新型免疫介导骨髓造血功能衰竭小鼠模型的建立及特性分析

目的:通过建立自身免疫介导的再生障碍性贫血(AA)小鼠模型,并分析该小鼠模型的免疫特性,探讨不同类型调节性T细胞(T-regs)在其发生、发展过程中可能的作用。方法:分离雄性B6(C57BL/6)外周淋巴结,并制成淋巴细胞制成悬液,予不同淋巴细胞数经尾静脉注入杂交1代(F1)小鼠(cBy/B6F1),不同时间段处死小鼠,分析外周血及骨髓,观察其造血功能的变化,在此基础上,采用直接免疫荧光标记,流式细胞术检测CD4+T-regs和CD8+T-regs亚群的变化。结果:实验证实注入雄性B6外周淋巴结淋巴细胞,可建立免疫介导的AA小鼠模型;输入淋巴细胞数量是决定受体小鼠骨髓抑制的关键,当输入淋巴细胞数达到10×106/只,可建立AA小鼠模型,淋巴细胞数达到40×106L-1时,受体小鼠因骨髓造血功能衰竭在14～20d内死亡;受体小鼠血细胞下降与雄性B6外周淋巴结淋巴细胞注入时间相关,随着时间的增加,血象呈进行性下降。同时还发现在AA小鼠模型中CD4+和CD8+调节性T细胞亚群有显著变化,与正常F1小鼠相比,受体小鼠CD4+CD25+细胞、CD8+CD25+及CD8+CD28-细胞群明显减少,分别为(1.7+0.9%vs14.4+9.3%)、(2+0.5%vs8.45+0.4%)和(16+0.6%vs39.45+1.2%)。结论:不同亚群在AA的发生、发展过程中起不同作用,CD4+CD25+/CD8+CD25+细胞可能在AA的启动阶段起作用,而CD8+CD28-则可能是小鼠骨髓损伤的细胞亚群。     

免疫治疗鼠获得性免疫缺陷综合征相关性淋巴瘤

目的本研究通过T细胞输注方法阻断程序凋亡因子-1(programmed death-1,PD-1)抑制性信号在CD8+T细胞上的表达,来治疗鼠获得性免疫缺陷综合征(MAIDS)相关性B细胞淋巴瘤。方法为了选择性阻断PD-1抑制性信号通路在CD8 T细胞中的表达,采用B6.PD-1-/-小鼠作为CD8 T细胞的供体,与来自野生型B6小鼠的CD8 T细胞做比较。定期测量肿瘤直径大小,来检测PD-1-/-和野生型的CD8 T细胞抗肿瘤功能。结果我们的研究结果发现未接受CD8 T细胞输注的Rag-1-/-受体小鼠,肿瘤持续增大。接受B6.PD-1-/-CD8 T细胞输注的受体小鼠肿瘤缩小/消失的时间和速度均比接受野生型B6 CD8 T细胞输注的受体小鼠显著性缩短和加快。结论阻断CD8 T细胞中的PD-1抑制性信号通路能够增强保护性CD8 T细胞抗肿瘤的功能,可达到控制和治疗逆转录病毒感染导致MAIDS相关肿瘤的发生和发展。     

免疫治疗鼠获得性免疫缺陷综合征相关性淋巴瘤

目的本研究通过T细胞输注方法阻断程序凋亡因子-1(programmed death-1,PD-1)抑制性信号在CD8+T细胞上的表达,来治疗鼠获得性免疫缺陷综合征(MAIDS)相关性B细胞淋巴瘤。方法为了选择性阻断PD-1抑制性信号通路在CD8 T细胞中的表达,采用B6.PD-1-/-小鼠作为CD8 T细胞的供体,与来自野生型B6小鼠的CD8 T细胞做比较。定期测量肿瘤直径大小,来检测PD-1-/-和野生型的CD8 T细胞抗肿瘤功能。结果我们的研究结果发现未接受CD8 T细胞输注的Rag-1-/-受体小鼠,肿瘤持续增大。接受B6.PD-1-/-CD8 T细胞输注的受体小鼠肿瘤缩小/消失的时间和速度均比接受野生型B6 CD8 T细胞输注的受体小鼠显著性缩短和加快。结论阻断CD8 T细胞中的PD-1抑制性信号通路能够增强保护性CD8 T细胞抗肿瘤的功能,可达到控制和治疗逆转录病毒感染导致MAIDS相关肿瘤的发生和发展。     

