rIL-2激活的骨髓细胞抗瘤活性——白血病自体骨髓移植净化

用rIL-2激活未分离的骨髓外胞(bone marrow cell,BMC)观察其抗瘤活性和造血功能。经rIL-2激活24h后的未分离BMC即表现对NK细胞敏感的K562和NK细胞不敏感的Raji细胞均有杀伤活性;在一定时间内,随激活时间延长抗瘤活性增强,在第4天达高峰。rIL-2激活的未分离BMC与分离BMC二者抗瘤活性无差异。5例急性非淋巴细胞白血病病人骨髓经rIL-2激活后,白血病祖细胞CFU-L生成能力下降;经rIL-2激活的骨髓细胞生成CFU-GM能力无下降。根据我们的研究结果提出,rIL-2激活未分离BMC可用于白血病ABMT中残留白血病细胞净化,其方法简便、实用。     

同基因骨髓混合H-2半相合异基因骨髓移植的抗白血病效应实验研究

为了诱发自体骨髓移植(ABMT)后的移植物抗白血病(GVL)效应,提高患者的长期无病存活,研究了同基因混合H-2半相合异基因骨髓移植(MBMT)后的GVL效应以及移植物抗宿主病(GVHD)的耐受情况。通过混合脾细胞移植对新生小鼠GVHD的作用和MBMT对成年小鼠GVHD的活体观察,证实受体小鼠接受亚致死剂量放射后行同基因骨髓移植时,能够耐受1/6有核细胞量的H-2半同基因骨髓而不发生或发生轻度GVHD。比较观察混合1/6有核细胞H-2半同基因的同基因BMT的抗白血病效应。结果显示,单用同基因BMT者移植后2周皆死亡,混合组5周后仅2只死亡,预计生存率为66％,存活小鼠未观察到明显的GVHD,单纯照射组及L615白血病细胞接种组者1周内全死亡,照射后未接种白血病细胞而行同基因BMT者5周仅1只死亡,提示接受1/6量H-2半同基因骨髓细胞的同基因骨髓移植有显著的抗白血病效应(P<0.01),而不发生GVHD。这一研究为临床ABMT后诱导GVL效应,提供了一个新的途径。     

抗CD44单克隆抗体IM7在体外诱导慢性髓系白血病干/祖细胞的凋亡

本研究探讨抗CD44单克隆抗体IM7在体外诱导慢性髓系白血病干/祖细胞(leukemic stem/progenitor cells,LSPC)凋亡情况及其可能的作用机制。取20例初诊的慢性髓系白血病(chronic myeloid leukemia,CML)病人骨髓液5-10ml,用免疫磁株分离法分离出CD34+、CD38-、CD123+LSPC。利用Annexin-V试剂盒检测IM7诱导CML-LSPC凋亡情况;荧光实时定量PCR及RT-PCR检测CML-LSPC中原癌基因c-myc、NF-κB表达水平;Western-blot检测IM7孵育后CML-LSPC中BCL-2蛋白表达变化。结果表明:经IM7孵育后CML-LSPC的早期凋亡率由对照组(5.42±1.84)(升高至(12.58±2.84)((p0.05)。IM7孵育后CML-LSPC中原癌基因c-myc、NF-κB mRNA表达水平明显降低,IM7能有效抑制CML-LSPC中BCL-2蛋白表达下调,CML-LSPC中NF-κB的活性受到抑制。结论:抗CD44单克隆抗体IM7能有效诱导CML-LSPC凋亡,其可能机制为NF-κB的活性降低,作为下游靶基因c-myc及bcl-2表达下调,从而诱导LSPC凋亡。     

抗人PCIA1单克隆抗体的制备及其生物特征的研究

目的:筛选制备抗人PCIA1的单克隆抗体,鉴定其生物学特征。方法:采用原核表达并纯化的PCIA1蛋白作为抗原,采用杂交瘤技术制备抗人PCIA1单克隆抗体的杂交瘤,用ELISA进行筛选和鉴定其亚类,并用细胞扒片免疫化学方法检测其反应性。结果:获得了32株分泌抗人PCIA1的McAb杂交瘤细胞,McAb-1A4的染色体呈双亲特点,该McAb与多种人肿瘤细胞株和正常肝细胞株有反应。结论:McAb-1A4可用于PCIA1的细胞内定位研究。     

