The Effect Of Mammalian Rapamycin Target Protein Signaling Pathway On The Activation Of Bone Marrow Mesenchymal Stem Cells Into Carcinoma-associated Fibroblasts In Myeloproliferative Neoplasm

Objective: To explore the basic biological characteristics of MPN MSCs and to preliminary investigate the signal pathway of mammalian target protein(mTOR)in the activation of bone marrow mesenchymal stem cells(MSCS)into carcinoma-associated fibroblasts in myeloproliferative neoplasms.Methods: Bone marrow specimens of 40 newly diagnosed MPN patients and 17 normal controls were collected from the Union Hospital of Huazhong University of Science and Technology for in vitro culture and identification.Under appropriate conditions,MSCs are induced to differentiate into osteoblasts.The formation of calcium nod ule was observed by alizarin red staining.The proliferation capacity of MSC was calculated by formula,and the proliferation curve of MSC was determined by cck-8 experiment.The cell cycle and apoptosis of MSC were detected by flow cytometry and he gene expression in MSC was analyzed by RT-PCR,and.In order to investigate whether MSCS from MPNs have carcinoma-associated fibroblasts-activated to methylsma,western blot and RT-PCR were used to detect the methylsma of MSCS from MPNs,and TGF-stimulated MPNs group as well as normal control BMMSC were utilized to detect the expression of the CAF-labeledα-SMA for 48 h and 72 h after intervention.Thus,in order to study the regulatory effect of mTOR signal on CAF of MSCs from MPNs under the stimulation of TGF-β1,we examined the expression of mTOR signal of MSCs from MPNs under the stimulation of TGF-β1.BMMSC obtained from MPNs was treated with the mTOR inhibitor rapamycin to detect the expression of CAF-labeled a-SMA and mTOR pathway proteins,and to further verify the regulatory effect of mTOR signal on the activation of MSC from MPNs stimulated by TGF-TGF on CAF.Results:(1)All MSCs in the control group could achieve in vitro growth,and five samples from MPN patients could not achieve the in vitro proliferation.The control group presented a thin and fibrous appearance,while the MPN-derived MSC presented a flat and polygonal appearance,whose volume was larger than that of the control group.(2)The proliferative ability of MSCs from MPNs was lower than that of the control group,and it still maintained the osteogenic and adipogenic differentiation ability in vitro.Compared with the control group,the number of calcium nodules formed after the induced differentiation of MSCs from MPN,as well as the expression levels of transcription factors Runx2 and Oterix that regulated osteogenic differentiation before differentiation were higher.(3)The proportion of MSCs derived from MPN in G0/G1 phase was higher than that in the control group,while that in S phase was lower than that in the control group(P<0.05).The apoptotic cells of MSCS from MPNs were significantly lower than those in the control group(P<0.05),and the difference was statistically significant.(4)MSCs derived from MPN have the ability to activate fibroblasts associated with tumors,and the ability to activate CAF under the stimulation of TGF-β that is enhanced to a certain extent,which is accompanied by the activation of mTOR signal.(5)After the treatment of MPN MSCs with rapamycin,the expression level of mTOR and α-SMA decreased(P < 0.05).Inhibition of mTOR signal can partially weaken the stimulation of TGF-β on α-SMA expression in MPN MSCs.Conclusions:(1)The biological characteristics of MSCs derived from MPN differ from normal controls,and the cell volume was increased.with the proliferation capacity decreased,the number of S phase cells decreased,the proportion of apoptotic cells decreased,while the osteogenic differentiation ability of MPN-derived MSCs increased.(2)MSCs derived from MPN have the ability to be activated into CAF.Compared with the normal control group,the ability to activate into CAF under TGF-β stimulation was significantly enhanced.(3)mTOR signaling protein has a regulatory role in the process of TGF-β1 stimulating the activation of MPN derived MSC into CAF,which can be partially weakened by mTOR inhibitors.Therefore,the functional changes of MSCs derived from MPN may play an important role in the development of MPN,and may become a potential therapeutic target.