Effect Of Parathyroid Hormone On The Pathogenesis And Chemoresistance Of Leukemia By Regulates Bone Marrow Microenvironment

Objective: Bone marrow mesenchymal stem cells are the basic cellular components that make up the bone marrow microenvironment.It is an important component supporting the formation of microenvironment cytokine networks.In patients with leukemia,the morphology and function of mesenchymal stem cells(MSCs)will change,which is related to the transformation from normal bone marrow microenvironment to leukemia bone marrow microenvironment.Parathyroid hormone can promote the transformation of MSCs into osteoblasts through bone morphogenetic protein(BMP)pathway.At the same time,the pathway interacts with connective tissue growth factor(CTGF),which can mediate the adhesion,migration and drug resistance of tumors.Based on the above reasons,our research wanted to confirm whether the change of bone marrow microenvironment can affect the drug resistance of leukemia.And the effect of parathyroid hormone on bone marrow MSCs whether can regulate the occurrence,progression and drug resistance of leukemia.The pathways involved in the regulation were preliminarily studied in order to find new targets for leukemia treatment and provide new theoretical basis for the treatment of leukemia.Methods: 1.Cell experiment: Primary cultured acute myeloid leukemia-derived mesenchymal stem cells(AML-MSCs)were co-cultured with K562/K562-ADM.The cells after co-culture were identified by morphological detection,peroxidase staining(POX),real-time fluorescence quantitative polymerase chain reaction(RT-PCR)and fluorescence in situ hybridization(FISH).The cell cycle changes of K562/K562-ADM under the action of bone marrow microenvironment were analyzed by flow cytometry.The changes of cytokine IL-6/IL-32 were detected by ELISA after co-culture.Parathyroid hormone was added to the cells after co-culture.The proliferation and apoptosis of K562/K562-ADM cells were detected and compared by flow cytometry.The changes of cytokine IL-6/IL-32 after dosing were detected by ELISA.Different expression levels of BMP4,CTGF,ABCB1 genes and proteins in co-culture group and co-cultured drug group were detected by RT-PCR and Western blotting.2.Clinical trial: During from the 2015-2017,56 cases of bone marrow samples from patients with acute myeloid leukemia were collected by the First Hospital of Lanzhou University.According to the NCCN guidelines,the patients were divided into a good prognosis group,a moderate prognosis group,a poor prognosis group,and a relapse resistant group.10 patients with non-malignant hematologic disease and 10 normal subjects were selected as the control group.The expression levels of BMP4,CTGF and ABCB1 were detected by RT-PCR.The contents of BMP4 and CTGF in bone marrow plasma and plasma were detected by ELISA.Results: 1.AML-MSCs promoted spindle-shaped transformation of K562-ADM,which is rare in the K562 co-culture group.2.AML-MSCs promote the proliferation of K562-ADM and inhibit apoptosis.AML-MSCs inhibit the proliferation of K562 and promote apoptosis.3.The soluble cytokines IL-6 and IL-32 decreased in the K562 co-culture group,but increased significantly in the K562-ADM co-culture group.4.In the co-culture group,the addition of parathyroid hormone can promote the differentiation of AML-MSCs,inhibit proliferation,inhibit the production of IL-6/IL-32,promote the apoptosis of K562/K562-ADM,and inhibit proliferation.The above effect of parathyroid hormone on K562-ADM is stronger than that of K562,which is most obvious at 24 hours.5.Co-cultured AML-MSCs and K562-ADM,BMP4 gene / p-Smad1 / 5 protein expression decreased,but co-culture with K562,BMP4 gene / p-Smad1 / 5 protein expression increased.6.Co-cultured AML-MSCs and K562/K562-ADM,CTGF and ABCB1/P-gp expression increased,K562-ADM co-culture group increased significantly.7.parathyroid hormone up-regulated the expression of BMP4 gene / p-Smad1/5 protein in AML-MSCs,down-regulated the expression of CTGF,inhibited the expression of ABCB1/P-gp on K562/K562-ADM.8.The expression of BMP4 is decreased and the expression of CTGF is increased,which is closely related to the poor prognosis of patients with acute myeloid leukemia.The expression of CTGF was positively correlated with ABCB1 expression(r=0.907,r>1);while the expression of BMP4 was negatively correlated with ABCB1(r=-0.827,r<1).The change in the content of BMP4 was consistent in the expression of bone marrow plasma and plasma;the change in the content of CTGF was inconsistent in the expression of bone marrow plasma and plasma.Conclusion: 1.K562-ADM spindle transformation induced by bone marrow microenvironment change is closely related to chemotherapy resistance of leukemia.2.Parathyroid hormone can promote the apoptosis of K562 and K562-ADM by inhibiting the proliferation of AML-MSCs,and it has a stronger effect on K562-ADM than K562.3.Parathyroid hormone may down regulate the expression of multidrug resistance genes and proteins of K562 and K562-ADM by regulated BMP and CTGF.It is involved in the occurrence of leukemia and multidrug resistance.4.Decreased BMP-4 expression and increased CTGF expression are related to multidrug resistance and poor prognosis of acute myeloid leukemia.