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Objective:To observe and analyze the morphological characteristic of bone marrow and peripheral blood in patients diagnosed with de novo acute leukemia.Methods:From October 1, 2015 to December 31, 2021, 1151 patients aged 47 (26, 62) years, consisting of 602 males and 549 females with newly diagnosed acute leukemia in the Department of Hematology, Affiliated Hospital of Xuzhou Medical University, were collected to preform the morphological analysis in bone marrow and peripheral blood smears. Based on the comprehensive diagnosis results of morphology, immunology, cytogenetics, and molecular biology, comparison between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), AML with RUNX1-RUNXITI gene, AML with CBFβ/MYH11 gene, acute promyelocytic leukemia (APL) with PML/RARA gene, AML with NPM1 gene, the rest of the AML, Ph+ALL and Ph-ALL were performed by Chi-square test along with analysis of the differences in the ratio of wood bundle cells, pseudo-Chediak-Higashi (PCH) inclusions, cytoplasmic small particles, nuclear notches, leukemia cells with cup-like changes (cup cells); as well as the differences in the micromeganuclei, early immature granulocytes, plasma cells, high eosinophils and other accompanying cells and the distribution of "grape-like" aggregation. Finally, the morphological characteristics of acute leukemia cells, the appearance and arrangement of accompanying cells were summarized.Results:Between AML and ALL, there were statistically significant differences in cytoplasmic Auer bodies[(45.5%, 0%), χ 2=211.400, P<0.01], PCH inclusion bodies[(28.9%, 0%), χ 2=114.100, P<0.01], cytoplasmic fine particles[(20.7%, 2.9%), χ 2=53.798, P<0.01], nuclear notches[(0.7%, 6.1%), χ 2=30.906, P<0.01], and goblet cells[(4.9%, 0.3%), χ 2=13.495, P<0.01], micromegakaryus [(22.4%, 0.3%), χ 2=80.398, P<0.01], plasma cells[(87.6%, 10.6%), χ 2=604.241, P<0.01], hyperacidophils[(15.3%, 1.0%), χ 2=46.116, P<0.01] showed significant differences in the "grape-like" aggregation distribution. In AML with RUNX1-RUNXITI gene, the changes of vacuoles and PCH inclusion bodies are more obvious; in AML with CBFβ/MYH11 gene, the increase of hypereosinophils is more obvious; in APL with PML/RARA gene, the increase of woodbundle is more obvious. The morphology of nuclei chromatin, nucleolus, and vacuoles were also different among the groups. Comparison between Ph+ALL and Ph-ALL showed that Ph+ALL was more prone to develop early immature granulocytes and plasma cells (all P<0.05). Conclusion:There are significant differences between AML and ALL in the characteristics of leukemia cells, the regularity of accompanying cells, and the aggregation and distribution patterns. The subtypes of AML with specific genetic abnormalities have their own characteristics in the appearance of vacuoles, PCH inclusions, hypereosinophils, woodbundle cells, and goblet cells. Ph+ALL is more prone to present early immature granulocytes and plasma cells.
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Objective:To explore an assay that can concisely, rapidly, and accurately quantify the amount of chimeric antigen receptor (CAR)-T cells in the bone marrow or peripheral blood of patients after CAR-T cell immunotherapy by morphological analysis and flow cytometry assay, providing timely and accurate feedback for clinical treatment.Methods:We analyzed the CAR-T cell detection results in peripheral blood and bone marrow of 256 patients who received CAR-T cell immunotherapy in the Department of Hematology, Affiliated Hospital of Xuzhou Medical University from August 2016 to August 2021. All 256 patients survived more than one month after CAR-T cell infusion. Among them, there were 118 patients with multiple myeloma, 68 patients with acute lymphoblastic leukemia, and 70 patients with lymphoma. The morphological characteristics, positive rate and detection rate of CAR-T cell in peripheral blood and bone marrow were analyzed by morphological methods. The positive rate and detection rate of CAR-T in peripheral blood and bone marrow were analyzed by flow cytometry protein L detection. χ 2 test was used to comprehensively analyze the difference between the detection rate of the combined analysis of the two methods and the detection rate of the single method. Results:CAR-T cells have significant morphological characteristics, and there are obvious morphological differences from normal lymphocytes. The detection rates of CAR-T cells in peripheral blood or bone marrow by morphological methods and flow cytometry were 88.28%(226/256) and 79.29% (203/256), respectively. When the two methods were combined, the detection rate of CAR-T cells can reach 99.22%, with statistically significant difference comparing to that of single method( P<0.05). Through the analysis of the detection results of peripheral blood at different time points, it was found that the average detection rates of morphology and flow cytometry in 118 patients with multiple myeloma were 9.50% and 10.23% on the 7th day, and 13.50% and 15.19% respectively on the 15th day. On the 21st day, the average detection rates of morphology and flow cytometry were 8.00% and 10.07%, respectively. The average detection rates of morphology and flow cytometry in 68 patients with acute lymphoblastic leukemia were 12.00% and 11.22% on the 7th day, and 21.00% and 23.10% respectively on the 15th day. On the 21st day, the average detection rates of morphology and flow cytometry were 13.50% and 10.91%, respectively. The average detection rates of morphology and flow cytometry in 70 lymphoma patients were 7.50% and 10.35% on the 7th day, and 9.00% and 10.35% respectively on the 15th day. The average detection rates of morphology and flow cytometry at 21 days were 6.50% and 5.69%, respectively. The number of CAR-T cells in samples from patients with different diseases reached a peak around the 15th day. Conclusion:The detection rate of CAR-T cells from peripheral blood or bone marrow was significantly higher with the combination of the 2 methods compared to the single method.
