| Section Ⅰ Single-cell sequencing analysis and cluster identification of CD34+stem/progenitor cells in acute myeloid leukemiaBackground:Acute myeloid leukemia(AML)encompasses a group of heterogeneous disorders characterized by the expansion of malignant clones of hematopoietic progenitor cells blocked at various stages of differentiation.Remission induction chemotherapy with cytarabine and anthracycline has been a standard treatment for newly diagnosed AML for more than 30 years.Although 70%of patients with newly diagnosed AML attain morphologic complete remission(CR)with intensive induction chemotherapy,approximately 30%of adults AML are not sensitive to chemotherapy,and at least 50%of those who achieve remission will relapse.The different outcomes of AML patients are due to the fact that AML is a heterogeneous and molecularly complex disease with variable hematologic phenotypes.AML hematopoietic stem/progenitor cells sustain the disease and display stem cell properties,such as self-renewal,quiescence,and heterogeneity.Therefore,the AML hematopoietic stem/progenitor cells with heterogeneous properties enable their ability to generate the heterogeneity of intra-or inter-patient,and then fuel different responses to induction chemotherapy during disease progression.Identification of the heterogeneity in AML hematopoietic stem/progenitor cells is of vital importance for prediction of AML prognosis.Recently,single cell RNA-sequencing(scRNA-seq)technologies have matured such that one can sequence and analyze thousands of cells per tumor.At this scale,it can derive significant insights into the cellular heterogeneity,characteristics of the molecular diversity and the biological features that distinguish different cell subpopulations.Savas et al found that CD8+TRM cells contributed to breast cancer immunosurveillance and were the key targets of modulation by immune checkpoint inhibition through scRNA-seq of T cells isolated from human breast cancers.Mathys et al discovered disease-associated cellular subpopulations in Alzheimer’s disease.Witkowski et al uncovered a role for non-classical monocytes subcluster in bone marrow microenvironment supporting B-ALL progression and treatment evasion.Here,we adapted 10× Genomics scRNA-seq technology to acquire transcriptional data for thousands of single cells from bone marrow CD34+cells of newly diagnosed AML patients.We showed a comprehensive and heterogeneous map of the CD34+cells in AML patients and healthy controls.We leveraged transcriptome wide features to distinguish malignant-like cells from normal-like cells,and malignant-like clusters from each other via several analytical strategies.Furthermore,by comparing sensitive AML patients with resistant patients,we discovered a poor-prognosis related subpopulation with specific gene signatures in CD34+cells.Moreover,we anchored two subpopulations with specific markers in AML CD34+cells that were associated with good response of patients to induction chemotherapy.Our discovery created a chance for prognosis or prediction in newly diagnosed AML patients.Objective:Using a new generation of high-throughput single-cell sequencing technology,the single-cell genomic map of AML was drawn to further understand the cytogenetic characteristics of AML,identify the clonal subsets related to chemotherapy response in AML cells,and determine their characteristic gene expression profiles and cell surface specific phenotypes.And then to formulate specific and targeted accurate treatment strategies,predict the prognosis of AML,solve the problem of drug resistance of AML,and find its "fate determining point",so as to provide a basis for individualized treatment and accurate treatment of AML patients.Materials and Methods:1.Samples of clinically diagnosed acute myeloid leukemia were collected and bone marrow mononuclear cells were isolated for single cell sequencing.(1)sample collection:fresh bone marrow samples were collected from 38 newly diagnosed AML patients,21 complete remission(CR)AML patients after the first cycle of induction chemotherapy,and 17 non-remission(NR)AML patients.(entry conditions:age range from 18 to 80 years old,no severe infection,no acute promyelocytic leukemia,no history of myeloproliferative syndrome,no tumor chemotherapy or radiotherapy records,white blood cell count<100×109/L.The percentage of CD34+cells in bone marrow was≥20%).(2)Sample preparation:bone marrow mononuclear cells were obtained from fresh bone marrow samples of AML patients by Ficoll gradient centrifugation.CD34+cells were selected by immunomagnetic beads.2.Single-cell sequencing 10×genomic technique was used to draw the single cell map of acute myeloid leukemia.