B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用

 目的: 探讨B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用。方法: 抗CD45RB抗体对BALB/c裸鼠进行预处理后制备脾脏单细胞悬液,与BALB/c小鼠T淋巴细胞和C57BL/6小鼠脾细胞混合培养,流式细胞术分析Th1、Th2、Treg和Tm淋巴细胞。以B6.μMT^-/-小鼠为受体、BALB/c小鼠为供体建立皮肤移植模型,移植后向受体鼠腹腔注射抗CD45RB单抗,监测脾淋巴细胞CD3⁺CD45RB^hi细胞比例。在混合淋巴培养过程中加入抗CD45RB单抗,分离B细胞,建立以BALB/c小鼠为供体、B6.μMT^-/-小鼠为受体的心脏移植模型,通过尾静脉注射B细胞给B6.μMT^-/-小鼠,观察受体鼠生存期和B细胞分布。结果: 在裸鼠体内用抗CD45RB抗体处理过的B淋巴细胞,与T淋巴细胞混合培养时,可使Treg和Th2淋巴细胞比例明显升高,Th1淋巴细胞的比例明显下降,Tm细胞无明显变化。在体内B淋巴细胞缺失的情况下,抗CD45RB抗体依然能够降低T细胞表面CD45RB的表达,与对照组B淋巴细胞存在组相比,抗CD45RB抗体对T淋巴细胞表面CD45RB下调更为快速,但最终CD3⁺CD45RB^hi T细胞比例无明显变化。体外抗CD45RB抗体处理过的B淋巴细胞可以延长受体鼠的生存时间。B6.μMT^-/-鼠在接受抗CD45RB抗体处理的B细胞并进行同种异体心脏移植后,B细胞可向胸腺迁移。结论: 在抗CD45RB抗体诱导的免疫耐受中,B淋巴细胞可能通过介导各T淋巴细胞亚群比例发挥着重要作用,且在中枢耐受中也起到一定作用,但是仅靠B淋巴细胞无法形成完全耐受。     

间充质干细胞可能通过调控免疫细胞促进再生障碍性贫血小鼠骨髓的造血功能

目的:探讨间充质干细胞(MSCs)影响再生障碍性贫血小鼠造血功能的可能免疫机制。方法:30只小鼠分3组,分别为单纯照射组、再障模型组、MSCs治疗组,建立再生障碍性贫血小鼠模型。单纯照射组仅经5Gy[60Co]γ射线照射,再障模型组经5Gy[60Co]γ射线照射后输注DBA/2小鼠胸腺淋巴结细胞1×106cell/只,MSCs治疗组经5Gy[60Co]γ射线照射后输注DBA/2小鼠胸腺淋巴结细胞1×106cell/只,3d后输注人骨髓MSCs1×106cell/只。观察各组小鼠外周血象、骨髓及外周血CD4+细胞、CD8+细胞、CD4+/CD8+、NK细胞变化及与造血的关系。结果:经5Gy[60Co]γ射线照射后,单纯照射组、MSCs治疗组外周血象第7d开始下降,21d血象恢复正常。再障模型组外周血象第7d开始持续性下降,至21d血象仍未恢复。照射后7d,各组小鼠间外周血CD4+细胞、CD8+细胞、CD4+/CD8+无明显差异,但MSCs治疗组NK细胞低于单纯照射组和再障模型组(P0.05)。照射后14d3组CD4+细胞比例均明显下降,CD8+细胞则表现不同,再障模型组与MSCs治疗组CD8+细胞比例明显高于单纯照射组,至照射后21d,MSCs治疗组CD8+、NK细胞与单纯照射组相近、而模型组则明显高于单纯照射组和MSCs治疗组;MSCs治疗组小鼠股骨病理切片中脂肪细胞比例明显低于再障模型组,与单纯照射组结果一致。结论:MSCs输注可能通过调控免疫细胞影响再生障碍性贫血小鼠骨髓造血功能。     