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Objective@#To explore the function of NLRP1 in noninfectious pulmonary injury (nonIPI) after allogeneic stem cell transplantation (allo-HSCT) .@*Methods@#In this study, we established the model of allo-HSCT with C57BL/6 and NLRP-/- mouse as recipients. Chimera rate was measured by flow cytometry. The HE staining was used to observe the pathology changes in the lungs. NLRP1 and relevant inflammatory proteins were measured by Western Blot.@*Results@#On the day 14 after allo-HSCT, the chimera rate was more than 96%, HSCs of donors had been successfully transplanted into recipients. HE staining showed that nonIPI occurred after allo-HSCT. The degrees of injuries reached the peak on day 21. In addition, the expressions of MPO, NLRP1, p20, Mature-IL-1β and Mature-IL-18 had same tends with the degrees of nonIPI. When we knocked out NLRP1 gene of recipients, the degrees of nonIPI reduced and the expressions of MPO, p20, Mature-IL-1β and Mature-IL-18 were less than in non-knockout group.@*Conclusion@#allo-HSCT could cause nonIPI and high expressions of MPO, p20, IL-1β, IL-18, NLRP1. Knocking out NLRP1 gene could alleviate the degrees of nonIPI and reduce the expressions of relevant inflammatory proteins, indicating that NLRP1 might be one of factors contributed to nonIPI after allo-HSCT.
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Objective@#To explore effects of allogeneic hematopoietic stem cell transplantation (HSCT) in combination with infusion of endothelial progenitor cells (EPC) on bone marrow inflammatory injury.@*Methods@#6-8 weeks BALB/c (H-2Kd) mice after lethal dose of irradiation (TBI) were subjected to allogeneic bone marrow transplantation (BMT group) or co-transplantation of EPC (EPC group) . Samples of bone marrow cells of mice in each group on days 7,14,21,28 after transplantation were obtained to detect EPC cultural and cell chimeric rates by flow cytometer. Mice were sacrificed on days 7, 14, 21 and 28 post HSCT to analyze bone marrow pathology by H&E staining, the infiltration of macrophages and neutrophils by Western blot, validation expression levels of inflammatory complexes nlrp1、nlrp6 and its downstream molecules casepase-1 by Q-PCR and Western blot.@*Results@#Cell chimeric rate on day 7 after transplantation in EPC group[ (91.65±2.77) %] was significantly higher than in BMT group[ (83.69±1.26) %]. Alleviated osteomyelitis injury and inflammatory cell infiltration in EPC group were observed when compared with BMT mice. Also significant reductions of the levels of nlrp1、nlrp6、casepase-1 transcription complexes in EPC mice were noted when compared with BMT ones.@*Conclusion@#Co-transplantation of HSC and EPC could alleviate inflammatory cell infiltration and activation of the complex to promote the repair of bone marrow.
