【作者】 王海燕；

【导师】 谢毅； 陈彤； 李佩；

【作者基本信息】 复旦大学 ， 内科学， 2009， 博士

【摘要】 淋巴细胞活化与BMMSCs向脂肪分化的相互影响及可能的临床意义研究背景再生障碍性贫血(再障，Aplastic Anemia，AA)发病机理较为复杂，近来认识到免疫机制紊乱尤其是淋巴细胞免疫功能紊乱在再障发病中的作用，己成为再障基础研究方面一个最活跃的领域。骨髓间充质干细胞(Bone marrowmesenchymal stem cells，BMMSCs)作为骨髓基质细胞的起源细胞细胞，BMMSCs及其分化成的细胞在再障发病过程中的作用鲜有研究报道。红髓脂肪化是再障特征性病理改变。但对于再障骨髓中的脂肪组织增多原因及作用尚未见到相关报道。一直以来，脂肪组织被认为是单纯的能量储存组织。然而随着一些脂肪细胞分泌的因子被人们发现，脂肪细胞的其他重要生理学功能被逐渐发掘出来。数十种脂肪细胞分泌因子(adipocyte-secreted factors)在近10余年来已被相继发现，并引起广泛重视。这些因子被统称为脂肪细胞因子(adipocytokines)或简称脂肪因子(adipokines)。目前认为脂肪组织是有独特组织类型，具有特定功能(例如，平衡能量储备、分泌激素和调节免疫)的真正器官系统。Leptin和Adiponectin是脂肪组织参与免疫调节反应的两个主要的脂肪因子。Leptin对T淋巴细胞、单核细胞、巨噬细胞都具有免疫调节作用。免疫细胞受到免疫刺激后，产生细胞因子(尤其是TNF-α)作用于脂肪细胞，使其分泌Leptin，从而达到使免疫信号扩大化的目的。Adiponectin是脂肪细胞表达最丰的激素。主要在天然免疫效应中发挥作用，能抑制髓单核细胞祖细胞的增殖，抑制巨噬细胞的活性。因此，与Adiponectin不同，对于造血免疫系统，Adiponectin被看作是一种负调控因素，可能参与炎症反应的终止。异常的免疫反应可能在损伤造血干细胞的同时对BMMSCs也具有损伤作用，对BMMSCs的脂肪分化能力产生一定的影响，骨髓内显著增多的脂肪细胞也可能对免疫系统有一定的作用。我们对此进行以下研究，以期从一个新的角度探索再生障碍性贫血的发病机制。目的(1)体外分离、培养、扩增小鼠BMMSCs，并研究其生物学特点。(2)培养、扩增再障患者和正常对照BMMSCs，比较两组BMMSCs生物学特性及脂肪分化能力的不同。(3)建立免疫相关骨髓衰竭动物模型，观察免疫紊乱条件下模型小鼠骨髓脂肪化程度的改变，并研究该模型BMMSCs的生物学特性。(4)研究同基因条件下，活化淋巴细胞对BMMSCs向脂肪分化能力的影响及BMMSCs向脂肪细胞分化后对单个核细胞(MNCs)增殖及活化状态的影响。方法(1)贴壁筛选法分离纯化BALB／c小鼠BMMSCs，胰酶消化传代，FCM鉴定细胞表型，并在特定诱导剂下诱导向脂肪细胞、成骨细胞及软骨细胞分化。(2)体外密度梯度离心法分离培养正常人和再障患者的BMMSCs，观察其形态学的异同，同时用流式细胞仪检测其分子表面抗原以及RT-PCR法检测成脂肪细胞的差异。(3)C57BL/6小鼠(父本)和BALB／c小鼠(母本)杂交产生CByB6F1小鼠，将F1小鼠γ射线亚致死量照射(4．5Gy)后尾静脉注射母本淋巴细胞，建立免疫相关骨髓衰竭动物模型。记录白细胞，红细胞，血小板及血红蛋白的变化，股骨切片HE染色观察模型小鼠骨髓脂肪化程度，贴壁筛选法分离小鼠BMMSCs，观察其生物学特性的改变。(4)将Percoll密度梯分离BALB／c小鼠脾脏MNCs，ConA活化后与BALB／c小鼠BMMSCs共培养72小时，Real-time PCR，Western Blot及ELISA检测BMMSCs脂肪分化相关基因和蛋白表达的变化。BMMSCs诱导分化成脂肪细胞，与脾MNCs共培养72小时，CCK-8检测MNCs增殖，流式检测淋巴细胞活化标志的变化。结果(1)BMMSCs呈梭形或三角形，长梭形，细胞排列整齐，低倍镜下部分呈平行状或旋涡状。流式检测BMMSCs高表达CD44，CD73，CD90和CD105，低表达或不表达CD14，CD34，CD45及HLA-DR。在特定的诱导条件下，BMMSCs能向脂肪细胞，成骨细胞及软骨细胞分化。