TGF-β1和/或TNF-α反义PS-ODNS对造血干/祖细胞体外扩增的作用

目的研究TGF-β1和/或TNF-α反义硫代寡核苷酸(PS-ODNS)对造血干/祖细胞体外扩增的调节作用.方法采用免疫磁珠分离技术从新鲜脐带血中分离CD34+细胞,用淋巴细胞分离液从骨髓血中分离单个核细胞,应用液体培养及造血祖细胞集落分析检测TGF-β1和/或TNF-α反义PS-ODNS对CD34+细胞数及多向性造血祖细胞(CFU-mix)、粒-单祖细胞(CFU-GM)、红系祖细胞(BFU-E和CFU-E)集落数的影响.结果培养体系中加入TGF-β1反义PS-ODNS后CD34+细胞数、CFU-mix、CFU-GM、BFU-E和CFU-E集落数分别是对照组(仅加细胞因子组)的4、2.6、2.7、1.8、2.1倍;加入TNF-α反义PS-ODNS后CD34+细胞数、CFU-mix、CFU-GM、BFU-E和CFU-E集落数分别是对照组的4、2.9、2.6、1.7、1.8倍;同时加入TGF-β1和TNF-α反义PS-ODNS后CD34+细胞数、CFU-mix、CFU-GM、BFU-E和CFU-E集落数分别是对照组的5.3、2.1、2.7、1.9、1.8倍.结论采用反义PS-ODNS阻断内源性TGF-β1和TNF-α是造血干/祖细胞体外扩增的有效措施之一.     

真性红细胞增多症患者骨髓单个核细胞植入再生障碍性贫血小鼠

背景：真性红细胞增多症患者骨髓的高增殖低凋亡特性使其造血干细胞植入NOD/SCID小鼠后成功分化出粒红系细胞,但其能否植入并改善再生障碍性贫血小鼠造血功能,目前国内外尚未有报道。 目的：探讨JAK2阳性真性红细胞增多症患者骨髓单个核细胞植入后对再生障碍性贫血小鼠造血重建的影响。 方法：应用注射用重组人γ-干扰素联合白消安的方法建立再生障碍性贫血小鼠模型,随机分为实验组(n=10)和对照组(n=10),给药结束后第5天实验组经尾静脉输注真性红细胞增多症患者骨髓单个核细胞悬液,对照组同法输注等容积生理盐水。输注后第14天检测小鼠外周血常规、骨髓细胞形态、骨髓组织病理变化以及小鼠外周血和骨髓内CD45+细胞的百分含量。 结果与结论：输注后第14天,实验组小鼠血细胞计数三系减少,骨髓涂片可见散在淋巴细胞和早期造血细胞,骨髓组织活检可见骨髓增生减低,少量粒系细胞及幼红细胞,未见巨核细胞,与对照组比较,造血功能无明显改善。对照组小鼠外周血及骨髓均未检测到CD45+细胞,实验组小鼠外周血及骨髓均可检测到人源化的CD45+细胞,且骨髓中CD45+细胞比外周血高,提示JAK2V617F阳性真性红细胞增多症患者骨髓单个核细胞能成功植入再生障碍性贫血小鼠体内,但血常规、骨髓涂片及活检结果显示未能明显改善骨髓造血功能。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

雷公藤多甙对aGVHD小鼠T细胞及相关细胞因子的影响

目的探讨雷公藤多甙对小鼠移植物抗宿主病的作用.方法供鼠BALB/C,雌性,受鼠C57BL/6,雄性.无菌取供鼠脾脏及双侧股骨骨髓,制成混合细胞悬液.受鼠在骨髓移植前24h内应用直线加速器全身照射,总剂量为1800cGy,剂量率为200Gy/min,将制成的混合细胞悬液经尾静脉输给受鼠0.5mL,含骨髓细胞1×106个,脾淋巴细胞1×107个,制成小鼠aGVHD模型.随机分成5组同基因移植组,异基因移植组,CsA+MTX组,GTT组,GTT+CsA组.CsA+MTX组受鼠骨髓移植后第1-10d,每日腹腔注射CsA2.5mg*kg-1*d-1,第1、3、5、9d腹腔注射氨甲蝶呤(7mg/M2).GTT组受鼠骨髓移植后第1-10d,每日腹腔注射GTT5mg*kg-1*d-1.GTT+CsA组受鼠骨髓移植后第1-10d,每日腹腔注射GTT2.5mg*kg-1*d-1、CsA2.5mg*kg-1*d-1,第11d处死各组受鼠,用ELISA法检测血清中细胞因子IL-2、TNF-α、IL-4、IL-10的浓度.用流式细胞仪检测脾T淋巴细胞及表面粘附分子的阳性表达率.结果(1)雷公藤多甙组小鼠11d生存率明显高于异基因移植组、CsA+MTX组.(2)雷公藤多甙组小鼠CD+3、CD+4、CD+8、CD+4CD11a+、CD+4CD18+、CD+8CD11a+、CD+8CD18+细胞的百分率明显低于异基因移植组.(3)雷公藤多甙组小鼠血清TNFα、IL-2浓度低于、IL-10高于异基因移植组.结论(1)雷公藤多甙可明显提高aGVHD小鼠的生存率.(2)其机制与①雷公藤多甙可降低T细胞及亚群数量及其相关粘附分子的表达有关.②雷公藤多甙降低促进aGVHD的细胞因子-TNFα、IL-2的表达,提高抑制aGVHD的细胞因子-IL-10的表达有关.     