8-溴-7-甲氧基白杨素的合成及其抗人急性细胞性白血病作用

目的 观察8-溴-7-甲氧基白杨素对人急性粒细胞性白血病(HL-60)细胞增殖抑制作用。方法 MTT比色法测定8-溴-7-甲氧基白杨素对人急性粒细胞性白血病(HL-60)细胞增殖抑制作用。HL-60细胞小鼠肾囊膜下移植瘤模型观察8-溴-7-甲氧基白杨素对人急性粒细胞性白血病的治疗作用。结果 8-溴-7-甲氧基白杨素对人急性粒细胞性白血病(HL-60)细胞增殖具有抑制作用，呈浓度依赖性。8-溴-7-甲氧基白杨素对人急性粒细胞性白血病细胞小鼠肾囊膜下移植瘤生长具有抑制作用，呈剂量依赖性。结论 8-溴-7-甲氧基白杨素是一种急性粒细胞性白血病治疗新候选药物。     

人源IL-21增强细胞因子诱导杀伤细胞抗白血病作用及其机制研究

目的 研究IL-21对外周血来源的细胞因子诱导杀伤细胞(CIK细胞)抗白血病作用及其机制.方法 采集分离正常人外周血单个核细胞,加用细胞因子诱导培养,用MTT法检测CIK细胞的增殖能力及其对自血病细胞系K562细胞的杀伤作用;用流式细胞术检测CIK细胞免疫表型及其表面IL-21受体(IL-21R)、FasL、细胞内穿孔素和颗粒酶B的表达;半定量RT-PCR法检测CIK细胞IFN-γ、TNF-α、TNF-β、穿孔素、颗粒酶A、颗粒酶B、FasL和NKG2D mRNA的表达;酶联免疫法检测培养上清中IFN-γ和TNF-α的表达;Western blot法检测CIK细胞JAK-STAT信号途径的变化.结果 在IL-21作用下,CIK细胞的产生由(17.5±4.7)％升至(26.5±2.1)％,对K562细胞的杀伤作用由(22.8±2.8)％升至(44.6±8.3)％,CIK细胞表面IL-21R的表达增加约2倍.CIK细胞IFN-γmRNA的表达由0.3760±0.2358升至0.7786±0.2493,TNF-α mRNA的表达由0.6557±0.1598升至1.3145 ±0.2136,穿孔素mRNA的表达由0.6361 ±0.1457升至0.9831 ±0.1265,颗粒酶B mRNA的表达由0.4084±0.1589升至0.7319±0.1639,FasL mRNA的表达由0.4015±0.2842升至0.7381±0.2568,差异均有统计学意义.CIK细胞表面FasL的表达水平,加IL-21组(0.19％)与未加IL-21组(0.04％)相比,差异有统计学意义.CIK细胞内穿孔素的表达由35.28％升至53.16％,颗粒酶B的表达由43.16％升至78.82％,培养上清中IFN-γ的表达由(25.8 ±6.1)ng/L升至(56.0 ±2.3) ng/L,TNFα的表达由(5.64±0.61) μg/L升至(15.14±0.93) μg/L.STAT3和STAT5b的表达增加.结论 人源IL-21可增强外周血来源CIK细胞抗白血病作用,此作用是通过增加其受体的表达,增加穿孔素、颗粒酶B、FasL、IFN-γ和TNFα的表达而实现的,JAK-STAT细胞信号途径的激活是此种作用的机制之一.提示IL-21在增强白血病免疫治疗中具有潜在的临床应用前景.     