(1)the cells were washed,and the cell count and activity were detected.Gel beads containing the Barcode sequence is mixed with a mixture of samples and enzymes and then combines with oil surfactants to form GEMs(Gel Bead-In-Emulsions,meaning oil droplets that wrap a mixture of Gel beads,samples,and enzymes).The GEMs is collected and flowed to the reservoir,the Gel beads dissolves and releases the Barcode sequence,and the sample is labeled.The products containing Barcode information in each droplet were mixed,and the number of amplification cycles was selected according to the number of cells.The cDNA amplification reaction was carried out to purify the cDNA amplification products,and the standard sequencing library was constructed.(2)Cell Ranger analysis process:data analysis for 10x Genomics 3’single cell transcriptional group sequencing,including cell number statistics,single cell reads number statistics;quality control analysis,through UMI analysis to determine PCR amplification efficiency;quantitative gene expression;selection of Seuratt for cluster analysis;difference analysis for genes in different cluster.3.Marker genes identification:differentially expressed genes were screened and specific phenotypes related to induced chemotherapy response were identified.(1)the analysis results were identified by TCGA and GEO database,and the first 100 genes with the smallest difference in average expression level were selected from RNA-seq data as background gene set.Then,the average expression value of the background gene set is subtracted from the expression of each marker gene,and the sum of the result values of each gene in the marker gene set is defined as the marker gene set score of AML patients.Patients were divided into high score group and low score group according to the median score,and then survival analysis was carried out.Diagnosis was made according to the diagnostic criteria of NCCN guidelines,and prognostic risk stratification was performed.Common AML mutations,including FLT3,RAS,IDH1/2 and NPM1,were also evaluated for the patients included in the study.(2)Clinical samples were used to identify the cell subsets sensitive to chemotherapy.CD34+CD52+and CD34+CD74+DAP12+were used to label the cell subsets sensitive to chemotherapy.Bone marrow mononuclear cells were isolated from bone marrow fluid samples of 32 newly diagnosed AML patients for flow cytometric analysis.17 patients reached CR after one cycle of induction chemotherapy,so they were classified as chemotherapy sensitive group.The other 15 patients developed NR after one cycle of induction chemotherapy,so they were classified as chemotherapy resistance group.The cell proportion of chemosensitivity related subsets in the two groups was compared and the correlation with clinical characteristics was analyzed.Results:1.Atlas identification of CD34+cells in newly diagnosed AML patients.(1)there were significant differences in CD34+cells between AML and healthy controls,and significant heterogeneity in CD34+ cells between individuals.Then,by means of unsupervised clustering,all 60402 CD34+cells were clustered into 17 different cell clusters according to the similarity of gene expression profiles.The results showed that CD34+cells had diversity and heterogeneity.(2)by comparing the marker genes of each cell cluster and the characteristic genes of stem/progenitor cell lineage,CD34+cells were classified into six hematopoietic stem/progenitor cell types.It includes hematopoietic stem cells(HSC),lymphocytic progenitor cells(MLP),megakaryocytic-erythroid progenitor cells(MEP),granulocyte-monocyte progenitor cells(GMP),pro-B cell(ProB)and earliest thymic progenitors(ETP).2.The proportion of granulocyte-monocyte progenitor cells(GMP)in CD34+cells of newly diagnosed AML patients was significantly increased.(1)the proportion of CD34+cell types in AML patients was different from that in normal controls.In terms of cell types,the proportion of GMP in AML patients was significantly higher than that in healthy controls,while the proportion of HSC and pro-B was significantly lower than that in healthy controls;for cell subsets,the proportion of GMP-1,GMP-2,GMP-3,GMP-5 and GMP-7 in AML patients was significantly higher than that in healthy controls,while HSC-1,HSC-2,Pro-B-1,Pro-B-2 and MEP-2 were significantly lower than those in healthy controls.