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<p><b>OBJECTIVE</b>To investigate the sensitivity of imatinib (IM) on Sup-B15 Ph+ acute lmphoblastic leukemia (ALL) cells indused by stromal cells OP9, and to further explore its mechanism.</p><p><b>METHODS</b>The study is divided into two group, Sup-B15 cells group and co-cultured with OP9 cells group (Sup-B15/OP9 group). The inhibitory effects of IM on leukemia cells were measured by CCK-8 test, and the apoptosis by Annexin Ⅴ/7-AAD dyeing and the percentage of CD 34+CD38- leukemia cells were determined by flow cytometry. ALDH1, CD144, and β-catenin mRNA were detected by real-time RT-PCR, protein levels by Western blot. Inmunoprecipitation was used to detect the level of β-catenin connected to CD144.</p><p><b>RESULTS</b>IM presented inhibitory effects on Sup-B15 and Sup-B15/OP9 cells at multiple concentrations from 10 μmol/L to 45 μmol/L. The IC50 of IM on Sup-B15/OP and Sup-B15 cells were 35.8 μmol/L and 6.3 μmol/L, respectively (P<0.05). After 24 h of 30 μmol/L IM treatment, the percentages of apoptosis cells in Sup-B15/OP9 and Sup-B 15 cell were (14.24 ± 2.11)% and (3.45 ± 0.68)%, respectively (P<0.05). The percentage of CD34+CD38- cells in Sup-B15/OP9 group was significantly higher than that in Sup-B15 group [(3.42 ± 0.28)% vs (0.16 ± 0.15)%, P<0.05]. As compared to Sup-B15 cells, the transcription of ALDH1 in Sup-B15/OP9 group was remarkably upregulated (0.097 ± 0.012 vs 0.046 ± 0.010, P<0.05), and the CD133 protein level was also upregulated in Sup-B15/OP9 group. The transcription of CD144 in Sup-B15/OP9 group was remarkably upregulated compared with Sup-B15 group (0.103 ± 0.015 vs 0.010±0.003, P<0.05), as well as the CD144 protein. β-catenin mRNA transcription has no obvious changes between Sup-B15 group and Sup-B15/OP9 group (P>0.05), while the whole β-catenin protein and the cell nucleus β-catenin significantly increased, as well as the β-catenin protein combined with CD144.</p><p><b>CONCLUSION</b>Co-cultured with OP9 cells, Sup-B15 cells show less sensitivity to imatinib. The raising activity of CD144 and CD144/β-catenin signaling may work in this procession.</p>
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Humans , Apoptosis , Cell Line, Tumor , Imatinib Mesylate , Philadelphia Chromosome , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Signal Transduction , Stromal Cells , beta CateninABSTRACT
<p><b>OBJECTIVE</b>To explore the function of nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammsomes in liver damage after allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>The study presented a murine (BALB/c-based) model of allo-HSCT. Chimera rate was measured by flow cytometry. The hematoxylin-eosin, Masson's trichrome, immunohistochemistry staining were used to observe the pathology changes in liver, then measured the degree of liver damage. Inflammation cells and NLRP3 were measured by Western blot, cytokines IL-1β, IL-18 and NLRP3 related genes were tested with real-time quantitative polymerase chain reaction (q-PCR).</p><p><b>RESULTS</b>Hematopoietic stem cells had been successfully transplanted, the chimera rate was geater than 97% on the 10th day. Liver damage occurred after allo-HSCT and suffered infiltration of inflammation cells, which reached the peak on day 15, then moved to moderate; the cytokines IL-1β, IL-18 had the similar trend with liver injury, and reached the highest level on day 15, their mRNA expressions increased by (1.19 ± 0.40) fold and (1.64 ± 0.76) fold, respectively; Meanwhile, caspase-1 had the similar trend, its mRNA expression increased by (3.51 ± 0.46) fold on day 15; the inflammasomes NLRP1, NLRP3, NLRC4 and NLRP5 expressed in liver on day 15 of post-allo-HSCT, and NLRP3 inflammasome expressed highest among them. The mRNA and protein level of NLRP3 inflammasomes were kept with the serious degree of the liver damage, its mRNA expression increased by (2.91 ± 0.41) fold on day 15.</p><p><b>CONCLUSION</b>NLRP3 inflammsome expressed in liver injury during allo-HSCT in mice, and may be one of the important factors contributed to liver injury.</p>
Subject(s)
Animals , Male , Mice , Carrier Proteins , Metabolism , Hematopoietic Stem Cell Transplantation , Inflammasomes , Metabolism , Liver , Metabolism , Pathology , Mice, Inbred BALB C , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Postoperative PeriodABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of the lentiviral vector mediated CXCR4 overexpressed mesenchymal stem cell (MSCs) on graft-versus-host disease (GVHD).</p><p><b>METHODS</b>Lentiviral vector containing CXCR4 was constructed. CXCR4 overexpressed MSC by lentiviral vector mediated were assessed. A major histocompatibility complex (MHC)-mismatched mouse model of bone marrow transplantation (BMT) from C57BL/6 donors to BALB/c recipients was constructed. Mice were divided into five groups: total body irradiation (TBI) group, mice received irradiation only; BMT group, mice were transplanted with bone marrow (BM) after TBI; GVHD group, mice were transplanted with BM and splencytes after TBI; CXCR4-MSC group, mice were transplanted with CXCR4-MSC, BM and splencytes after TBI; EGFP-MSC group, mice were transplanted with EGFP-MSC, BM and splencytes after TBI. The survival, body weight and clinical score of GVHD in transplanted mice were monitored. Liver, intestine and skin from mice in each group were obtained for histological examination. Plasma concentrations of inflammation factors such as interleukin (IL)-2, IFN-γ and TNF-α were also determined using a cytometric bead array cytokine kit.