| BackgroundChemotherapy is a routine treatment for various kinds of hematological system tumors and other solid tumors.Chemotherapy drugs not only destroy and inhibit tumor cells,but also cause certain damage to healthy cells in the body,and can cause side effects such as nausea,vomiting,hair loss,oral pain,skin redness and so on.Myelosuppression is the most serious side effect induced by chemotherapy.Due to the damage caused by chemotherapy drugs to the hematopoietic function of bone marrow,the number of peripheral blood red blood cells,white blood cells and platelets will be extremely reduced,so the body will appear anemia,infection,bleeding and other symptoms,seriously threatening the life safety of patients.Previous studies have shown that myelosuppression is mainly due to hematopoietic stem cells(HSCs)being severely damaged in chemotherapy.Recent studies have found that the hematopoietic microenvironment of bone marrow may also be damaged during chemotherapy,resulting in decreased support for HSCs and thus affecting hematopoietic function.This injury factor should not be ignored.Mesenchymal stem/stromal cells(MSCs)are important cellular components constituting the hematopoietic microenvironment of bone marrow.They can support and regulate the self-renewal,differentiation and development of HSCs by interacting with HSCs and secreting a variety of soluble cytokines.Bone-associated mesenchymal stem cells(BA-MSCs)are derived from the endosteum of bone marrow.They are important components in the formation and maintenance of hematopoietic microenvironment and play a role in supporting and regulating hematopoietic activities of the body.During HSC transplantation,if the host hematopoietic microenvironment is severely damaged by radiotherapy or chemotherapy,HSC hematopoietic reconstruction will fail.However,at present,there are few relevant studies on the damage of BA-MSCs by chemotherapy,and the degree of damage of BA-MSCs in chemotherapy and its related mechanism remain unclear.This study explored the damage and mechanism of BA-MSCs induced by chemotherapy.5-Fluorouracil(5-FU)is a derivative with a structure similar to that of uracil.It affects the replication process of cell DNA by inhibiting thymidylate synthetase.It is a classic and efficient anti-tumor chemotherapy drug commonly used in clinic.In this study,5-FU was used as a stimulus factor to establish cell and animal models of chemotherapyinduced BA-MSCs damage,to explore the damage effect of chemotherapy on BA-MSCs and related mechanisms,and to further explore the repair effect of platelet-derived mitochondria on BA-MSCs damage caused by 5-FU chemotherapy.ObjectiveTo elucidate the damage of BA-MSCs caused by 5-FU chemotherapy and preliminarily reveal the damage mechanism,and then to explore the repair effect of platelet-derived mitochondria on the damage of BA-MSCs caused by 5-FU chemotherapy.MethodsThe cell and animal models of BA-MSCs damaged by chemotherapy were established with 5-FU.Firstly,the apoptosis and cell cycle changes of BA-MSCs and their hematopoietic support ability were detected by flow cytometry under the treatment of 5-FU.The expression of BA-MSCs hematopoietic regulatory factor genes was detected by q RTPCR.The levels of SCF and CXCL12 secreted by BA-MSCs were detected by ELISA.Then,the mechanism of BA-MSCs damage induced by 5-FU was further explored from the perspective of mitochondrial damage.Mitochondrial morphological changes were detected by transmission electron microscopy.The expression of mitochondrial fusion and fission genes was detected by q RT-PCR.The expression levels of mitochondrial fission protein Drp1 and mitochondrial inner membrane fusion protein Opa1 were detected by Western blot.Mitochondrial membrane potential(MMP)was measured by flow cytometry and ATP content was measured by chemiluminescence to evaluate mitochondrial function.Then platelet-derived mitochondria were used to repair the damage of BA-MSCs caused by 5-FU in vitro,and the changes of apoptosis and hematopoietic support ability of BA-MSCs as well as the changes of mitochondrial morphology were detected,so as to evaluate the repair effect of platelet-derived mitochondria on the damage of BA-MSCs caused by 5-FU chemotherapy.Finally,the expression of mitochondrial fusion and fission genes in BAMSCs was further detected by q RT-PCR,so as to preliminarily analyze the therapeutic mechanism of platelet-derived mitochondria.Results1.It was confirmed that 5-FU damaged BA-MSCs and their mitochondria at the cellular levelFirst,mouse primary BA-MSCs were isolated and identified.In order to study the damage of BA-MSCs induced by 5-FU,a cell model of 5-FU induced damage of mice BAMSCs was established in vitro.After 12.5 μM 5-FU stimulated BA-MSCs for 48 h,flow cytometry detected that the proportion of apoptotic cells increased and cell cycle arrest occurred under the stimulation of 5-FU.In order to analyze the hematopoietic regulatory function of BA-MSCs treated with 5-FU,we co-cultured BA-MSCs with Bone marrow