体外阻断CD_(40)-CD_(40)L共刺激途径减轻小鼠异基因骨髓移植中移植物抗宿主病的研究

目的　在异基因骨髓移植移植物抗宿主病 (GVHD)小鼠模型中 ,观察抗CD4 0 L单克隆抗体 (单抗 )体外预处理供鼠T细胞输注 ,观察其减轻GVHD的作用并探讨其作用机制。方法　供鼠(C5 7BL 6 H 2b)脾T细胞作为反应细胞 ,受鼠 (BALB cH 2d)脾细胞作为刺激细胞 ,分别加抗CD4 0 L单抗或不加单抗进行混合培养 ,培养第 5天的细胞作为经体外诱导后的脾T淋巴细胞 ,分别混合供鼠骨髓细胞后移植给接受 8.0Gy全身照射预处理的受鼠。比较受鼠GVHD发生和造血重建。在移植后不同时间点采用流式细胞仪检测受鼠脾细胞中T细胞表型的改变 ,用ELISA法检测外周血血清中Th1和Th2类细胞因子的水平。结果　移植后对照组受鼠均于 2 5d内死于GVHD ,实验组GVHD的发生率为2 0 % ,与对照组小鼠相比 ,存活率和存活时间明显增高和延长 (P <0 0 1) ;存活的实验组小鼠 (8只 )第4 0天时骨髓细胞中H 2Db 阳性细胞为 (93.5 4± 2 .32 ) %。实验组小鼠CD3+ CD4+ 、CD3+ CD8+ 、CD4+CD2 5+ 、CD4+ CD6 9+ 、CD8+ CD2 5+ 、CD4+ CD4 0 L+ 和CD8+ CD6 9+ T细胞比例明显低于对照组 (P <0 .0 5 ) ,CD8+ CD4 0 L+ 和CD4+ CD4 5RA+ T细胞比例在两组中变化差异无显著性 (P >0 .0 5 )。实验组受鼠血清中细胞因子水平明显低于对照组 (P <0 .0 5 )。结论　应     

人B7．1真核表达载体的构建、鉴定及其在白血病细胞上表达的实验研究

本研究构建包含人B7．1cDNA的重组真核表达载体pDisplay-hB7．1并鉴定,实现其在白血病细胞株HL-60细胞上的表达,获得HL60-hB7．1。应用分子克隆、PCR法扩增人B7．1基因片段,采用ApaⅠ、Sall分别双酶切PCR产物和表达载体pHook,纯化后的酶切产物由T4DNA连接酶连接,转导大肠杆菌DI-15et;应用ApaⅠ、SalⅠ双酶切和DNA序列分析技术进一步鉴定阳性克隆pDisplay-hB7．1,应用脂质体转化技术将B7．1转染HL-60细胞,同一标本分别采用直接免疫荧光法、SABC法及FACS法鉴定hB7．1在HL-60细胞上的表达,阳性细胞命名为HL60．hB7．1。结果表明,PCR法扩增出约620bp的基因片段;连接产物转导感受态细胞后,共筛选出5个阳性克隆,均可经ApaⅠ、SalⅠ双酶切出大小约620bp的基因片段,与人B7．1基因片段大小相符,提示重组载体构建成功。进一步DNA序列分析证明人B7．1基因序列和读码框架正确,与文献报道人B7．1cDNA序列一致,无基因突变。直接免疫荧光法鉴定表明,HL60-hB7．1阳性细胞数为70％,SABC法鉴定为65％,FACS法鉴定为92．7％。经以上鉴定证实hB7．1cDNA已成功转染HL-60细胞株并在其膜上高效表达,获得了HL60-hB7．1。结论：应用分子克隆方法可成功构建人B7．1（CD80）重组真核表达载体,并稳定、高效表达B7．1-于HL-60细胞胞膜上,推测应用这种方案制备的瘤苗可能具有免疫治疗和免疫保护作用。     

抗VEGF抗体联合重组人可溶性TRAIL诱导白血病细胞K562凋亡的研究

本研究探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)和抗血管内皮生长因子(VEGF)抗体对人慢性髓系白血病细胞K562的生长抑制和诱导凋亡的协同作用。将TRAIL和抗VEGF抗体单独及联合作用于K562细胞,应用CCK-8法检测各组的细胞抑制率和用流式细胞仪检测各组的细胞凋亡率。结果表明:以TRAIL 75、100和150ng/ml的浓度作用于K562细胞48小时的凋亡率分别为(4.26±0.67)%、(8.91±0.55)%、(11.71±0.78)%;抗VEGF抗体2.5、5和7.5μg/ml的浓度作用于K562细胞48小时的凋亡率分别为(3.95±0.69)%、(7.98±0.74)%和(10.26±0.83)%。以抗VEGF抗体2.5μg/ml+TRAIL 75 ng/ml或抗VEGF抗体5μg/ml+TRAIL100 ng/ml或抗VEGF抗体7.5μg/ml+TRAIL150 ng/ml作用于K562细胞48小时的细胞凋亡率分别为(22.16±0.93)%、(36.32±1.31)%和(49.19±0.71)%。抗VEGF抗体与TRAIL联合作用于K562细胞后,细胞抑制率及凋亡率显著高于单独应用TRAIL组或抗VEGF抗体组(p0.05)。结论:TRAIL和抗VEGF抗体对K562细胞在诱导凋亡过程中具有协同作用。     