</p><p><b>RESULTS</b>All mice in TBI group died within 14 days, while all of BMT group survived. The mean survival times for GVHD, EGFP-MSC and CXCR4-MSC groups were (17.0 ± 2.3) d, (21.7 ± 4.8) d and (30.1 ± 9.1) d, respectively. Treatment with CXCR4 over-expressing MSCs could decrease the mortality rate. All mice in each group developed clinical signs such as hunched posture, dull fur, diarrhea and weight loss. Meanwhile, histopathological findings in target organs were confirmed the presence of GVHD. While, clinical GVHD scores and histopathological scores in CXCR4-MSC group were significantly lower than that of GVHD group. Moreover, compared with control groups, the plasma IL-2, IFN-γ and TNF-α level in recipients infused with CXCR4-MSC were significantly decreased (P<0.05).</p><p><b>CONCLUSION</b>The results revealed that CXCR4- transduced MSCs could effectively control the occurrence of mouse GVHD following allogeneic BM transplantation.</p>
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Animals , Mice , Bone Marrow Transplantation , Cytokines , Genetic Vectors , Genetics , Graft vs Host Disease , Lentivirus , Genetics , Mesenchymal Stem Cells , Allergy and Immunology , Receptors, CXCR4 , Genetics , Allergy and Immunology , Transplantation, HomologousABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of bromdomain protein 4 (BRD4) inhibitor GSK525762A on the proliferation, apoptosis of B-cell acute lymphoblastic leukemia cell line RS4;11 cells, and to further explore the mechanism.</p><p><b>METHODS</b>Compared with Jurkat leukemia cells, the activity of BRD4 on RS4; 11 cells were inhibited by the inhibitor GSK525762A. The inhibitory effects of BRD4 on RS4; 11 cells were measured by CCK-8 test and the apoptosis of those cells was determined by AnnexinV/7-AAD dyeing using flow cytometry. The transcripts of anti-apoptotic genes c-myc, Bcl-2, CDK6 and proapoptotic genes Bad, Bak, Bax were detected by quantitative PCR, and the expression of Bcl-2 and Bak proteins were detected via Western blot.</p><p><b>RESULTS</b>Proliferation of RS4;11 cells could be inhibited by GSK525762A in a time- and dose-dependent manner, and the inhibitory IC50 at 48 and 72 h was 6.174 and 1.996 μmol/L, respectively. Compared with DMSO in control group, the levels of c-myc, Bcl-2 and CDK6 mRNA transcripts in RS4; 11 cells were reduced in GSK525762A treated group, while the levels of Bad, Bak, Bax mRNA transcripts were enhanced,moreover, Bcl- 2 protein levels decreased and Bak protein levels increased. However, the inhibitory effect of GSK525762A on Jurkat cells proliferation was not obvious.</p><p><b>CONCLUSION</b>GSK525762A can inhibit the proliferation of RS4; 11 cells and promoted cells apoptosis. The possible mechanisms underlying this phenomenon might be achieved via downregulation of Bcl-2 protein induced apoptosis of leukemia cells.</p>
Subject(s)
Humans , Apoptosis , Benzodiazepines , Pharmacology , Caspase 3 , Metabolism , Cell Line, Tumor , Cell Proliferation , Leukemia, B-Cell , Metabolism , Pathology , Proto-Oncogene Proteins c-bcl-2 , MetabolismABSTRACT
Objective To establish a reproducible mouse model of hepatic veno-occlusive disease (HVOD) after allogeneic bone marrow transplantation (aallo-ABMT) and explore its pathogenesis.Methods Balb/c mice were randomly divided into three groups:(1) normal saline (NS) control group; (2) total body irradiation (TBI) group; (3) allogeneic bone marrow transplantation (allo-BMT) group.Liver weight,total bilirubin (TBil),tumor necrosis factor α (TNF-a),interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were detected on the day 0,5,10,15 and 20 after transplantation.Hepatic vein and sinusoid congestion,infiltration of inflanmatory cells,and damage to hepatic cells and vascular endothelial cells were observed under the light microscopy after HE staining.Fibrosis of hepatic sinusoids and venule was observed under the light microscopy after Masson staining.Results Liver weight and TBil levels were elevated at 5th day and reached the peak at 15th day after all-ABMT.The changes of hepatic congestion and edema were obviously observed and there was infiltration of inflammatory cells at 5th and 10th day after alloABMT.At 15th and 20th day,hepatic congestion,edema and necrosis were reduced and liver damage was mainly presented with liver fibrosis and inflammatory infiltration.All mice died within 10 days after TBI,and hepatic congestion and edema were aggravated.As compared with NS control group,TNF-α,IL-6 and MCP-1 concentrations were significantly increased after all-ABMT.Conclusion A reproducible mouse model of hepatic veno-occlusive disease after all-ABMT was successfully established,and the pathogenesis was closely related to endothelial damage caused by total body irradiation,inflammatory cell infiltration and increased concentrations of cytokines.