Cx43在造血调控及重建中的作用

目的:采用条件性基因敲除技术构建造血系统间隙连接蛋白43(Cx43)基因敲除(Cx43~(-/-))小鼠模型,并探讨Cx43在维持造血细胞自我更新及功能稳定中的作用。方法:将引进的2对转基因小鼠Cx43 loxP/loxP和Lyz-Cre/+杂交,选取F1雌性子代Cx43 loxP/-_Lyz-Cre/+与雄性Cx43 loxP/loxP合笼回配,提取所获得子代小鼠鼠尾组织基因组DNA,采用PCR方法鉴定小鼠基因型,RT-PCR方法筛选Cx43~(-/-)小鼠,同时分析小鼠不同器官中Cx43基因的表达差异;该类小鼠经5-氟尿嘧啶(5-FU;125 mg/kg)处理,在化疗前及化疗后第5、10和15天经眼球取血分析其血象变化。Cx43~(-/-)及Cx43~(+/+)小鼠予7.5 Gy(~(60)Co-γ)的致死量照射,剂量率1 Gy/min,照射后6 h分别给予事先准备就序的骨髓细胞,每只3×10~6细胞于尾静脉注入,2周后处死小鼠检测造血是否重建:分离股骨切片后,收集骨髓细胞进行细胞表型分析(选用的单抗为CD45R、Gr-1、CD4、 CD8a、TCRαβ、Mac-1、抗sIgM、TER119、Sca-1及CD117);同时进行体外造血细胞集落实验观察造血细胞的体外增殖能力。结果:本研究通过2种转基因小鼠间杂交和回交,成功获得造血系统选择性Cx43基因敲除小鼠;该类小鼠骨髓及外周血细胞无Cx43表达,参与造血的组织,如肝脏和脾脏中Cx43表达也显著下调(P0.01),而心脏和肾脏的Cx43表达则无影响,小鼠成年后外周血象分析并无明显异常,但应急代偿能力下降,经5-FU处理后,其造血功能恢复显著减缓,处理15 d后,Cx43~(+/+)小鼠造血功能已接近正常水平,而Cx43~(-/-)小鼠仍无明显的恢复迹象,血红蛋白、白细胞及血小板仍处低位,2者差别有统计学显著性(P0.01);体外集落试验也证实Cx43~(-/-)小鼠造血干/祖细胞的增殖能力下降,其CFU-GM或CFU-E集落数均明显少于Cx43~(+/+)小鼠(P0.01),但流式细胞术结果显示,Cx43~(-/-)小鼠骨髓中Lin~-/c-Kit~+/Sca-1~+细胞亚群数量与Cx43~(+/+)小鼠相比差异并无统计学显著性;Cx43~(-/-)小鼠在化疗或移植后其骨髓造血功能重建均延迟,且化疗15 d后骨髓切片及涂片均证实其骨髓中造血细胞增生程度明显降低,脂肪组织显著增多,而且T、B细胞发育也有异常。此外,其外周血中CD4~+CD8~+细胞比例比野生型小鼠增多(P0.05),但CD4~+T细胞显著减少(P0.01),尤其是TCRαβ亚群细胞减少最为明显(P0.01)。同样,Cx43~(-/-)小鼠外周血中CD45R~+sIgM~-细胞亚群比例与野生型小鼠相比显著减少(P0.01)。结论:骨髓中Cx43基因表达在造血干/祖细胞发育(尤其是应急状态时)具重要作用,敲除Cx43基因后造血干/祖细胞增殖减缓,造血及免疫重建功能受损。     

人类和小鼠T11(CD2)基因的外显子-内含子组织和顺序比较

50KDa T11(CD2)表面糖蛋白是T淋巴细胞与绵羊细胞结合的受体,抗人类T11分子特定表位(T11_1)的单抗能阻断T细胞和羊红细胞的花环形成。CD2分子在T细胞活化过程中也起重要作用。本文作者构建了含人和小鼠编码CD2分子基因的DNA克隆。人和小鼠T11基因的限制性内切酶和     

Immunoarchitecture of normal human bone marrow: a study of frozen and fixed tissue sections.

