The Interaction Between Activated Lymphocytes And Adipogenic Differentiation Of BMMSCs And Its Significance

The interaction between activated lymphocytes and adipogenic differentiationof BMMSCs and its significanceBackgrounds Aplastic Anemia(AA) is a heterogeneous disease characterized byfailure of bone marrow hematopoiesis resulting in varying degrees of pancytopenia witha markedly hypocellular bone marrow.Despite the exact causes of AA are unknown,various lines of laboratory evidence point towards an immune-mediated inhibition ofhematopoiesis.Bone marrow mesenchymal stem cells (BMMSCs) arenonhematopoietic progenitor cells of the bone marrow stromal cells and the progenitorof most cell components in the hematopoietic microenvironment.But it is unclear thatthe effect of BMMSCs and its differentiated cells in hematopoietic microenvironment inAA.Hypocellular with prominent adipocytes is the most characteristic pathology of AA.There is evidence that MSCs are responsible for creating adipocytes and osteoblastswithin the bone marrow microenvironment.Until recently,adipose tissue has been considered to be a mere storagecompartment of triglycerides.It is now clear that adipocytes are highly active endocrinecells that play a central role in overall energy homeostasis and are importantcontributors to some aspects of the immune system.They do so not only by influencingsystemic lipid homeostasis but also through the production and release of a host ofadipocyte-specific and adipocyte-enriched hormonal factors,cytokines,andextracellular matrix components (commonly referred to as"adipokines").Littleattention has been given to the role of adipose tissue in infectious disease.However,thestrong proinflammatory potential of adipose tissue suggests an important role in thesystemic innate immune response.Leptin and Adponectin are two adipokines important in the modulation of the hostresponse to infection.Leptin modulates the proliferation and activation of peripheral Tlymphocytes in mice and in humans and can enhance the production of cytokines.Adponectin is a secretary protein synthesized exclusively by adipocytes.It serves as ananti-inflammatory cytokine and antagonizes the effects of TNF-α.In acquired aplastic anemia,abnormal immune mechanism is responsible for thedestruction of the hematopoietic cell compartment.The abnormal immune mechanism also has the ability to destruct BMMSCs,and alter their capacity to differentiate intoadipocyte.Moreover,the prominent adipocytes have abnormal effects on lymphocytes.The study is to reveal whether these deficiencies exist,thus to offer a new standpoint inthe theoretical basis for the pathogenesy of AA.Objective (1) To establish a method for isolation,cultivation and expansion of miceBMMSCs,and study their biological features.(2) To culture AA patients and controlsBMMSCs,and compare BMMSCs of AA patients morphologies and the process ofadipogenic differention to the controls.(3) To establish the model of immune-mediatedbone marrow failure in vivo,and study the biological features of BMMSCs in thismodel.(4) To investigate the effect of activated lymphocytes from the homogenic miceon BMMSCs adipogenic differentiation;to investigate effect of adipogenic cellsdifferentiated from BMMSCs of the homogenic mice on peripheral bloodmononuclears.Mathods (1) BMMSCs of BALB/c mice were separated and amplified in vitroby wall sticking method.The cells was digested by 0.25% trypsin and passaged whencells fused 80-90%.The phenotypes of BMMSCs were examined with flow cytometry.Their differentiation potential were examined by specific induction conditions.(2)BMMSCs in patients with aplastic anemia(AA)and the control group were separatedwith Percoll(1.073g/m L) and cultured in low glucose DMEM.Then,observing theirmorphologies,checking their molecule surface antigen by flow cytometry andexamining the process of adipogenic differention by RT-PCR.(3) Male B6 (C57BL/6)mice and female BALB/c mice were used at the ages of 6-16 weeks,then hybrid thecByB6F1 mice.The lymphnode and spleen cells of normal BALB/c mice were injectedto hybrid CByB6F1 recipients with a sublethal dose ofγirradiation (4.5Gy) to establishthe model of immune-mediated bone marrow failure.The number of white blood cells,red blood cells,platelets and hatmatoglobin bere recorded.HE stain of bone was used tounderstand the relationship between bone marrow adipogenesis and immue abnormality.BMMSCs of the animal models were separated and amplified in vitro by wall stickingmethod to compare their difference to the normal ones.