| Objective:To explore the mechanism of CCN1 on osteoblasts derived from MM patients and the expression level of Axl in MM cells and its role on proliferation of myeloma cells,macrophages and MDSCs.Content:(1)Experimental methods such as primary cell culture,in vitro cell model enhancement or inhibition of CCN1 were used to observe the effect of CCN1 on osteoblasts derived from MM patients.The study is to verify that CCN1 regulates the proliferation and differentiation of osteoblasts through the PI3K/Akt signaling pathway as well as clarify its molecular mechanism to promote MSC differentiation to OB.To determine whether CCN1 plays a protective role in MBD bone damage and its related mechanism,and preliminary explore whether CCN1 can directly inhibit MM cells or synergistic effect with related chemotherapy drugs at the same concentration.(2)Exploring the expression level of Axl in MM cells by detecting the expression level of Axl in MM patients,healthy control groups,and different human MM cell lines.The changes of Axl expression level in MM cells were detected after using the traditional chemotherapy drug Melphalan and expolre the role of Axl in the drug resistance of MM cells.By observing the changes of apoptosis and proliferation of MM cell line after using melphalan alone and combination with Axl inhibitor R428,we can explore the effect of Axl inhibitors in MM treatment as well as the effect Axl inhibitors on immune microenvironment,such as MDSCs and macrophages.Methods:Part I.Mechanism of CCN1 on osteoblasts derived from MM patients1.OBs derived from MM patients were induced in vitro,and the effects of CCN1 on OBs were observed.2.AKT signaling pathway antibody kit was used to detect OBs derived from MM patients and OBs from healthy control groups,and screen out the possible signaling pathways for CCN1 to act on OBs.3.After preliminary verification of the relevant signaling pathways of CCN1 acting on OBs screened by Western Blot,we found the key sites on the relevant signaling pathways.Then we added corresponding inhibitors or agonists and used Western Blot to verify the signal pathway of CCN1 on OBs derived from MM patients.4.Observe the effects of CCN1 alone and in combination with different concentrations of Bortezomib(Bz)on MM cell lines at different time points.The proliferation rate and the apoptosis rate were detected by CCK8 and FCM.Part II.Preliminary exploration of the role of AXL in myeloma1.The expression level of Axl in JJN3 and U266 was too low to be detected,but the expression levels of Axl in both cell line were significantly increased after MM chemotherapy.After 24 hours of melphalan treatment,the relative expression level of Axl in JJN3 increased compared with the control group,but did not reach statistical difference.After 24 hours of bortezomib treatment,the relative expression level of Axl in JJN3 increased significantly compared with the control group.Except for the low concentration group of 0.625 n M,the other concentration groups including 1.25n M(p=0.023),2.5 n M(p=0.029),5 n M(p=0.015)and 10n M(p=0.001)were all reached statistical differences.And as the concentration of bortezomib increased,the expression level of Axl showed an upward trend.U266 and JJN3 both showed obvious Axl expression after 24h treatment of high-concentration melphalan group(Mel 5?M),but they did not show obvious Axl expression in the low-concentration melphalan group.2.The relative activities of JJN3 and U266 in the Melphalan group were significantly higher than those of the Melphalan+Axl inhibitor R428(1?M)group(Mel 0.625?M group p=0.025?1.25?M group p=0.021?2.5?M group p=0.025).At the same time,the apoptosis rates of JJN3 and U266 in the melphalan group were significantly lower than those in the melphalan+Axl inhibitor R428(1?M)group(Mel 0.625?M group p=0.001?1.25?M group p=0.003).The combination of melphalan and R428 can significantly improve its ability to promote MM cell apoptosis,and there is a synergistic effect when R428 and melphalan are used in combination.3.The expressions of Axl in macrophages M1,M2,and TAM cells derived from the bone marrow of 5T33MM mice were obviously positive,but not in the 5T33vv cells(CD11b~-).R428 had inhibitory effect on MM mouse-derived MDSCs cells and MM cells,and the inhibitory effect of R428 on MDSCs is stronger than that on MM cells.This difference was most obvious at the concentration of 0.5?M.4.The expression of Axl in the MM patient group was 500.31±111.47 pg/ml,which was significantly lower than that of the healthy control group,which was 643.38±84.35 pg/ml(p=0.001).The serum Axl expression level in the MM patient group was lower than that in the healthy control group.Conclusions:1.CCN1 can stimulate growth and promote differentiation of osteoblasts derived from MM patients.2.CCN1 stimulates the differentiation and proliferation of osteoblasts derived from MM patients by enhancing the PI3K/AKT/GSK3?signaling pathway,and its inhibitory effect on GSK3?is similar to the GSK3?inhibitor TWS119.Neither CCN1used alone(at the same concentration 30ng/ml)nor in combination with bortezomib could inhibit MM cells significantly.3.Axl expressions were not detected in the human MM cell lines JJN3,U266,but the expression levels of Axl in the cell lines were significantly increased after MM chemotherapy drugs.The combination of Axl-specific inhibitor R428 and melphalan can enhance the effect on inducing MM cell apoptosis.4.The expressions of Axl in M1,M2,and TAM cells in the bone marrow of 5T33MM mice are obvious.R428 has inhibitory effect on mouse MM cells and MDSCs,and R428 has a stronger inhibitory effect on MDSCs than on MM cells.5.The expression level of Axl in the serum of MM patients was lower than that in the healthy control group.It can be speculated that Axl plays an important role in the proliferation of MM cells. |
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