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Objective To (e)xplore inhibition of endothelial progenitor cells (EPCs) against hepatic vein thrombosis after allogeneic bone marrow transplantation (BMT).Methods Balb/c mice were randomly divided into three groups: (1) BMT group [Balb/c mice were injected intravenously with 5 × 106 bone marrow cells after total body irradiation (TBI)]; (2) EPCs co-transfusion with bone marrow cells group: 5 × 105 EPCs were infused into recipient mice simultaneously; (3) Normal control group.Liver index was detected on the day 0,5,10,15 and 20 after transplantation.Hepatic vein thrombosis,hepatic cells and vascular endothelial damage were observed under the light microscopy after H&E staining.The injury of liver cells,liver veins,hepatic sinusoidal endothelial cells (SECs)and platelet adhesion conditions were observed under a transmission electron microscope (TEM).The proportion of activated platelets and TNF-α concentration in peripheral blood were detected by using flow cytometry.Results On the day 0,5,10,15 and 20 after transplantation,the proportion of activated platelets,liver index and TNF-α concentrations in BMT group and EPCs co-transfusion group showed an upward trend,peaked on the 15th day,and then decreased.However,they were still significantly higher than those in normal control group (P＜0.05).The above parameters in EPCs co-transfusion group at each time point were significantly lower than those in BMT group (P＜0.05).As compared with BMT group,platelet adhesion decreased,hepatic vein thromboses were reduced,hepatocyte swelling and necrosis were alleviated,and liver damage repaired rapidly in EPCs co-transfusion group.Conclusion EPCs co-transfusion with bone marrow cells could inhibit the hepatic veins thrombosis and ameliorate liver damage significantly.
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Objective To explore prevention of cyclosporine A (CsA) combined with Cobalt protoporphyrin (CoPP) against murine graft versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods C57BL/6 (H-2Kb) mice were used as donors and BALB/c (H-2Kd) mice as recipients,which were randomly divided into 4 groups.The mice in total body irradiation group (TBI group) were lethally irradiated and injected intravenously with PBS; The mice in Allo-HSCT group (BS group) were lethally irradiated and injected intravenously with bone marrow cells and spleen cells; The mice in CsA intervention group (CsA group) were injected with CsA intraperitoneally after allo-HSCT; The mice in CsA combine with CoPP intervention group (combination group) received both CsA and CoPP intraperitoneally after alloHSCT.Recipients were monitored for condition,survival rate and weight.The liver,small intestine and skin in the recipients were gained and pathological changes of GVHD were assessed.The kidney was stained with Masson staining dye to observe the tissue fibrosis.The expression levels of renal HO-1 mRNA in the recipients were detected.Results In contrast to BS and CsA groups,GVHD degree in combination group was mild,with less reduction and quick recovery of weight.On the day 30 after HSCT,survival rate in BS group was 36.8％,and that in combination group and CsA group was 69.6％ and 53.5％ respectively (P＜0.05).In comparison with BS and CsA groups,pathological changes in combination group were mild,cellular edema and degeneration degree of the liver,small intestine and skin were slight,and few necrosis and infiltrated inflammatory cells were observed.Tubulointerstitial fibrosis hardly occurred in combination group,but it occurred in CsA group abundantly.As compared with BS group,the expression levels of HO-1 mRNA was increased in combination group,while decreased in CsA group (P＜0.05).Conclusion CsA combined with CoPP enhanced the protective effect of CsA against GVHD,moreover,CoPP could alleviate the side effects of CsA,which might be related with up-regulation of the expression levels of HO-1.
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Objective To observe the effect of bortezomib on acute graft-versus-host disease (aGVHD) in an aGVHD model of mice and investigate the related mechanism. Methods Male C57BL/6( H-2Kb)mice were used as donors and female Balb/c (H-2Kd) mice used as recipients. Balb/c mice received total body irradiation (TBI) by 7.0 Gy X-radiation, and randomly divided into five groups. normal (group A), TBI (group B), TBI + bortezomib (group C), TBI + bone marrow cells (BMC) + spleen cells (SC) (group D) and TBI + bortezomib + BMC + SC (group E). The physical signs and the pathological damage of aGVHD, mean survival time, and chimerism were observed in recipients. The NF-κB p65 levels in nuclei of the liver and small intestine tissues of groups A,B and C were analyzed by Western blot. Results ( 1 ) The clinical aGVHD score in group D was (7.37±0. 32), significantly higher than in group E (5.85 ± 0.40) (P＜0. 05). Histopathology of the gut, liver and skin illuminated that the Ⅲ-Ⅳ degree GVHD occurred in group D. The occurrence of aGVHD in group E was later than in group D. The symptoms and the pathological damage of aGVHD in group E were milder than in group D. The average survival time in group E was significantly longer than that in group D (P＜0.05). The percentage of donor-derived cells in recipient mice was above 90% at day 12 after transplantation; (2) NF-κB p65 levels in nuclei of the liver and small intestine tissues in group B was significantly higher than in group C on the day 1,3 and 5 (P＜0. 05). Conclusion Bortezomib can inhibit the activation and expression of NF-κB,which may be the underlying mechanism for it to relieve aGVHD.