(4) Homogenic spleen-derivedMNCs isolated by Percoll density centrifugation were actvited by ConA and co-culturedwith BMMSCs.The adipognesis gene and protein of BMMSCs were analyzed byReal-time PCR,Western Blot and ELISA.The action of adipogenic differentiatedBMMSCs on MNCs proliferation was investigated by using Cell Counting Kit-8 (CCK-8).The percent of activated lymphocytes,co-cultured with adipogenicdifferentiated BMMSCs for 72 hours,was assayed by flow cytometry.Results (1)Both primary and passage BMMSCs appearance presentedcambiform or triangular-shaped,fibroblasts-like and vortex-like.The third generationwas showed by FACS that expression of CD44,CD73,CD90 and CD105 was strongpositive in the cultured cells,and expression of CD14,CD34,CD45 and HLA-DR wasnegative.Under suitable conditions,BMMSCs have the similar ability ofdiferentiatation into adipocyte,osteoblast and chondrocyte.(2)The cell clones formedby the initial culture of AA patients were fewer than that of the controls[(19.3±4.77)/5×10~5MNCs vs (47.72±3.46)/5×10~5MNCs,(P＜0.05)].But there was nosignificant difference between control group and AA patients in the morphology ofBMMSCs.And their capacity of adipogenic differentiation in AA patients is earlier thancontrols.(3)Infusion of lymphnode and spleen cells of a parent animal into sublethallyirradiated recipients produced severe BM failure.Marrow cavities were essentiallyempty in these affected mice in comparison to untreated control mice.Residual BMcontained a very large proportion of adipocytes in the immune-related bone marrowfailure mice while few adipocytes existed in the controls bone marrow.BMMSCs ofthese immune-related bone marrow failure mice had lower proliferation capacity thancontrols.(4)When BMMSCs co-cultured with ConA actvited homogenicsplenic-derived MNCs in the ratio of 1:5 for 72 hours,the Leptin gene of BMMSCswere highly expressed while the Adiponectin gene were slightly decreased compared toBMMSCs co-cultured with normal MNCs by Real-time PCR analysis.Though not soobvious,Western Blot analysis showed the same tendency.Leptin protein in thecoculture supernatant was undetectable by ELISA while the Adiponectin protein had anotable decrease in this circumstance.When homogenic MNCs were added toBMMSCs or adipogenic differentiated BMMSCs in vitro,the inhibitory effect ofadipogenic differentiated BMMSCs to MNCs proliferation was significantly lower thanthat of BMMSCs in the ratio of 1:0.5 (BMMSCs:MNCs)(P＜0.05);in the ratio of1:1(BMMSCs:MNCs),the effect of adipogenic differentiated BMMSCs to MNCs wasslightly proliferation while BMMSCs had a inhibition effect (P＞0.05);the proliferationeffect of adipogenic differentiated BMMSCs to MNCs was significantly higher thanthat of BMMSCs in the ratio of 1:2.5,1:5,1:10(BMMSCs:MNCs),the undifferentedBMMSCs to MNCs vs the adipogenic differentiated BMMSCs to MNCs was(-3.9±8.3)% vs (34.2±17.1)%,(1:2.5,P＜0.05);(9.4±8.8)% vs (40.8±16.3)% (1:5, P＜0.05);(20±14.9)% vs (65.8±12.5)% (1:10,P＜0.05).After co-cultured withadipogenic differentiated BMMSCs in the ratio of 1:5 in vitro for 72 hours,lymphocytes that became CD69+ and CD25+ activated cells were up-regulatedcompared to the lymphocytes co-cultured with undifferentiated BMMSCs [adipogenicdifferentiated co-cultures vs undifferentiated co-cultures:CD25+ (50.9±7.7)% vs(16.2±5.7)%,(P＜0.05);CD69+ (47.3±5.8)% vs (13.3±4.6)% (P＜0.05)].Conclusions (1) BMMSCs could be isolated and purified by adhesivenessdelection.Derived cells could be cultured stably and expanded quickly.Expanded cellswe harvested had the characteristic markers of BMMSCs,and had the potential todifferentiate to adipocyte,osteoblast and chondrocyte.(2)There was no significantdifference between AA and control group in the morphology of BMMSCs.In AApatients,the amount of BMMSCs was fewer and adipogenic differentiation was earlierthan controls.(3)Infusion of lymph node and spleen cells of a parent animal into sublethally irradiated recipients produced immune-related BM failure.In immune-relatedBM failure mice,residual BM contained a very large proportion of adipocytes,andBMMSCs of these mice had lower proliferation capacity than the controls.(4)Theactivated homogenic lymphocytes could promote the adipogenic differentiation ofBMMSCs,and the induction of mRNA level of Leptin was increased while Adiponectinreduced.Therefore,in this circumstance,adipogenic differentiated BMMSCs haddisturbed adipokines secretion.After BMMSCs differentiated into adipocytes,theinhibitory effect to homogenic MNCs was decreased while the proliferation effect wasincreased.The adipogenic differentiated BMMSCs had the capacity to up-regulate thepercentage of activated lymphocytes.(5)This study had suggested that the activatedlymphocytes could facilitate BMMSCs adipogenic differentiation.Furthermore,theadipogenic differentiated BMMSCs could activate lymphocytes.This could probably avacious cycle in the morbility of aplastic anemia.