| Background and objectiveAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is nowadays the mosteffective therapies for malign diseasesof the hematopoietic system. While Acutegraft-versus-host disease(aGVHD) is the main complications after allo-HSCT for lowquality of life or even mortality. Except for the traditional aGVHD organs, the majorityof aGVHD patients accompany with leucopenia, oligocythemia, thromobocytopenia,even panhematopemia. However, the mechanism of aGVHD on normal HSCs has notbeen definitively investigated. Hematopoietic stem cell functions are the supported andregulated by the hematopoietic microenvironment (niche,). Yusuke Shono et al.mentioned the concept of the bone marrow of aGVHD, in their opinion, the aGVHDhematopoietic dysfunction stems from aGVHD,aGVHD attacks niche, results indysfunction of niche supporting hematopoietic function, which affects hematopoieticstem cells (Hematopoietic stem cell, HSC), the function [3]. Niche is composed of avariety of stromal cells, extracellular matrix, cell factor,. a class expression of nestin(nestin) stromal cells are mesenchymal stem cells (Mesenchymal stem cells, MSC), forthe sympathetic regulation of signaling receptors (of β3adrenergic receptor), does notmark the CD45expression, the vascular endothelial marker CD31and the hematopoieticcells, c-Kit, the bone marrow Nestin+mesenchymal stem cells. the bone marrow Nestin+mesenchymal stem cells express of support for HSC self-update and owned by the nestfactor (such as of CXCL12, c-kit of ligand, IL-7Expression of angiopoietin-1, VCAM-1)[4], description of the bone marrow Nestin+mesenchymal stem cells support andregulate HSC the function has an important role. To investigate the hematopoieticfunction in acute graft-versus-host disease (aGVHD), the mechanism of inhibition studyaGVHD the bone marrow Nestin+mesenchymal stem cells count change and changemechanism to find the reason of aGVHD induced hematopoietic dysfunction, and for thefurther search for aGVHD treatment means to enhance hematopoietic stem celltransplantation efficacy of the theoretical basis. Materials and methodsMice C57BL/6J mice were used for breeding CB6F1mice, purchased from ShanghaiInstitute of Experimental Animal Center,8weeks, male.BALB/C mice were used asbreeding CB6F1mice, purchased from Shanghai Institute of Experimental Animal Center,8weeks, female.CB6F1mice (leukocyte antigen phenotype CD45.2), male C57BL/6Jmice and female BALB/C mice, hybrid generation, paired by the laboratory breeding,6-8weeks, body weight16-18g, female.B6.SJL mice (leukocyte antigen phenotypeCD45.1) were purchased from the Chinese Academy of Medical Sciences, Institute ofHematology, Professor Cheng Tao laboratory gifts and paired breeding in the laboratory,8-10weeks, body weight18-22g, male.All mice were keeping the room sterilesquirrel-cage reared in the animal house of the Second Military Medical University ofSPF grade, positive pressure air clean room sterile squirrel cage, bedding, feed and waterwere sterilized in high temperature and pressure, changed weekly.aGVHD modelinduction Donor: B6.SJL (leukocyte antigen phenotype CD45.1),8-10weeks, weight18-22g, male.Receptor: CB6F1(leukocyte antigen phenotype CD45.2), male of C57BL/6mice and female BALB/C mice of hybrid generation,6-8weeks, weight16-18g,female. Pre-transplant weight stratified randomization, required no difference in weightbetween the groups (P>0.05). Total receptor mouse were divided into two groups:experimental group (aGVHD group),the control group (the BMT group). Lethallyirradiated recipient mouse were placed in the the breathable plexiglass box,received asingle133Cs-ray irradiation to a total dose8Gy, dose rate69.4cGy/min. Radiation sourcewas provided by Fudan