长期骨髓细胞培养净化白血病细胞与临床疗效关系的研究

应用Dexter长期培养体系进行了17急性非淋巴细胞白血病例(ANLL)骨髓的长期培养,按照培养4周后基质细胞CFU-ANLL和CFU-GM的生长情况,将其分为3型:Ⅰ型(11/17例)为培养净化有效型;Ⅱ型(3/17例)为部分净化有效或需4周以上培养观察其净化效果的类型;Ⅲ型基质细胞缺陷。体外培养中各种类型细胞均同步死亡。Ⅰ型病人化疗有效率较Ⅱ、Ⅲ型高。该结果提示临床应用Dexter体系净化进行自体骨髓移植(ABMT)时,应按其体外生长类型选择病人。比分类与所处临床状态无关,其净化作用可能与临床化疗之间无选择性交叉抗肿瘤关系。     

纳米材料复合人骨髓成骨细胞培养的研究

目的　探讨纳米材料作为组织工程骨基质材料的可行性。方法　分离培养人骨髓间充质干细胞 ,诱导为成骨细胞后作为种子细胞 ,与纳米晶羟基磷灰石胶原材料体外联合培养 ,通过对复合物光镜及扫描电镜观察 ,了解细胞在材料中生长情况。结果　人骨髓间充质干细胞可以诱导为成骨细胞 ,体外复合培养 2周 ,分布于支架材料上的细胞大量分化增殖。结论　纳米晶羟基磷灰石胶原材料是一种构建组织工程骨的较好的支架材料     

THE PROLIFERATION OF PLASMA CELLS FROM MOUSE BONE MARROW IN VITRO : I. THE ROLE OF THYMUS

An in vitro proliferative system for immunoglobulin-G-forming plasma cells from the bone marrow of mice was established by the addition of antigenic protein and thymic cells or their homogenate to bone marrow cultures. The promoting activity of the thymus on plasma cells was independent from mouse strain, but it differed in strength with the variations of donor strains. Synthesis of immunoglobulin-G in proliferating plasma cells and its antibody reactivity against the administered antigen were demonstrated by immunocytological analyses.     

成纤维细胞及成纤维细胞条件培养液对L_(833)白血病细胞增殖与分化的影响

本文通过连续传代的方法建立纯化的成纤维细胞层,观察成纤维细胞及成纤维细胞条件培养液(F-CM)对L_(833)白血病细胞增殖与分化的影响。并对F-CM进行了初步的生物学活性鉴定。结果提示:成纤维细胞对L_(833)细胞增殖有抑制作用;其条件培养液有刺激L_(833)细胞增殖并部分诱导分化作用;成纤维细胞对L_(833)细胞增殖的抑制作用强于混合基质细胞;F-CM促分化效应强于F_0-CM及rmGM-CSF。经琼脂隔离后的成纤维细胞对L_(833)细胞增殖的抑制作用消失。     

人胚骨髓基质干细胞的分离纯化及向神经样细胞的诱导分化

目的　培养纯化人胚骨髓基质干细胞并向神经系统方向诱导。方法　提取人胚胎股骨、肱骨骨髓 ,Percoll分离液分离 ,将含有MSC的中间白膜层培养于DMEM +10 %的胎牛血清培养基中 ,并进行扩增。用 2 0 0 μm的BHA对其进行诱导分化 ,并用免疫组化染色检测特异性神经元烯醇化酶 (NSE)和胶质纤维酸性蛋白 (GFAP)。结果　成功进行了人胚骨髓基质干细胞原代和传代培养 ,并成功完成了向神经样细胞的诱导。结论　人胚骨髓基质干细胞可以比较容易的获得并能向神经样细胞分化 ,是一个研究神经再生的较好的标本来源     

