after 6 days of IGF-1 treatment was significantly lower than that of BMSCS group(P<0.05,P<0.01).It indicates that the long-term culture of BMSCS in the abnormal microenvironment with high expression of IGF-1 significantly increases the anti-senescence ability and has the risk of malignant transformation.8.Western Blot analysis suggested that BMSCs treated with different concentrations of IGF-1 for 5 days,IGF-1 at concentrations of 150 ng/mL and 300 ng/mL could stimulate the high expression of p-Akt protein in BMSCs,and the ratio of phosphorylated Akt(pAkt/ Akt)was significantly higher than BMSCs in the control group(P<0.05,P<0.01).There was no significant difference in the expression of p-Akt between BMSCs treated with other concentrations of IGF-1 and the control group.9.The CCK-8 cytotoxicity experiment showed that when the working concentration of LY294002 is 5 ?mol/L,the relative cell survival rate is more than 85%,and this concentration can simultaneously ensure cell viability and inhibition.The cells were treated with the same method.The optimal working concentration of MK2206 was 5 ?mol/L.10.Observed under fluorescence microscope,the apoptosis rate of IGF-1 stimulation group treated with Hoechst33342 stained cells was significantly lower than that of blank control group(P < 0.05);apoptosis was increased in blocker group.The rate was significantly higher than the IGF-1 stimulation group(P < 0.05).11.Annexin V-FITC/PI double staining assay showed that the apoptosis rate of IGF-1 stimulation group was lower than that of blank control group(P<0.05).The apoptosis rate of blocker group(LY294002,MK2206)was higher than that of IGF-1 stimulation group(P<0.01).12.RT-PCR results showed that there was no significant difference in the expression of Akt1 in each group of cells.The relative expression levels of Bad,Bcl-xl,c-Myc,STAT3,and P53 mRNA in the IGF-1 stimulated group were significantly higher than those in the blank control group.The difference was statistically significant(P<0.05);The relative expression levels of Bad,Bcl-xl,c-Myc,STAT3,and P53 mRNA in the two groups of cells treated with blocker group were significantly higher than those of the IGF-1 group.The difference was statistically significant(P < 0.05).13.Western Blot results suggest that the relative expression levels of p-Akt,p-Bad,Bad,Bcl-xl,c-Myc,p-STAT3,STAT3,and P53 in IGF-1 stimulated cells were significantly higher than those in blank control.In the group,the difference was statistically significant(P<0.05,P<0.01).Both PI3 K inhibitor LY294002 and Akt inhibitor MK2206 could block the up-regulation of Akt,downstream apoptosis-related proteins and proto-oncogene induced by IGF-1 stimulation,and the difference was statistically significant(P<0.05,P<0.01).Conclusion: 1.IGF-1 can promote the proliferation and migration of BMSCs through the cell cycle node,and activate the PI3K/Akt signaling pathway,upregulate the apoptosis regulatory proteins such as Bad,Bcl-xl and inhibit the apoptosis of BMSCs.The genes c-Myc,STAT3,and P53 are also abnormally activated and have a concentration-dependent manner within a certain range,suggesting that BMSCs tend to have malignant transformation in the abnormal microenvironment in which IGF-1 is highly expressed.2.Both Akt phosphorylation inhibitors and PI3 K inhibitors can reverse the proliferation and apoptosis-related biological characteristics of BMSCs stimulated by IGF-1 stimulation,which suggesting that PI3K/Akt signaling pathway is the process of IGF-1 induced BMSCs transformation.Key signal transduction pathways.