University, Shanghai Medical College of Radiology.4-6hoursafter exposure, the mouse tail vein infusion of cells:(C57BL/6×BALB/c) CB6F1micewere transplanted with haplo-MHC matched BM from B6.SJL mice (5×106) with spleencells from B6.SJL mice (6×107).The mice in control group received BM from B6.SJLmice alone(5×106).System assessment of aGVHD Survival was monitored daily.Signsand symptoms of aGVHD mice of experimental group were observed daily aftertransplantation, namely, activity, arched, hair loss, diarrhea, anorexia; aGVHD targetorgan pathology were purchased+14days after transplantation, each group of mice organs (small intestine, liver soak, lung, spleen), placed in10%neutral formalin solution,sent to the Shanghai Ninth people's Hospital to do the biopsy, and analysis of HE staining.Blood test was measured weekly of peripheral blood, the mice tail vein20μL blood plus140μL heparin, submission of the Second Military Medical University, ChanghaiHospital, Emergency Clinical Laboratory.Flow cytometric analysis and cell sortingMice BM cells were obtained from flushing ilias, femurs and tibias. Purified fluoresceinPE-cy7-Ter119,PE-cy7-Gr1,PE-cy7-Mac1,PE-cy7-B220,PE-cy7-CD3,PE-cy7-CD4,PE-cy7-CD8,FITC-Sca-1,APC-c-Kit,FITC-CD45,Alexa-Fluro647-Nestin,Percp-cy5.5-c-Kit,PE-MHC-I,PE-MHC-II,PE-cy7-CD44,PE-CD29,PE-VFGFR3,FITC-CD44,PE-cy7CD45,PE-CD29were purchased from BD Bioscience,or eBioscience. Fixation and permeabilizationPerm, wash Buffer were purchased from BD Bioscience.Cells were stained withapproppriate concentrantion of mAbs.After incubation with mAbs,data were collected withBD FACS AriaI,and then analyed with Flowjo software.The purity of sorted cells wasroutinely more than98%.Statistical analysis Experimental results were presented as mean±standard deviation.The non-parametric test was used for measurement data (SPSS16.0software package), and the Log-rank test was used for survival data (graphpad prism5software).A P value of＜0.05was considered statistically significant.ResultsGeneral biological characrerristics of CD45-Nestin+cells The bone marrow CD45-Nestin+cells are a class of rare cells in bone marrow,which accounted (0.08±0.00)%ofbone marrow nuclear cells,(4±0.6)%of CD45-cells, absolute number of about (1.6±0)×10~5per mice. CD45-Nestin+cells highly express CD44(98.70±1.49)%, CD29(84.88±13.57)%, Sca-1(89.9±3.50)%, do not express MHC-II,expression of vascularendothelial marker VEGFR3,the hematopoietic stem/progenitor cell marker c-Kit. Thehaploidentical aGVHD model a simple model of the bone marrow transplant to buildthe success of aGVHD group mice on day+14after transplantation, the gradual emergenceof arched, lack of exercise, hair loss, diarrhea, and weight loss typical of aGVHDsymptoms. aGVHD mice, median survival time of21days (15-27days), bone marrowtransplant alone group in the30-day observation period, all survived. aGVHD group targetorgans liver, lung, spleen, small intestine and other organs pathology seen significantinflammatory cell infiltration, and organizational structure was partially destroyed, simple basic integrity of the organizational structure of the organs of bone marrow transplantgroup. aGVHD group mice after the transplantation of hematopoietic dysfunction+7days,two groups of three lines of mouse peripheral blood reached the bottom, then graduallyrecovered group of three lines of aGVHD recovery than a simple bone marrowtransplantation group, but in the day+21after transplantation three lines of aGVHD groupdecreased significantly, significantly lower than that of bone marrow transplant group.After transplantation+7days,+14days,+21days aGVHD group of bone marrow nuclearcell count less than pure bone marrow transplant group [+7after transplantation:(.454±0.066)×10~8vs (.616±0.167)×10~8. P=0.094, day+14after transplantation:(0.632±0.124)×10~8vs (1.48±0.16)×10~8, P=0.009;+21days after transplantation:(0.3±0.088)×10~8vs (3.694±0.61)×10~8, P=0.009]. Transplant day+14,+21days aGVHD group ofhematopoietic stem cells (HSC) and hematopoietic progenitor cell (HPC), the absolutenumber is lower than a simple bone marrow transplant group, the HSC: after the transplantday+14:(1.05±0.30)×10~5vs (3.28±0.41)×10~5, P=0.009;+21days aftertransplantation:(0.39±0.126)×10~5vs (7.43±0.676)×10~5, P=0.009; the HPC: aftertransplantation+14days:(1.96±.776)×10~5vs (10.554±1.412)×10~5, P=0.009;+21days after transplantation:(0.598±0.162)×10~5vs (19.138±2.46)×10~5. P=0.009.groups for aGVHD when the bone marrow contains mesenchymal stem cells (CD45-c-kit-CD44+CD29+gating) in the bone marrow cells does not decrease, but the absolutedecrease in the number of transplant+7days,+14days,+21days by flow cytometry ofbone marrow contains mesenchymal stem cells in cell population (CD45-c-kit-CD44+CD29+gating) in bone marrow nucleated cells, absolute number, the results showed thataGVHD group of bone marrow contains mesenchymal stem cells, cell groups (CD45the-c-Kit-CD44+CD29+set the door) in the bone marrow have nucleated cells, theproportion with simple bone marrow transplant group without significantly with thedifference [after transplantation+7days:(0.801±0.14)%vs (1.36±0.35)%, p=0.064;day+14after transplantation:(0.7±0)%vs (0.86±0.11)%, p=0.066;+21days aftertransplantation:(0.6±0.36%vs0.7±0.28)%, p=0.71]. aGVHD group bone marrowcontains mesenchymal stem cells in cell groups (CD45-c-Kit-CD44+CD29+gating) theabsolute number is less than pure bone marrow transplantation group [+7days aftertransplantation:(3.63±0.66)×10~5vs (7.75±2.08)×10~5, P=0.05; day+14aftertransplantation:(4.4±0)×10~5vs (12.8±1.73)×10~5, P=0.034;+21days aftertransplantation:(1.8±1.08)×10~5vs (25.47±9.63)×10~5, P=0.05]. aGVHD when the bone marrow of Nestin+mesenchymal stem cell ratio, the absolute number of reducingtransplant+7days,+14days,+21days, analyzed by flow cytometry of bone marrow ofNestin+mesenchymal stem cells in the bone marrow nucleated cells in the proportion andabsolute number of results the aGVHD group of bone marrow of Nestin+mesenchymalstem cells in the bone marrow nucleated cells in less than a simple bone marrow transplantgroup [+7after transplantation:(0.0366±0.004)%vs (0.071±0.01)%, P=0.008; day+14after transplantation:(0.027±0.007)%vs (0.054±0.013)%, P=0.008;+21days aftertransplantation:(0.019±0.004)%vs (0.053±0.006)%, P=0.008], of aGVHD in bonemarrow of Nestin+mesenchymal stem cells absolute number is less than a simple bonemarrow transplant group [+7after transplantation:(1.62±0.192)×10~4vs (4.37±0.65)×10~4, P=0.009,+14days after transplantation:(.564±0.14)×10~4vs (19.316±2.31)×10~4,P=0.008]. aGVHD when the bone marrow for the body-source of CD4+and CD8+Tlymphocytes FASL expression were increased, and the transplantation of early CD4+Tlymphocytes FASL expression higher than CD8+T cell transplantation after+3days+7days,+14days of flow cytometry detection of aGVHD mice, simple bone marrowtransplantation mice bone marrow donor source of CD4+/CD8+T lymphocytes FASLexpression, the results show aGVHD group of CD4+/CD8+T lymphocytes FASLexpression was higher than the BMT group of CD4+T lymphoid Cell:+3days aftertransplantation:(92.2±0.8)%vs (68.63±6.55)%, p=0.003;+7days after transplantation:(72.2±8.61)%vs (31.35±7.84)%, p=0.007; day+14after transplantation:(72.8±12.48)%vs (45.32±5.06)%, p=0.002],[CD8+T-lymphocytes: Transplantation+3days:(62.6±1.44)%vs (15.73±1.88)%, p=0.015;+7days after transplantation:(65.6±5.77)%vs (14.7±3.2)%, p=0.008; transplantation+14days after:(56.3±5.44)%vs(16.2±6.31)%, p=0.002];+3days