cells(BMCs),and used flow cytometry to detect the differentiation and development of BMCs at different stages as well as cell cycle changes.The results showed that HSCs and hematopoietic progenitor cells(HPCs)differentiated downstream and BMCs cell cycle was inhibited,indicating that 5-FU impaired the hematopoietic regulatory function of BA-MSCs.After 5-FU stimulation of BA-MSCs,q RT-PCR detected that the expression of hematopoietic regulatory factor genes Scf,Cxcl12,Csfg,Csfm,Csfgm and Il6 was downregulated.ELISA results showed that the secretion levels of hematopoietic regulatory factors SCF and CXCL12 decreased.These results indicate that 5-FU seriously damages the hematopoietic regulatory function of BA-MSCs in vitro.In order to further study the mitochondrial damage of BA-MSCs caused by 5-FU,increased mitochondrial fusion was observed in 5-FU damaged BA-MSCs by transmission electron microscopy in vitro.According to the results of q RT-PCR,the expression of mitochondrial fusion genes Opa1,Mfn1 and Mfn2 was significantly up-regulate,as was the expression of mitochondrial fission genes Drp1 and Fis1.Western blot analysis of the mitochondrial fission protein Drp1 and the mitochondrial inner membrane fusion protein Opa1 showed that the stimulation of 5-FU to BA-MSCs increased the expression of the mitochondrial inner membrane fusion protein Opa1.The MMP and ATP content of BAMSCs stimulated by 5-FU were detected,and the MMP and ATP content were significantly decreased.These results suggest that the mitochondrial dynamics of BA-MSCs are affected by the effect of 5-FU in vitro,and the mitochondrial function of BA-MSCs is seriously damaged.2.It was confirmed that 5-FU damaged BA-MSCs and their mitochondria at the animal levelIn order to study the damage caused by 5-FU to BA-MSCs in vivo,a 5-FU induced mouse model of myelosuppression was established.After 6 days of intraperitoneal injection of 5-FU,BA-MSCs and BMCs were isolated for damage analysis.The results showed that apoptosis of BA-MSCs increased,cell cycle was inhibited,and the number of HSCs and HPCs in BMCs decreased significantly.The results of q RT-PCR showed that the expression of hematopoietic regulatory factor genes Thpo,Csfg,Csfm,Csfgm and Il6 was significantly down-regulated.These results indicate that 5-FU chemotherapy seriously damages the hematopoietic regulatory function of BA-MSCs in vivo.In order to further study the damage of 5-FU on mitochondria of BA-MSCs in vivo,q RT-PCR detection revealed that the expression of BA-MSCs mitochondrial fusion genes Opa1 and Mfn1 in 5-FU-induced myelosuppression mice was significantly up-regulated.The expression of mitochondrial fission genes Drp1,Mff,Fis1 and Mid51 was also significantly up-regulated.The MMP and ATP content of BA-MSCs stimulated by 5-FU in vivo were detected,and the levels of MMP and ATP content were significantly decreased.These results suggest that 5-FU induces changes in mitochondrial dynamics of BA-MSCs in vivo,and seriously damages mitochondrial function of BA-MSCs.3.The study confirmed that platelet-derived mitochondria could alleviate the damage of BA-MSCs induced by 5-FUIn order to explore whether platelet-derived mitochondria can alleviate the damage of BA-MSCs caused by 5-FU,the morphology,purity and function of platelet-derived mitochondria were identified in this study,proving that platelet-derived mitochondria with high purity and biological function were successfully isolated.In vitro studies,plateletderived mitochondria were co-cultured with 5-FU-damaged BA-MSCs for 24 h,and it was found that platelet-derived mitochondria could be successfully transferred to BA-MSCs.Apoptosis of 5-FU damaged BA-MSCs was alleviated and hematopoietic regulatory function was improved after treatment with platelet-derived mitochondria.The results of in vitro co-culture experiment showed that BA-MSCs with the platelet-derived mitochondrial transfer promoted the proliferation of BMCs and improved the ability of BA-MSCs to support the self-renewal,differentiation and development of BMCs.Further studies showed that after platelet-derived mitochondrial therapy,BA-MSCs regulated mitochondrial dynamics by up-regulating the expression of mitochondrial fusion genes Opa1,Mfn1 and Mfn2,thereby reducing the apoptosis of BA-MSCs damaged by 5-FU and improving their hematopoietic support ability.These results indicate that platelet-derived mitochondria can alleviate 5-FU induced apoptosis of BA-MSCs and improve the hematopoietic regulatory function of damaged BA-MSCs.ConclusionIn vivo and in vitro studies have confirmed that 5-FU chemotherapy induces severe damage to BA-MSCs and their mitochondria,and platelet-derived mitochondria can alleviate apoptosis of BA-MSCs and improve the damage of their hematopoietic regulatory function,thus contributing to the recovery of bone marrow hematopoietic function after chemotherapy.This finding provides a new experimental basis for exploring therapeutic strategies for BA-MSCs damage caused by chemotherapy. |
PDF Full Text Request