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Objective To assess the functional role of TH 17 cells in allogeneic hematopoietic stem cell transplantation (allyHSCT).Methods Bone marrow monocytes and splenic T cells were enriched from C57/BL6 donors.Recipient Balb/c mice were irradiated with 7.5 Gy total body irradiation (TBI) and injected with 5 105 splenic T cells and 5 106 bone marrow monocytes.Survival was monitored daily,clinical graft-versus-host disease (GVHD) was assayed three times a week,and detailed histopathologic analyses of lung were performed at day six after Allo-HSCT.Flow cytometry analysis was performed using CD3-FITC,CD4-PE,CD45-PerCP-CyS.5 monoclonal antibodies.Cells were stained for intracellular cytokines using mouse TH 1/TH2/TH 17 cytokine kit.Results All the experimental animals showed GVHD manifestations on the day 6 after transplantation.Animals from BMT and HF groups were scarified and histological analysis of lung was performed.Absence of TH 17 cells induced severe pathologic pulmonary lesions.The histopathology of the lung tissue was characterized by disorganization,epithelia cell damage,interstitial fibroplasias,and monocytes infiltration.The proportion of TH1 and TH 17 in BMT group was (5.53 ± 0.11 ) ％ and ( 1.04 ± 0.34)％ respectively,both significantly different from that in HF group.The levels of IL-17A and IFN-γin BMT group were (2.81 ±0.19) and (42.97 ± 0.23) pg/mL respectively.IL-17A could not be detected in HF group,yet the level of IFN-y was only (9.89 ± 0.51 ) pg/mL.IL-10 in both HF and BMT groups was not detectable.Conclusion Lung is on target of aGVHD.IL-17A may play a key role in the lung injury after transplantation.
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Objective To determine the conditioning regimen suitable for mice allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods Twelve BALB/c mice were randomly divided into 2 equal groups to undergo X-ray irradiation by linear accelerator at the dose of 7.0 Gy (pure X-ray group) or 60Co source irradiation at the dose of 7.0 Gy (pure γ-ray group).Thirty mice were randomly divided into 2 equal groups to undergo X-ray irradiation and then infusion of bone marrow from donor mice via caudal vein (X-ray + transplantation group) or γ-ray and then infusion of bone marrow via caudal vein (γ-ray + transplahtation group).3,5,7,10,15,20,and 30 d later peripheral blood samples were collected to calculate the number of white blood cells (WBCs) and detect the chimeric rates of lymphocytes by flow cytometry.5,10,and 20 d after irradiation 15 mice were killed with their lung,liver,small intestine,spleen,and femurs taken out to undergo pathological examination.Results The survival rates during the period 5-15 days of the γ-ray + transplantation group were all significantly higher than those of the X-ray + transplantation group.The pathological changes of organs of the X-ray +transplantation group were all more severe than those of the γ-ray + transplantation group.Since the fifth day after transplantation cells originating from the donor began to appear in the peripheral blood.The chimeric rate of the γ-ray + transplantation group 10 days after transplantation was (95.53± 2.57) %.The chimeric rates 5,10,and 20 days after transplantation of the γ-ray + transplantation group were all significantly higher than those of the X-ray + transplantation group (t = 15.263,3.256,P < 0.05).The WBC count of both irradiation groups decreased to the lowest level 5 d later and began to increase 10 days after transplantation and the WBC counts of the γ-ray + transplantation group 10 and 20 days aftertransplantation were both significantly higher than those of the X-ray + transplantation group (t = 3.624,6.695 ,P < 0.05).The chimeric rats of the peripheral lymphocytes 10 and 20 days after transplantation of the γ-ray + transplantation group were both significantly higher than those of the X-ray + transplantation group (t = 12.317,8.295,P < 0.05).The homogeneity rate of transplantation of the γ-ray +transplantation group was better than that of the X-ray + transplantation group.Conclusions As a conditioning regimen in allogeneic hematopoietic stem cell transplantation γ-ray irradiation causes milder injury and accelerated reconstitution of hematopoiesis and immunity,in comparison with X-ray irradiation.