To provide baseline information on the immunoarchitecture of normal bone marrow, we studied cryostat-cut, frozen, and paraffin-embedded, fixed tissue sections prepared from 21 core biopsies of normal bone marrow obtained during bone marrow harvests for transplantation. A large panel of antibodies was applied that included, for frozen tissue, Leu-6 (CD1), T11 (CD2), Leu-3a (CD4), Leu-1 (CD5), Leu-2a (CD8), J5 (CD10), My7 (CD13), Leu-11 (CD16), B4 (CD19), B1 (CD20), B2 (CD21), Tac (CD25), My9 (CD33), T200 (CD45), NKH-1 (CD56), kappa and lambda chains, beta F1, Ki-67, HLA-DR, TQ1, and keratin, and for fixed tissue, leukocyte common antigen (CD45), L26 (CD20), LN1 (CDw75), LN2 (CD74), LN3, LN4, LN5, MB1 (CD45R), MB2, MT1 (CD43), MT2 (CD45R), UCHL1 (CD45R0), BM1, Ki-1 (CD30), Leu-M1 (CD15), lysozyme, KP1 (CD68), actin, S100, neuron-specific enolase, vimentin, and keratin. On fresh-frozen sections CD19 and CD2 were the most reliable and sensitive markers for B and T cells, staining 5% and 9% of marrow cells, respectively. Immunoglobulins generally showed heavy background staining, which frequently precluded an accurate assessment. The CD4 to CD8 ratio in the bone marrow was reversed from that of peripheral blood. On fixed tissues, leukocyte common antigen was found in 14% of the marrow cells, corresponding roughly to the lymphocyte population. L26, a pan-B-cell marker, stained 3% of the marrow cells. Among the other B-cell markers, LN1 and MB2 stained a large number of cells (40% to 70%), indicating reactivity with cells of the myeloid or erythroid series in addition to lymphocytes. Among the T-cell markers, UCHL1 and MT1 stained 66% and 50% of the cells, respectively, which could be explained by their cross-reactivity with myeloid cells. Nonspecific myelomonocytic markers (Leu-M1, KP1, and lysozyme) also showed reactivity in a high percentage of cells. No particular architectural distribution patterns of B or T lymphocytes were noted in either frozen or fixed bone marrow specimens. The results of this study provide normal baseline data for the immunohistologic application of hematopoietic and lymphoid markers on frozen or fixed bone marrow biopsy specimens.     

Promoting effects of serotonin on hematopoiesis: ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5-hydroxytryptamine (5-HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony-forming unit-fibroblast (CFU-F) formation, and antiapoptosis of megakaryoblastic M-07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony-forming unit-mixed [CFU-GEMM]) and multilineage committed progenitors (burst-forming unit/colony-forming unit-erythroid [BFU/CFU-E], colony-forming unit-granulocyte macrophage, colony-forming unit-megakaryocyte, CD61+ CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU-E, and CFU-GEMM engraftment in BM of animals 6 weeks post-transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor stimulated BM CFU-F formation. In M-07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase-3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5-HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation. Disclosure of potential conflicts of interest is found at the end of this article.     

