| Objective：Aplastic anemia (AA) is one of lethal heterogeneous diseasesin children. In aplastic anemia, the basic component of the marrow becomesabnormal: cells responsible for generating blood cells (hematopoietic cells) aregreatly decreased in number and are replaced by adipocytes. Bone marrowtransplantation and immune inhibitor remain the most successful treatment foraplastic anemia. Survival of pediatric patients with aplastic anemia after ISTwith ATG/ALG and BMT is improved. However, long-term follow-up onthese patients has indicated a high frequency of hematologic complications,including PNH, myelodyspasia, ANL, and recurrent aplasia, and it is notapparent if aplastic anemia is aggressive adipogenesis or a failure ofhematopoietic stem cells. which indicate that immune disorder can not beresponsible for all the cases of aplastic anemia.In recent years much emphasis has been put on the immune attack to theHSC by the T lymphocytes. The microenvironment has been thought of beingimportant to the hematopoiesis for many years, and recently many kinds ofniche has been assumed to be responsible for the hematopoiesis, while fewefforts have been put on the microenvironment of the hematopoiesis. themicroenvironment or niche is a place in which the stem cell resides, recently ithas been shown that the stem cells’ remarkable ability to undergo bothself-renewal and to give rise to progeny that can differentiate, is greatlydependent on the microenvironment or the niches.Multiple stem cell niches have been proposed include the osteoblastic,vascular and CXCL12-abundant reticular (CAR) niches in the bone marrowamong them the osteoblast stem cell niche seems to be the most important onesince it seems to have the ability to control the hematopoietic stem cells fate: to keep the quiescent state or to enter the cell cycle for self-renew. Since themicroenvironment is so import to the hematopoiesis we want to know whetherthe development of the hematopoiesis failure disease-AA partly associateswith the microenvironment change. As it is well known the red bone marrowwill turn into yellow bone marrow after the development of aplastic anemiabecause adipocyte increases greatly in the AA patient bone marrow. Since theadipocyte comes from the MSC in the bone marrow and the osteoblast sharedthe same ancestor with adipocyte, it is likely that the increased adipocyte mayaffect the produce of osteoblast. In order to know this we analysis theosteoblast on the bone slice of the AA patient with HGE staining. As it hasbeen described previously, osteoblast is an important niche in the bone marrow,the reduce of the steoblast may in turn affect the self-renew or differentiationof the HSC, whether this will cause the reduce of mature blood cells andfinally cause the pancytopenia is still leave to be decided.In this study, retrospective analysis of the information from24casesof severe aplastic anemia confirmed with bone marrow pucture and (or)bone marrow biopsy in pediatrics ward of Second Affilited Hospital ofHebei medical University from June1996to November2010. According toChina Medical Association Pediatric Branch, Hematology Group,recommendations of pediatric SAA diagnosis and treatment evaluationstandards. We collected24SAA patients’ clinical data and evaluated theirclinical effect in order to find the clinical factors that may contribute todifferent outcomes of this regimen. Besides, we took the side effects inconcern so as to find way to prevent or treat them. We use blast AA BMMSCsas the research object, completed culture purification and identification of AApatients, such as porliferation, differentiation, apoptosis and other aspects,made K562cells co-cultured with AA BMMSCs, contrast to normal BMMSCs,observed the influence of proliferation and apoptosis on AA BMMSCs toK562cell, discussion the interaction mechanism of BMMSCs.Methods:1Retrospective analysis of the information from24cases of severe aplastic anemia confirmed with bone marrow pucture and (or) bone marrowbiopsy in pediatrics ward of Second Affilited Hospital of Hebei medicalUniversity from June1996to November2010, which were treated byATG[4-5mg/（kg·ｄ）×5] plus CSA[3-5mg/（kg·d）] therapy. We collectedtheir clinical data such as age, sex, the course of disease, and we monitered thechange of their symtom, the curve of peripheral blood cell counts, and theincidence of different side effects.2Isolated and cultured the BMMSCs of AA children patients, observedthe cultured and passaged characteristics of the AA children patients’BMMSCs,used the normal as the control group.3Selected the good condition P3-generation cells.Compared thedifference of morphology between AA children patients and the normal peopleafter Wright’s Giemsa staining.4Measured the growth curve of AA children patients with MTT.5Compared the capacity of adipogenic differentiation between the AAchildren patients with normal people after used Differentiation-inducingliquid.6Used the RT-PCR and immunohistochemistry detection the gene andprotein level of PPAR2gene,and compared the diferent expression betweenthe AA children patient and normal people.PPAR2gene is the marker genesof adipogenic differentiation.7Establish BMMSCs and K562cells co-culture system, detected theproliferation of K562cells affect by BMMSCs used the direct countingmethod and MTT assay。