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Objective To study the repair function of united endothelial progenitor cells (EPC)transplantation on injured liver endothelium by bone marrow transplantation (BMT) conditioning.Methods C57BL/6 mice were divided into four groups randomly: normal control group, without any treatment; irradiation alone group, administered a total body irradiation(TBI) pretreatment, without BMT; (3) BMT alone group: C57BL/6 mice were infused with bone marrow mononuclearcells (MNC) 5 × 106/only through caudal vein not more than 4 h after the same TBI pretreatment as the irradiation alone group; united transplantation group: receiving the same way as the BMT alone group, but C57BL/6 mice were infused with EPC 5 × 105/only at the same time. Two, 4, 7, 14, and 21 days after the TBI, the changes of the liver weight were observed regularly. The histopathological examination of liver was done at the 4th, 7th, 14th, and 21st day after the TBI. Results In irradiation alone group, BMT alone group and united transplantation group the liver weight began to increase significantly on the day 2 and peaked at 14th day after the TBI, and the peaks were respectively (1.65±0. 15) times (P＜0. 05), (1.61 ±0.06) times (P＜0.05), and (1.11 ±0.40)times (P＜0. 05) of those in normal control group. At the day 14, the liver weight in irradiation alone group, BMT alone group and united transplantation group began to decrease, and on the day 21 the liver weight in united transplantation group had been completely restored to normal level, however the liver weight in irradiation alone group and BMT alone group were still significantly heavier than that in normal control group (P＜0. 05). Liver histopathological examination revealed that there were obvious sinusoidal endothelial cells (SEC) injury, hepatocyte edema and severe inflammatory cell infiltration in irradiation alone group, and on the day 7 the hepatocyte edema and necrosis were significantly worse than before, and almost no alive SEC were found. On the day 14 the injury of SEC in BMT alone group was lighter than before, but on the day 21 the injury had not returned to normal. On the day 7 the injury of SEC, hepatocyte edema and necrosis were alleviated in united transplantation group as compared with irradiation alone group and BMT alone group, and on the day 14 the injury had returned to normal basically. Conclusion The transplantation conditioning could damage recipient liver endothelium and the injury would persist, and united EPC infusion could repair the injured SEC following BMT.
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Objective To explore a proper dose of endothelial progenitor cells (EPCs)administration that can achieve optimal hematopoietic improving effectiveness in a murine allogeneic hernatopoietic stem cell transplantation (allo-HSCT) model.Methods Female Balb/c mice were lethally irradiated with 60Co source,and then were injected intravenously with 5 106 bone marrow cells from C57BL/6 mice (bone marrow transplantation group).In co-transfer experiments,5 × 104,1 ×105,5 × 105 or 1 × 106 donor EPCs (EPCs treated groups) were injected simultaneously with bone marrow cells.The recipients were monitored for survival,peripheral white blood cells,hematopoietic stem cells (HSCs) and bone marrow histology.Results Compared with bone marrow transplantation group,all EPCs treated groups had accelerated recovery of peripheral white blood cells (P＜0.05),platelets (P＜0.05) and HSCs (P＜0.05).When infused with less than 5 × 105 EPCs,these effective hernatopoietic improving phenomena showed a positive correlation with the administrated doses of EPCs.However,when infused with 1 × 106 EPCs,the mice showed lower survival rate (P＜0.05)and slower recovery of peripheral white blood cells (P＜0.05),platelets (P＜0.05) and HSCs (P＜0.05) than 5 × 105 EPCs treated grpup.Bone marrow histopathology analysis confirmed the above findings.Conclusion Co-transfer with donor EPCs can improve survival rate and hematopoietic reconstitution of recipient mice in allo-HSCT,and 5 × 105 EPCs should be a proper dose to achieve the best effectiveness.
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Objective To explore the influence of the lentiviral vectors-mediated mouse genetic engineering regulatory T cells (Treg) infused after allogeneie bone marrow transplantation (allo-BMT)on graft-versus-host disease (GVHD) in mice.Methods Lentivirus-mediated expression of Forkhead box P3 (Foxp3) converted CD4~+ CD25~- T cells from Balb/c mice into engineered Tregs in vitro.An allo-BMT model of Balb/c→C57BL/6 mice was established.Mice were randomly assigned into four groups:(1) The recipients in engineering Treg group were injected with 5×10~6 donor bone marrow cells and 5×10~6 splenoeytes plus 5×10~6 genetic engineering Treg;(2)The recipients in transplantation control group were iniected with 5×10~6 donor bone marrow cells and 5×10~6 splenocytes;(3) The recipients in radiation group were injected with 0.2 ml RPMI 1640;(4)The recipients in empty vector control group were injected with 5×10~6 donor bone marrow cells and 5×10~6 splenocytas plus 5×10~6 empty vector transduced CD4~+ CD25~- T cells.Survival time,clinical GVHD Score or histopathological analysis(skin,liver and small intestine) were observed after allo-BMT.Chimerism of bone marrow cells from recipients survived for 60 days after transplantation was measured Results The mean survival times in radiation group, transplantation control group,erIgineering Treg group and empty vector control group were (8.8±0.6),(36.7±2.5),(51.6±4.0) and (34.1±2.3)days respectively.The survival time in engineering Treg group was signiticantly prolonged as compared with other groups as judged by the log-rank test(P<0.05).Histopathological ahalysis in several target organs (skin,liver and small intestine)confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD were observed in recipients in engineering Treg group and clinical GVHD scores in this group were significantly decreased compared to transplantation control group and empty vector control group. Conclusion Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD during allo-BMT in mice