(2)If more than 60%of the CD34+cells in AML patients come from healthy controls,we define the subsets as normal cell subsets(Normal-like cluster)and the rest as malignant associated cell subsets(Malignant-like cluster).Therefore,the cell subsets GMP-1-7,MEP-1,MLP and ETP-1 were classified as malignant related cell subsets,and the other cell subsets were classified as normal cell subsets.According to the results of KEGG and GO cell pathway enrichment analysis,the gene expression profiles of malignant related cell subsets were significantly enriched in cell cycle,oxidative phosphorylation,apoptosis and cancer-related pathways.3.The malignant related cell subsets of CD34+cells in newly diagnosed AML patients have obvious heterogeneity.(1)AML patients were divided into sensitive group and tolerant group.According to the response of AML patients to standard induction therapy(cytarabine and anthracycline),6 AML patients were divided into two groups.AML02,AML05,AML06 and AML07 reached complete remission(CR)after the first course of induction therapy,so they were classified as chemotherapy sensitive(Sensitive)group,while AML01 and AML03 were not relieved after the first course of induction therapy,so they were classified as chemotherapy resistant(Resistant)group.(2)The malignant related cell subsets in the chemotherapy resistant group were significantly different from those in the sensitive group.The transcriptional differences between the two groups were compared in each malignant related cell cluster,and the differentially expressed genes were functionally enriched.The functional enrichment results of KEGG,GO and GSEA showed that the malignant related cell subsets GMP-1,2,3,5,7 and MLP in the chemotherapy resistant group were significantly enriched in acute myeloid leukemia,cell cycle,oxidative phosphorylation and uncontrolled transcription compared with those in the AML chemotherapy sensitive group,so there were significant differences in gene expression profiles in GMP-1,2,3,5,7 and MLP between the two groups.4.Cellular subsets with CRIP1highLGALS1highS100Ashigh molecular characteristics are associated with poor prognosis of AML.(1)in order to dig out the CD34+cell clones related to the adverse response to AML treatment,we clustered the above six cell subsets(GMP-1,2,3,5,7 and MLP),and compared the cell proportion of each cluster in the AML chemotherapy resistant group,the chemotherapy sensitive group and the healthy control group.The results showed that 9 cell subsets with different labeling characteristics were obtained after clustering GMP-1,and the proportion of cells in AML chemotherapy resistant group was significantly higher than that in chemotherapy sensitive group and control group in cluster GMP-1-0.cell cluster GMP-3 included 8 clusters,which was the same as GMP-1-0 in cluster GMP-3-0.(2)the molecular characteristics of malignant clones associated with poor prognosis were determined.The cell subsets with cluster GMP-1-0 or GMP-3-0 labeled gene(CRIP1highLGALS1highS100Ashigh)were malignant clones related to poor prognosis.(3)the malignant clones associated with poor prognosis were identified.Based on the RNA-seq data of 134patients with newly diagnosed AML in TCGA database,the patients were divided into two groups according to the score:high score(n=67)and low score(n=67).Through the survival analysis of the high score group and the low score group the results showed that the total survival(OS)of the high score group was significantly lower than that of the low score group(P<0.0195)which indicated that the malignant clones with this specific marker gene expression profile were related to the poor prognosis of AML.5.Another way of re-grouping also confirmed that CRIP1highLGALS1highS100Ashigh subsets are associated with poor prognosis of AML.(1)for the reclassification of CD34+ cells in AML patients,in order to eliminate the interference of healthy control samples,we removed the healthy control samples and reanalyzed the single cell data of CD34+cells in AML patients.The CD34+cells in AML patients were clustered into 19 clusters.In clusters 2,3,5 and 6,more than 60%of the cells came from the chemotherapy resistance group.By matching the marker genes of subsets 2,3,5 and 6 with stem/progenitor cell lineage characteristic genes,we found that clusters 2,5 and 6 were similar to GMP,and cluster 3 was similar to MEP,so we defined the cell types of these clusters as GMP-like and MEP-like,respectively.(2)the marker gene profiles of cluster 2 were perfectly matched with those of GMP-1-0 and GMP-3-0 clusters,indicating that the cells clustered by cluster 2 and these two clusters are the same cell clone cluster,so cluster 2 is a group of cells related to poor prognosis.In order to further understand the function of specific marker genes in cluster 2,we carried out gene expression enrichment analysis and found that up-regulated genes were enriched in cell adhesion,cell proliferation and S100 protein-related pathways.