A transmembrane chemokine, CXC chemokine ligand 16, expressed by lymph node fibroblastic reticular cells has the potential to regulate T cell migration and adhesion

Stromal cells in lymphoid tissues provide microenvironmental fields required for the triggering of efficient immune responses. Fibroblastic reticular cells (FRCs) are one of the integral constituents of such stromal fields; they construct the reticular network and are considered to regulate immune cells' behavior. However, the factors that mediate the interaction between lymphocytes and FRCs are poorly understood. Here we show that a mouse lymph node (LN)-derived FRC cell line, BLS4, expresses a transmembrane chemokine, CXC chemokine ligand (CXCL) 16, in response to tumor necrosis factor alpha (TNFalpha) and IFNgamma. TNFalpha-induced expression of CXCL16 depends on NFkappaB, p38 MAPK and PKA. Matrix metalloproteinase activity is required for producing soluble CXCL16 in the culture supernatant, likely via shedding at the juxtamembrane region of the extracellular domain. IL-12 enhances the expression of CXCR6 in anti-CD3/CD28-stimulated CD8+ T cells and their adhesion to the BLS4 cell surface in a TNFalpha-dependent fashion. The adherence is significantly inhibited in the presence of both anti-CXCL16 and anti-vascular cell adhesion molecule 1 (VCAM-1) antibodies. CXCL16 expression is also detected in the FRCs in LN sections and in gp38+VCAM-1+ FRCs isolated from LNs. Taken together, these findings suggest that CXCL16 is an important mediator of lymphocyte-stromal interaction within lymphoid tissues.     

Significance of VLA-4–VCAM-1 interaction and CD44 for transendothelial invasion in a bone marrow metastatic myeloma model

In previous work, we established the B9/BM1 syngeneic murine bone marrow metastasis model. Interleukin (IL)-6-dependent, IL-1-producing B9/BM1 cells, which colonize the vertebral and femoral marrow after i.v. injection, show great similarity in cell surface phenotype to human myeloma cells, especially the expression of 3 adhesion molecules, CD44, VLA-4 and ICAM-1. Here we investigated the function of these adhesion molecules by binding and transendothelial invasion assays using a newly established bone marrow-derived endothelial cell line (BMEC). A combination of monoclonal antibodies against CD44 and VLA-4 significantly inhibited the adherence of B9/BM1 cells to BMEC and anti-CD44 mAb especially blocked B9/BM1 transendothelial invasion of unstimulated BMEC cells. Results of additional experiments, in which the cells were treated with anti-CD44 and hyaluronidase, demonstrated that the interaction of CD44 molecules on B9/BM1 cells with hyaluronan on BMEC cells was a critical factor in both adhesion and transendothelial invasion in this model. However, stimulation of BMEC with TNFα resulted in increased invasion by B9/BM1 cells, which was completely suppressed by anti-VCAM-1 mAb, implicating a significant role of this adhesion molecule in this process during inflammation. This revised version was published online in July 2006 with corrections to the Cover Date.     

再生障碍性贫血患者淋巴细胞表型变化

目的：研究再生障碍性贫血（AA）患者骨髓（BM）及外周血（PB）淋巴细胞及其活化相关分子的表达及临床意义。方法：采用单色和双色免疫荧光标记法，流式细胞仪分析AA患者的BM和PB中淋巴细胞膜分子的表达。结果：AA患者BM和BP中CD8^ 细胞增加，CD4／CD8比例下降，BM在CD25^ 细胞和HLA－DR^ 细胞增多，急性AA增加尤为显著（P＜0．01），BM中CD16^ 或CD56^ 细胞也明显增多（P＜0．05），双标记分析提示T细胞主要为CD8^ 细胞：急性AA患者CD8^ -CD25^ 细胞显著增多（P＜0．01），AA患者BM中淋巴细胞活化相关分子表达增多，尤其4－1BB^ ,CD95L^ 和CD40L＋细胞显著增多（P＜0．01），结论：AA患者BM中淋巴细胞活化相关膜分子增多，是AA免疫功能异常及最终导致造血功能衰竭的原因之一。     

Regulation of cell survival during B lymphopoiesis: increased pre-B cell apoptosis in CD24-transgenic mouse bone marrow

CD24 (heat-stable antigen) is expressed in a developmentally regulated fashion by B cell precursors in mouse bone marrow (BM), but its role in B lymphopoiesis remains obscure. A slight overexpression of CD24 in transgenic (Tg) mice leads to depletion of B lymphoid cells in BM. The present study examines whether CD24 is involved in apoptotic selection of B lineage cells under normal microenvironmental conditions in vivo. Double immunofluorescence labeling and flow cytometry have been used to quantitate the apoptotic rates of phenotypically defined B cell populations in BM of CD24-Tg mice. Apoptosis of pre-B cells expressing cytoplasmic mu heavy chains of IgM but lacking surface (s)IgM was increased both ex vivo and in short-term culture, while the number of pre-B cells was halved compared to BM of normal mice. In contrast, B220+mu- pro-B cells and sIgM+ B lymphocytes showed no significant change in either apoptosis or number. The findings provide evidence that CD24 can play a role in vivo in modulating pre-B cell apoptosis, a quality control checkpoint in B cell development.     