(2)在起始培养细胞数相同条件下，初次换液时再障患者形成的克隆数明显少于正常对照[(19．3±4．77)／5×10~5 MNCs vs(47．72±3．46)／5×10~5 MNCs，(P＜0．05)]，再障组和正常对照BMMSCs细胞形态和表面标志无明显差别，体外再障患者BMMSCs向脂肪细胞诱导分化较正常组早。(3)CByB6F1小鼠亚致死量照射后，骨髓衰竭为可逆性，外周血象在第10天开始恢复，约6周恢复至正常水平，而亚致死量照射后经尾静脉注射母本小鼠淋巴细胞的小鼠，外周血象降低为不可逆性，造成不可逆性免疫相关骨髓衰竭。骨髓病理学检查证实骨髓衰竭，免疫相关骨髓衰竭组小鼠髓腔空虚，脂肪组织明显增多，而正常对照组和单独照射组骨髓内均无明显脂肪组织形成。体外培养证实，与对照组相比，免疫相关骨髓衰竭组小鼠BMMSCs增殖能力降低，不能传代。(4)体外培养，BALB／c小鼠BMMSCs与经ConA活化的BALB／c小鼠脾脏的MNCs按1：5比例共培养72小时，Real-time PCR检测发现，BMMSCs的Leptin基因表达明显增加而Adiponectin基因表达轻度降低，Western Blot检测该两种蛋白表达也有近似的趋势。ELISA定量检测共培养上清中这两种蛋白的表达发现，Leptin低于检测下限而Adiponectin则明显减少。将BALB／c小鼠的脾MNCs与其BMMSCs或脂肪分化的BMMSCs按一定比例共培养，BMMSCs：MNCs为1：0．5时，脂肪分化的BMMSCs组的淋巴细胞增殖抑制作用显著低于正常对照组BMMSCs[正常对照组vs脂肪分化组为-(77．8±20．7)％vs-(47．8±14．3)％，(P＜0．05)]；而其比例相当(BMMSCs：淋巴细胞为1：1)时，脂肪分化的BMMSCs组对淋巴细胞的作用为轻度促增殖，但与正常对照组的轻度抑制增殖作用相比，差异无显著性[正常对照组vs脂肪分化组为-(28．3±5．2)％vs(3．3±4．9)％，(P＞0 05)]；当淋巴细胞数量逐渐增多，超过BMMSCs数量时，脂肪化的BMMSCs组就表现为促淋巴细胞增殖作用[正常对照组vs脂肪分化组为1：2．5时(-3．9±8．3)％vs(34．2±17．1)％，(P＜0．05)；1：5时(9．4±8．8)％vs(40．8±16．3)％(P＜0．05)；1：10时(20±14．9)％vs(65．8±12．5)％(P＜0．05)]，而且该作用在一定比例范围内，随着淋巴细胞数量的增多而逐渐增强。脂肪分化的BMMSCs与MNCs共培养72h后，却能使淋巴细胞的活化标志CD25和CD69表达与正常对照组相比明显增增加[脂肪分化BMMSCs共培养组vs正常BMMSCs共培养组：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)]。结论(1)全骨髓贴壁筛选培养法可简便、高效分离、纯化BMMSCs。所获得的细胞具有BMMSCs特征性的细胞表型及向脂肪细胞、成骨细胞及软骨细胞分化能力。(2)再障患者BMMSCs形态与正常对照相比无明显变化，但其增殖能力较正常对照降低，而且脂肪细胞分化早于正常对照。(3)CByB6F1小鼠在亚致死量照射后，骨髓衰竭为可逆性，经尾静脉注射母本小鼠淋巴细胞可诱导不可逆性骨髓衰竭，形成免疫相关骨髓衰竭动物模型，模型动物骨髓脂肪组织明显增多，BMMSCs增殖能力降低，不能传代，对照组却无此种改变。(4)ConA活化的淋巴细胞能促进BMMSCs脂肪分化相关基因的表达，且脂肪因子分泌失调，Leptin增加而Adiponectin减少。当BMMSCs诱导分化为脂肪细胞后，与MNCs共培养，可检测到淋巴细胞早期活化标志CD25和CD69表达的增加，提示BMMSCs脂肪分化后，具有刺激淋巴细胞活化的作用。(5)我们的实验提示，活化淋巴细胞能促进BMMSCs脂肪分化，BMMSCs脂肪分化后能刺激淋巴细胞增殖及活化，二者形成恶性循环。此现象可能在再障的发生发展中起一定的作用，有待后期实验进一步研究。更多还原

【Abstract】 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.更多还原