(3)the molecular characteristic of the cluster associated with poor prognosis was an independent prognostic factor.Two patients in the chemotherapy resistance group(AML01 and AML03)had MLL rearrangement,but no significant enrichment of MLL related genes was found in the cluster characterized by CRIP1highLGALS1highS100Ashigh.Therefore,we speculate that the molecular characteristics(CRIP1highLGALS1highS100Ashigh)of subsets associated with poor prognosis are independent prognostic factors.6.Two groups of cell subsets were found to be associated with good response to AML chemotherapy,and showed the characteristics of hematopoietic stem cell(HSC)cell types.(1)when comparing the proportion of AML chemotherapy resistant group and sensitive group in each cell cluster,we found that the proportion of chemotherapy sensitive group was much higher than that of chemotherapy resistant group in cluster 0 and 8.By matching the marker gene of cluster 0 or 8 with the characteristic gene of stem/progenitor cell lineage,we found that cluster 0 and 8 showed characteristics similar to HSC,so the cell type of cluster 0 and 8 was defined as HSC-like.In order to understand the function of these two subsets,we carried out gene expression enrichment analysis.The results showed that the up-regulated genes in the marker gene expression profiles of the two subsets were mainly involved in signal pathways such as helper T cell differentiation and immune response.(2)to determine the molecular characteristics of the clone subpopulation associated with good response to chemotherapy,cluster 0 was marked by CD52 in cluster 0 marker gene spectrum,and cluster 8 was marked by CD74 and DAP 12 in cluster 8 marker gene spectrum.7.Two groups of cell subsets associated with good response to AML chemotherapy were verified by clinical specimens and database.(1)The results of flow cytometry showed that the proportion of CD52+cells and CD74+DAP12+ cells in CD34+ cells of CR(chemosensitive group)was significantly higher than that of NR(chemotherapy resistant group)at the first visit.The CR rate of AML patients with more than 10%CD52+cells in CD34+cells after induction chemotherapy was significantly higher than that of patients with CD52+cells less than 10%,and the CR rate of patients with CD74+DAP12+ cells more than 30%was also higher than that of patients with less than 30%CD74+DAP12+cells.The correlation between the clinical characteristics and the response to induced chemotherapy in 32 patients with AML was analyzed.The results showed that the percentage of bone marrow primordial cells,WBC,hemoglobin,the proportion of CD52+cells in CD34+cells and the proportion of CD74+DAP12+ cells in CD34+ cells were statistically correlated with the response to induced chemotherapy in AML patients.(2)the clusters associated with good response to chemotherapy were identified by TCGA and GEO database.The RNA-seq data of 134patients with AML in TCGA database and 268 patients with AML in GEO database(GSE165430)were analyzed,and the scores of characteristic genes of cluster 0 and 8 were calculated respectively.The 134patients in TCGA database were divided into three groups according to prognostic risk.Good prognosis group(n=17),moderate prognosis group(n=89)and poor prognosis group(n=26).The results showed that the gene characteristic score of cluster 0 or cluster 8 in the poor prognosis group was significantly lower than that in the good prognosis group or the medium prognosis group.Then,the gene characteristic scores of recurrent patients in GSE165430 and patients with CR≥ 3 years were compared.The genetic characteristic scores of recurrent patients were lower than those of patients who maintained CR.Conclusion:1.The heterogeneity of CD34+cells in newly diagnosed AML patients was revealed by high-throughput single cell transcriptome sequencing.2.It provided a new field of vision for understanding the abnormal gene expression in AML CD34+cells,revealed the significant differences between the chemotherapy resistant group and the sensitive group,and identified specific cell subsets related to chemotherapy resistance and chemotherapy sensitivity.3.CD34+cell subsets associated with different responses to induction chemotherapy were found in newly diagnosed AML patients,which provides a new theoretical support for predicting the response to induction therapy in AML patients at the initial diagnosis stage.Section Ⅱ Roles and Mechanisms of Bone Marrow Adipocyte Disdifferentiation in MRD of T-ALLBackground:T-cell acute lymphoblastic leukemia(T-ALL)is an aggressive malignant blood disorder caused by clonal expansion of variant T cell progenitors,which