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Objective To study the relationship between graft-versus-host disease (GVHD) and endothelium injury following hematopoietic stem cells transplantation in mice. Methods C57BL/6 mice as donors and Balb/c mice as recipients were randomly divided into 4 groups: control group, bone marrow transplantation group, GVHD group, GVHD mitigation group. The clinical manifestations,circulating endothelial cells and tissue pathological changes were observed at different time points after transplantation. Results No manifestations of GVHD were found in each group at the day 5, while those were found in GVHD group at the day 9 and all died within 15 days. The counts of endothelial cells in peripheral blood showed no significant difference at the day 5 between GVHD group (7. 34 ±1.26 cells/μl) and bone marrow transplantation group (11.51 ± 7. 40 cells/μl) or GVHD mitigation group (7. 36 ± 0. 16 cells/μl), while among three groups there was statistically significant difference at the day 9 (GVHD group: 153. 64 ± 35. 35 cells/μl vs bone marrow transplantation group: 10. 49 ±5. 61 cells/μl and GVHD mitigation group: 47. 82 ± 4. 69 cells/μl). The scores of pathological aGVHD had no significant difference at the day 5 between GVHD group (4. 33± 1. 53) and bone marrow transplantation group (3. 33 ± 0. 58) or GVHD mitigation group (4. 00 ± 1.73), while among three groups there was statistically significant difference at the day 9 (GVHD group: 10. 0 vs bone marrow transplantation group: 3. 33 ± 1.15 or GVHD mitigation group: 4. 33 ± 0. 58) and at the day 14 (GVHD group: 10. 33 ± 2. 58 vs bone marrow transplantation group: 2. 33 ± 1.25 or GVHD mitigation group 3. 33 ± 1.15). Conclusion Occurrence of GVHD causes endothelial damage again and injured endothelium worsens the GVHD.
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Objective To explore the influence of the lentiviral vectors-mediated mouse genetic engineering regulatory T cells (Treg) infused after allogeneic bone marrow transplantation (alloBMT) on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect in mice.Methods Lentivirus-mediated expression of Forkhead box P3 (Foxp3) transformed CD4 + CD25- T cells from Balb/c mice into engineered Tregs in vitro. An allo-BMT model of Balb/c→C57BL/6 mice was established. The recipients were given lethal X-ray total body irradiation before transplantation.Mice were randomly assigned into five groups and each group contained 10 recipients: (1) The recipients in radiation group were injected with 0.2 ml RPMI 1640; (2) The recipients in leukemia control group were injected with 5 × 106 donor bone marrow cells and 500 mouse T-cell leukemia/lymphoma cells (EL4 cells); (3) The recipients in transplantation control group were injected with 5 × 106 donor bone marrow cells and 5 × 106 splenocytes plus 500 EL4 cells; (4) The recipients in engineering Treg group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 genetic engineering Treg; (5) The recipients in empty vector control group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 empty vector-transduced CD4+ CD25- T cells. Survival time, clinical GVHD score or histopathological analysis (skin, liver and small intestine) were observed after allo-BMT. Chimerism of bone marrow cells from recipients survived for 60 days after transplantation was measured. Results The mean survival time in radiation group, leukemia control group, transplantation control group,engineering Treg group and empty vector control group was ( 10. 3 ± 1.5), (20. 7 ± 1.9), (26. 0 ±4.3), (49. 0 ± 17. 7) and (24. 4 ± 4. 1 ) days respectively. The survival time in engineering Treg group was significantly prolonged as compared with other groups as judged by the log-rank test (P＜0. 05).Histopathological analysis in several target organs (skin, liver and small intestine) confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD or leukemia were observed in recipients in engineering Treg group and clinical GVHD scores in this group were significantly decreased as compared with transplantation control group and empty vector control group. Conclusion Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD without affecting GVL activity during allo-BMT in mice.
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Objective This study aimed to establish the leukemia mouse model by using EL4/DsRed cell line expressing red fluorescent protein (DsRed) and to evaluate the model. Methods After total body irradiation with X-ray of 7.0 Gy, C57BL/6 mice were inoculated 5×106 bone marrow cells mixed different numbers of EL4/DsRed cells via tail vein. The model was evaluated by flow cytometry (FCM), reverse transcriptase-polymerase chain reaction (RT-PCR), and histopathology. Results The incidence of leukemia was 100 %. The presence of EL4/DsRed cells was found in liver, spleen, bone marrow and peripheral blood of recipients by FCM two weeks after transplantation. Pathological section revealed that all recipients had several organs infiltration apparently. With the increase in the number of inoculated tumor cells, the survival time of recipients was reduced and the infiltration of leukemia cells in organs was more serious. Conclusion Mouse leukemia model was successfully established when C57BL/6 mouse was intravenously transplanted with ≥5×102 EL4/DsRed cells. The model could be employed usefully in the future research such as the pathogenesis of leukemia and minimal residual disease (MRD).