8Observed the morphological and structural differences between AApatients and normal persons used Hoechest33342staining and transmissionelectron microscopy。Result:1The total effective rate was66.7%in the24cases of patients whoreceived immunosuppressive treatment. the onset time is3-7months.Themain adverse reactions is allergy、serum sickness and infection, the incidence rate was83.3%,54.1%and70.8%, no forced withdrawal of cases. Threecases death,two patients died of severe sepsis after one month with the ATGtreatment,one patient died of a brain hemorrhage after two months with theATG treatment. Relevant statistical analysis of the efficacy show: fromdiagnosis to accept the ATG treatment, duration≤6months was significantlyhigher than duration>6months, the total effective rate respectively were92.3%, and50%(P <0.05); After ATG treatment, absolute lymphocyte (ALC)decrease>2×109/L was significantly higher than those absolute lymphocyte(ALC) decline≤2×109/L, the total effective rate respectively were92.3%and50%(P <0.05); The gender, age, severity of illness, the occurrence ofserum sickness, and T cell subsets (CD4/CD8ratio) of children had norelated with efficacy; there were not clonal disease such as PNH、MDS andAL occured during follow-up.2The fat cells increased in AA patient and the hematopoietic cellsdecreased in AA patient.3The BMMSCs of AA patients shows: growth slower、Clonogenic less、adherent and fusion longer compared with normal control. After to P3generation, the cells’ capacity of proliferation enhanced, the cellmorphology homogeneous, fibrous growth, vortex-like arrangement, smallbody, dense nucleus after Regis stain. there was almost no difference in themorphology of the light microscope between the AA patients’ BMMSCs andnormal controls’ BMMSCs.4The proliferation of AA patients’ BMMSCs is much weaker than thenormal control group5Compared with normal controls, children with aplastic anemiaBMMSCs more easily cultured in vitro with a lipid (78.6%±4.1%vs26.33%)(P <0.001).6In vitro cultured showed Compared with normal controls, children withaplastic anemia BMMSCs more easily Transformed Into lipid (78.6%±4.1%vs26.33%)(P <0.001).7BMMSCs effect on K562cell proliferation: In order to investigate the effects of abnormal differentiation ofBMMSCs on cell proliferation，we selected good condition BMMSCswithout adipocyte differentiation，after cultured with the adipogenic fluid，use oil red O stain confirmed the cell is fat cells, to build support cell mode。Used the cell mode co-culture with K562cells,then detected by MTT, theresults showed the Growth rate of k562cells cultured in BMMSCs cultured byAdipose differentiation inducer (0.651±0.06) was significantly lower than inthe normal MSC proliferation rate (0.879±0.03), there was statisticallysignificant (P <0.001), the growth rate of K562cells in both groups werehigher than normal controls (0.533±0.05, P <0.001).8There were not found apoptosis both in normal control group and theobservation group after Hoschest stain.9Compared to nuclear internal structure AA patient and normal controlgroup have no different observed by electron microscopy。looked at fromcytoplasm ingredient we found in normal control group between the nucleusand the cell membrane filled with a large number of mitochondria andvesicle-like structures, gathered into the film, and in the cells of patients withaplastic anemia, a relatively small number of mitochondria and vesicle-likestructures.Conclusions:1The development of AA is closely related with the disorder of theimmune system, according to the first part of our work, most of the AA patientcan gain a reasonable results after treated with strong immune suppressor likeATG combined with CSA, however, not all the AA patients can end up with agood results after treated with IST, so there must be some other reasons thattake part in the development of AA.2The BMMSCs in AA patient are easy to differentiate into adipocytes.Further study showed that the key regulator of MSCs differentiationPPAR-gamma2was up-regulated in AA BMMSCs in both gene level andprotein level, which can indicate that its function may be enhanced in AApatient. Since this regulator controlled the differentiation of MSCs into adipocyte, we can infer that the enhanced expression of PPRA-gamma2increased the adipocytes in the AA patient bone marrow.3The supporting function of BMMSCs after adipocyte induction issignificantly lower than those without induction treatment according to theproliferation rate of the up layer K562cells. So in this aspect we can infer thatthe increased adipocytes in AA patient bone marrow may affect thehematopoiesis supporting function, and then take a role in the development ofAA beside the immune problem. This may in some aspect explain the theoryof SOIL raised in the past years. Besides this, we also found some specialparticles in both Geimsa staining and HOCHEST staining. And under electricmicroscope the mitochondria inside the cytoplasma of AA BMMSCs wasdecreased, maybe this difference can showed a function decreasing in the AABMMSCs, which may in turn cause a decreased secretion function ofcytokines that important in hematopoiesis. All in a word, in our work weshowed some change in the AA patient bone marrow environment and maybuilt in the mechanism of AA development in theory. |
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