accounts for approximately 25%of ALL cases in adults.Due to the use of intensive combination chemotherapy(at least a glucocorticoid,vincristine,and an anthracycline)and modulation of treatment tailored to patient response,complete remission rates are high,but relapse often occurs.Adults younger than 60 years have five-year survival rates of only 40-50%,and older patients display an even worse outcome.Relapse is believed to develop from undetectable leukemia cells that survive in the bone marrow microenvironment(BMM)after chemotherapy.The BMM is the primary site for minimal residual disease(MRD)and supports survival,proliferation and drug resistance of leukemia cells,presumably contributing to therapeutic resistance and disease relapse.The BMM comprises cells of multiple lineages,including fibroblasts,osteoblasts,endothelial cells,adipocytes and mesenchymal stem/progenitor cells.Recent studies have shown that bone marrow adipocytes participate in the development of malignancies by providing energy to solid tumor cells or secreting derived adipokines to protect multiple myeloma cells against chemotherapy-induced apoptosis and modulating bone metastasis.Additionally,the BMM transitions from adipocyte depletion in primary B-ALL to a fully adipocyte-reconstituted state upon remission.However,no report has shown whether adipocytes are increased in T-ALL patients after chemotherapy and how adipocytes in the BMM influence the survival of T-ALL cells.Furthermore,bone mesenchymal stem cells(BMSCs)are precursor cells of adipocytes,and some chemotherapeutic drugs enhance BMSC differentiation into adipocytes.For example,in acute myeloid leukemia(AML),BMSCs decrease their self-renewal capability and are prone to differentiate into adipocytes and chondrocytes after treatment with cytarabine(Ara-C).It is important to clarify the causes of BMSC differentiation into adipocytes in T-ALL patients after chemotherapy and to identify intervention targets for improving MRD in T-ALL.In our study,we found an increase in adipocytes in the BMM after exposure to chemotherapeutic drugs by utilizing matched BM biopsies taken from five adult T-ALL patients at initial diagnosis and after induction therapy and then confirmed that the BMSC-derived adipocytes attracted T-ALL cells by CXCL13 and supported T-ALL cells via the DLL 1/Notch1 signaling pathway.Finally,SREBF1 was screened out and proven to be a key molecule of dexamethasone(DEX)-induced adipogenic differentiation.Our findings revealed that the adipogenesis of BMSCs induced by DEX in the T-ALL BMM contributed to the protection of residual T-ALL cells,which identified a potential therapeutic target for BMM reconstitution to reduce the recurrence rate.Objective:To explore the changes of adipocytes in bone marrow microenvironment of T-ALL patients before and after chemotherapy;to clarify how bone marrow adipocytes affect the survival of T-ALL cells and what signal pathway they act on T-ALL cells;to explore the mechanism of regulating the changes of bone marrow microenvironment induced by chemotherapeutic drugs in T-ALL patients.Materials and Methods:1.It was confirmed that the number of adipocytes in bone marrow microenvironment of T-ALL patients after chemotherapy was significantly higher than that before chemotherapy.One-to-one corresponding bone marrow biopsies of 5 newly diagnosed T-ALL patients before and after chemotherapy were collected,and the proportion of adipocytes in bone marrow microenvironment of T-ALL patients before and after chemotherapy was compared by Hype staining.2.To study the effect of chemotherapeutic drugs on adipogenic differentiation of BMSCs in patients with T-ALL.(1)isolation and culture of BMSCs from patients with T-ALL:bone marrow fluid from patients with T-ALL was collected and bone marrow mononuclear cells were isolated by Ficoll method.Bone marrow mononuclear cells were routinely cultured in BMSCs medium and passaged twice.(2)BMSCs were continuously treated with demethoxy daunorubicin(IDA),vincristine or DEX,and cultured with 10%fetal bovine serum α-MEM containing 0.5mmol/L 3-isobutyl-1-methylxanthine(IBMX)and 10 μg/ml insulin to induce adipogenic differentiation of BMSCs.On the 7th,14th and 21st day,BODIPY fluorescence staining was used to detect the difference of adipogenic differentiation ability of BMSCs before and after treatment with chemotherapeutic drugs.3.To study the effect of adipocytes derived from BMSCs on the biological behavior of T-ALL cells after chemotherapy.(1)BMSCs of T-ALL patients after chemotherapy were isolated and induced to differentiate into adipocytes.(2)the BMSCs and adipocytes mentioned above were directly co-cultured with T-ALL cell lines Jurkat and SupTl,respectively.