The distribution of CD10 (NEP 24.11, CALLA) in humans and mice is similar in non-lymphoid organs but differs within the hematopoietic system: absence on murine T and B lymphoid progenitors

Prior studies in the human have implied an important function for CD10 (CALLA, neutral endopeptidase 24.11) in early lymphoid development. To examine the role of this ectoenzyme in an experimental system, a rat mAb specific for mouse CD10, termed R103, was generated. Immunohistological and flow cytometric analyses indicate that the distribution of CD10 in non-lymphoid anatomical compartments is virtually identical in human and mouse. However, CD10 expression within the hematopoietic system is strikingly different. In contrast to human spleen, lymph node and thymus, the corresponding mouse organs contain no detectable CD10⁺ cells. Mouse granulocytes, unlike human granulocytes, also lack CD10 expression. Five-color flow cytometric studies of adult bone marrow (BM) from C57BL/6 and BALB/c mice with mAb specific for CD43, B220, HSA, BP-1 and immunoglobulin M fail to detect any significant number of CD10⁺ cells at pro-B, pre-B or B cell stages. In addition, lymphoid cells in both (rIL-7) independent and rIL-7-dependent in vitro pro-B cell cultures lack CD10 expression. Consistent with this result, CD10 mRNA is not detected. Unlike the AA4.1⁺ population from day 13 and 14 fetal liver, the CD10⁺ subset is unable to reconstitute T and B lymphoid compartments in RAG-2^?/? mice. Nevertheless, mouse CD10 is readily found on BM stromal elements known to support early B lineage lymphoid development. Given the common expression of CD10 on human and mouse BM stromal elements, this enzyme may have an important function in the stromal cell-dependent phase of hematopoiesis.     

A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells

In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.     

系统性红斑狼疮患者血清IgG对骨髓造血干/祖细胞抑制和凋亡的研究

目的：观察系统性红斑狼疮（SLE）患者血清IgG类抗体体外造血抑制活性及其与造血细胞过度凋亡的关系.方法：应用甲基纤维素集落培养法、原位末端标记及流式细胞术观察系统性红斑狼疮患者血清IgG在体外抑制正常骨髓造血干/祖细胞的增殖及促进正常CD34＋细胞的凋亡.结果：活动期SLE患者血清IgG体外可明显抑制正常骨髓粒-巨噬系祖细胞（CFU-GM）和红系祖细胞（CFU-E）的增殖.这种IgG可特异吸附于正常CD34＋细胞表面,加速CD34＋细胞凋亡,上调CD34＋细胞Fas抗原表达水平.结论：活动期SLE患者血清IgG在体外可抑制正常骨髓造血干/祖细胞的增殖.机制与其上调CD34＋ Fas抗原表达水平而促进CD34＋细胞凋亡有关.     

Ontogeny, distribution and function of CD38-expressing B lymphocytes in mice

Analysis of expression of CD38, CD45R (B220), IgM and IgD on splenic B lymphocytes from mice of different ages demonstrated CD38 on both immature (B220(+), BCR(-)) and mature (B220(+), BCR(+)) B lymphocytes. Similarly, CD38 is expressed as early as B220 on the surface of progenitor B cells in the bone marrow. In spite of expressing of CD38 and IgM, neonatal B cells, in contrast to the adult, failed to proliferate to either anti-CD38 or anti-IgM cross-linking when IL-4 was present. They did, however, respond to LPS and anti-CD40, and by 2 weeks of age they began to respond to anti-CD38 and anti-IgM, reaching adult B cell levels by 4 weeks. Although the distribution of CD38 on adult B cells from most different lymphoid compartments was broadly similar, significantly higher levels of CD38 were expressed on peritoneal B lymphocytes. A detailed analysis, using IgM / IgD ratio and staining with anti-CD5 confirmed that B1 lymphocytes were expressing a high level of CD38. Interestingly, both immature B cells and peritoneal B1 lymphocytes were unresponsive to anti-CD38. However, they were activated by LPS or anti-CD40.