of CD4+T lymphocytes FASL expression than CD8+T lymphocytes. aGVHD when the bone marrow of Nestin+mesenchymal stem cellsFAS expression increased after transplantation+3days+7days,+14days of flowcytometry to detect expression of FAS bone marrow of Nestin+mesenchymal stem cells,expression of FAS results show that the aGVHD group higher than the BMT group [+3days after transplantation:(94.2±5.6)%vs (2.56±0.82)%, p=0.001;+7days aftertransplantation:(89.42±4.41)%vs (3.45±0.84)%, p=0.002; day+14aftertransplantation:(87.3±3.48)%vs(3.32±0.46)%, p=0.002]. aGVHD when containingpopulations of cells of mesenchymal stem cells (CD45-c-kit-CD44+CD29+gating)chemokine CXCR4expression+7days after transplantation reduce,+14days,+21days, analyzed by flow cytometry of bone marrow containing between the charge and quality ofstem cells in the cell groups (CD45-c-kit-CD44+CD29+gating)) chemotactic factorCXCR4of expression, the results show mesenchymal stem cells in the cell groups(CD45-c-kit-CD44+CD29+gating), CXCR4expression is lower than a simple bonemarrow transplant group [+7after transplantation:(19.76±3.9)%vs (19.44±3.21)%, p=0.89, day+14after transplantation:(5.98±3.75)%vs (17.44±7.85)%, p=0.006,;+21days after transplantation:(16.25±3.63)%vs (49.93±7.12)%, p=0.003]. of Nestin+mesenchymal stem cell chemokine CXCR4expression on day+7after transplantationreduce aGVHD when the bone marrow on day+14,+21days, analyzed by flow cytometryof bone marrow of Nestin+mesenchymal stem cell chemokine expression of CXCR4, theresults show the bone marrow of the aGVHD group nestin+mesenchymal stem cellchemokine expression of CXCR4was lower than the simple bone marrow transplant group[on day+7after transplantation:(38.74±9.76)%vs (51.26±10.11)%, p=0.095; aftertransplantation+14days:(17.84±4.48)%vs (54.61±8.97)%, p=0.008;+21days aftertransplantation:(27.71±1.08)%vs (67.38±2.09)%, p=0.002]. aGVHD when the bonemarrow of Nestin+mesenchymal stem cell adhesion molecule CD44expression+7daysto reduce transplant day+14,+21days, analyzed by flow cytometry of bone marrow ofNestin+mesenchymal stem cell adhesion molecule expression of CD44, the results showthe bone marrow of the aGVHD group nestin+mesenchymal stem cell adhesion moleculeCD44expression gradually increased after transplantation, but still below a simple bonemarrow transplant group [+7after transplantation:(35.34±6.31)%vs (53.88±7.32)%, p=0.016, n=5; day+14after transplantation:(41.22±13.53)%vs (84.26±7.81)%, p=0.008;+21days after transplantation:(71.95±5.22)%vs (92.498±0.98)%, p=0.008].ConclusionsOur study demonstrates that the marrow Nestin+mesenchymal stem cells are rare inbone marrow,the marrow Nestin+mesenchymal stem cells are MSCs.The proportionthe CD45~-Nestin~+cells in the bone marrow nucleated cells and the absolute number bothreduced in the aGVHD environment, while the proportion of the MSC cell groups (to thecells of CD45-c-Kit-of CD44+for CD29+set the door) in the bone marrow did notreduce. CD4+and CD8+T cells have high expression of FASL during aGVHD andCD4+T lymphocytes FASL expression levels is higher than that of CD8+Tlymphocytes at early time after transplantation.The bone marrow Nestin+mesenchymalstem cells have high expression of FAS.Donor T lymphocytes attack the marrow Nestin +mesenchymal stem cells through the FAS-of FASL pathway mediated immune attack,resulting apoptosis of Nestin+mesenchymal.The the marrow Nestin+mesenchymalstem cells decreased may reduce when the expression chemokines of CXCR4andadhesion molecules CD44in the marrow Nestin+mesenchymal stem cells reduced,sothat the bone marrow Nestin+mesenchymal stem cells were not in the correctpositioning of the bone marrow microenvironment. |
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