(3)to study the effect of co-culture on the biological behavior of T-ALL cells:①after being treated with Ara-C for 48 hours,the cells were collected,the apoptosis rate of T-ALL cells was detected by AnnexinV/7AAD staining and flow cytometry.② the cells were collected after 48 hours,and the colony formation ability of T-ALL cells was detected by methylcellulose semi-solid medium.③48 hours later,each group performed the same operation of cleaning the upper layer cells,and the adhesion of remaining T-ALL cells to BMSCs or adipocytes was observed under microscope.4.To explore the mechanism of protective effect of BMSCs-derived adipocytes on T-ALL cells after chemotherapy.(1)adipocytes derived from BMSCs after chemotherapy recruit T-ALL cells around themselves by releasing CXCL13 chemokine.1)the above direct co-culture system showed that the number of T-ALL cells gathered around adipocytes was significantly higher than that around BMSCs.Transwell chemotactic assay verified the recruitment effect of adipocytes on T-ALL cells.2)in order to explore the mechanism of this phenomenon,qRT-PCR analysis and the expression level of chemokine-related molecules showed that chemokine CXCL13 was significantly increased in adipocytes,which was verified by ELIA.3)BMSCs and adipocytes were directly co-cultured with T-ALL cell line.24 hours later,the cells were collected and stained with CXCR5 antibody.The expression of CXCL13 receptor CXCR5 in T-ALL cells was detected by flow cytometry.4)after blocking it with CXCL13 blocking antibody,Transwell chemotactic assay was used to detect the effect of chemokine CXCL13 on adipocyte recruitment.5)different concentrations of human recombinant CXCL13 chemokine were added,and the chemotactic effect of CXCL13 on T-ALL cells was detected by Transwell chemotactic assay.(2)after chemotherapy,adipocytes derived from BMSCs mediate the protective effect of T-ALL cells through DLL1/Notchl pathway.1)the adipocytes were co-cultured with T-ALL cells,and the expression of Notchl,Hes1,Heyl and NICD in T-ALL cells before and after co-culture was detected by qRT-PCR and Western Blot.2)the changes of Notchl ligands JAG1,JAG2,DLL1,DLL3 and DLL4 during the differentiation of BMSCs into adipocytes were detected by qRT-PCR,and the expression of DLL1 was detected by Western-Blot.3)T-ALL cells were treated with Notch1 surface receptor blocking agent MHN519,and the above T-ALL cells were directly co-cultured with adipocytes or BMSCs;①after being treated with Ara-C for 48 h,the cells were collected,AnnexinV/7AAD staining and flow cytometry were used to detect the apoptosis rate of T-ALL cells;② after 48 h,the cells were collected and the colony formation ability of T-ALL cells was detected by methylcellulose semi-solid medium.③ 48 hours later,each group performed the same operation of cleaning the upper layer cells,and the adhesion of remaining T-ALL cells to BMSCs or adipocytes was observed under microscope.5.To explore the mechanism of chemotherapeutic drugs promoting adipogenic differentiation of BMSCs in patients with T-ALL.(1)to screen and identify differentially expressed genes during adipogenic differentiation of BMSCs from T-ALL patients before and after treatment with chemotherapeutic drugs,and to isolate and culture BMSCs of T-ALL patients in vitro,which were divided into two groups:①BMSC group(BMSCs):BMSCs was cultured with α-MEM containing 10%fetal bovine serum.②BMSC group after adipogenic differentiation induction(Induced-BMSCs):10%fetal bovine serum α-MEM with final concentration of 0.5mM 3-isobutyl-1-methylxanthine(IBMX),10μg/ml insulin and 1 μM dexamethasone(DEX)was added to induce adipogenic differentiation of BMSCs.On the 21st day,cells were collected,RNA was extracted and RNA-Seq was detected to screen the differentially expressed genes before and after adipogenic differentiation of BMSCs.qRT-PCR and Western-Blot were used to verify the results.(2)to study the effect of intervention of SREBF1 expression on adipogenic differentiation of BMSCs in T-ALL patients.The expression of SREBF1 increased gradually with the increase of liposuction degree.After BMSCs was down-regulated by lentivirus or treated with SREBF1 inhibitor(Fatostatin HBr),adipogenic differentiation of BMSCs was induced.On the 7th,14th and 21st day,oil red O staining was used to detect the effect of SREBF1 on adipogenic differentiation of BMSCs.6.To study the effect of inhibition of adipogenic differentiation of BMSCs on the biological behavior of T-ALL cells.(1)the adipogenic differentiation of BMSCs was continuously intervened by SREBF1 inhibitor(Fatostatin HBr)to induce adipogenic differentiation of BMSCs for 21 days.(2)the adipocytes and adipocytes inhibited by SREBF1 were directly co-cultured with T-ALL cell line to explore the changes of drug-induced apoptosis,proliferation and adhesion of T-ALL cells:①after treatment with Ara-C for 48 hours,the cells were collected,and the apoptosis rate of T-ALL cells was detected by AnnexinV/7AAD staining and flow cytometry;②after 48 hours,the cells were collected and the colony formation ability of T-ALL cells was detected by methylcellulose semi-solid medium.③48 hours later,each group performed the same operation of cleaning the upper layer cells,and the adhesion of remaining T-ALL cells to BMSCs or adipocytes was observed under microscope.7.To explore the effect of inhibiting adipogenic differentiation of BMSCs on T-ALL xenograft mice.(1)To construct T-ALL mouse model and to transfect human T-ALL cell line.Jurkat cells with lentivirus carrying green fluorescent protein(GFP).After flow sorting,GFP+cells were amplified and cultured.Severe immunodeficient mice aged 6-8 weeks were exposed to 1.5Gy radiation.Jurkat cells of GFP+were injected into each mouse through tail vein to establish human T-ALL xenotransplantation model.The content of GFP+cells in peripheral blood of mice was continuously monitored by flow cytometry.When the proportion of GFP+cells in peripheral blood could be monitored,the mice were divided into three groups and began to give drugs.The first group:Control group,given control solvent therapy;the second group,DEX group,was given 1.5mg/kg/d DEX treatment;the third group:DEX+SREBF1 inhibitor group,treated with 1.5mg/kg/d DEX combined with 15mg/kg/d SREBF1 inhibitor(FatostatinHBr(FH)).Take continuous administration for 5 days a week for 2 days as a cycle,a total of 5 cycles,a total of 35 days.(2)Detection of therapeutic effect of peripheral blood,spleen and bone marrow in mice.①The proportion of GFP+cells in peripheral blood of mice was detected by flow cytometry at the end of each cycle,②Euthanasia was given to mice after the fifth cycle of administration,and the animals were dissected to evaluate the infiltration degree of T-ALL cells in spleen and bone marrow of the three groups.(3)To explore the changes of adipocyte content in bone marrow of mice after the third cycle and the fifth cycle,euthanasia was given to the mice,and the femur of the mice was extracted.The histopathological section of the femur of the mice was performed,and the difference of histopathology and the content of adipocytes were observed by Helie staining.Results:1.It was confirmed that adipocytes in bone marrow microenvironment of T-ALL patients after chemotherapy were significantly more than those before chemotherapy,and adipocytes in bone marrow biopsies of T-ALL patients increased significantly after chemotherapy.Quantitative analysis showed that the number of adipocytes and the area of lipid vesicles in bone marrow biopsies of T-ALL patients increased significantly after chemotherapy.2.The effect of standard induction chemotherapy drugs on adipogenic differentiation of BMSCs in T-ALL patients,there was no significant difference in the proportion of adipogenic differentiation of BMSCs in IDA and Vincristine groups compared with the control group.When BMSCs was treated with different concentrations of DEX,the intracellular fat droplets increased significantly with the increase of DEX concentration,and some of them fused into vesicles,suggesting that the adipogenic differentiation ability of BMSCs was significantly enhanced in T-ALL patients treated with DEX.3.Adipocytes derived from BMSCs after chemotherapy recruit T-ALL cells around themselves by releasing chemokine CXCL13.(1)Adipocytes can recruit T-ALL cells.1)By observing the distribution of T-ALL cells in the direct co-culture system,it was found that the number of T-ALL cells gathered around adipocytes was significantly higher than that around BMSCs.2)The number of T-ALL cells migrated into the lower chamber in the adipocyte group was significantly higher than that in the BMSCs group and the blank control group.(2)After chemotherapy,adipocytes derived from BMSCs attract T-ALL cells by releasing chemokine CXCL13.1)The expression of CXCL13 in adipocytes was significantly increased by qRT-PCR and ELISA experiments.2)After co-culture with adipocytes,the expression of CXCR5 on the surface of T-ALL cells increased significantly.3)After blocking chemokine CXCL13,the recruitment effect of adipocytes on T-ALL cells was weakened.4)Recombinant human CXCL13 promotes the chemotaxis of T-ALL cells.4.Effects of BMSCs-derived adip... |
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