干细胞复合纳米晶羟基磷灰石胶原修复骨缺损的研究

目的　研究纳米材料作为组织工程骨基质材料的特点。方法　分离培养人骨髓间充质干细胞 ,与纳米晶羟基磷灰石胶原材料 (nHAC/PLA)于体外联合培养 ;通过大体观察、组织学分析及电镜观察了解成骨情况 ,进一步临床应用于修复骨缺损。结果　人骨髓间充质干细胞在体外可以大量扩增 ,复合细胞的材料植入骨缺损处后 ,X光摄片动态观察可见骨缺损处连接良好。结论　骨髓间充质干细胞具有成骨细胞作用 ,纳米晶羟基磷灰石胶原材料是一种很好的构建组织工程骨的支架材料。     

骨髓基质细胞的造血支持作用等生物学特性的研究

采用静置贴壁细胞培养法,体外长期培养了胎儿,儿童、成人骨髓基质细胞,时间为６个月,可传至１０代,并观察了成纤维肌样细胞,内皮细胞和巨噬细胞;通过免疫细胞化学染色法,证明了１－３代的骨髓基质肌样细胞Ｖｉｅｍｅｎｔｉｎ为阳性,第Ⅷ因子阴性;采用流式细胞仪检测法,证明了儿童髓基质细胞的细胞表型为ＣＥ＾－３３,ＣＤ＾－３４、ＣＤ＾－３８、ＣＤ＾＋Ｗ９０,成人骨髓基质细胞为ＣＤ＾－３３、ＣＤ＾－３４、ＣＤ＾     

THE RHYTHMIC RANGE OF THE WHITE BLOOD CELLS IN HUMAN,PATHOLOGICAL LEUCOPENIC AND LEUCOCYTIC STATES,WITH A STUDY OF THIRTY-TWO HUMAN BONE MARROWS

In a study of twenty clinical cases with a wide range of diagnoses, repeated total counts of the white cells at 15 minute intervals reveal a large fluctuation at various levels comparable to that found for the normal (1, 2). The granulocytes seem to follow a more or less hourly rhythm, the most marked shift to the left in the Ameth pattern and the moment of greatest percentage of motility coinciding with the peaks. The independence found existing between the peripheral blood concentrations of individual strains of white cells and the red cells, as determined by total and differential counts, their differential response to pathological and pharmacological stimuli, and their normal relative relations, all indicate some separate physiological mechanism of control for each type of cell, either working through, or independently of, their sources of origin. The many factors to which the circulation of the blood, as such, is subject, the complexity of the influences on origin, maturation, delivery, longevity, and destruction of each cell group, the limitations inherent in the present involved, indirect technics of counting, combine to make any single observation subject to grave misinterpretation. The value to the clinician must come in repeated observations, at times when the diagnosis or a therapeutic procedure is in doubt, at frequent intervals, at other times over longer or shorter periods, but always with the relation between consecutive counts, rather than the absolute values, the important point for consideration. Both the red and the white cells probably change their relative concentrations in the peripheral blood from time to time over a considerable range that is quite within normal physiological limits, so that, in theoretical considerations and in practical functional estimations, a zonal concept with adequate individual extremes should always be kept in mind for both physiological and pathological states. A cytological analysis of thirty-two bone marrows from human biopsy and autopsy material shows the striking reciprocity found to exist between the myelocytes and the mature polymorphonuclear leucocytes. This, together with the observed focal uniformity of maturation found in bone marrow, and the periodicity of the fluctuations of the neutrophils in the peripheral blood, leads to the formulation of the hypothesis of a constant functional withdrawal of granulocytes from the peripheral blood with a periodic delivery of new cells from the marrow, which in leucopenia and in leucocytosis represents a depression or a stimulation, respectively, of the normal mechanism. The nature and degree of the response are an approximate index of the cellular factor in the complex of the "resistance" of the particular individual.     

CONTRIBUTION OF A THYMIC HUMORAL FACTOR TO THE DEVELOPMENT OF AN IMMUNOLOGICALLY COMPETENT POPULATION FROM CELLS OF MOUSE BONE MARROW

The hypothesis that cells located in mouse bone marrow can acquire immunological competence by a process that involves interaction with a noncellular component of the thymus was tested using an in vitro assay of graft-versus-host reactivity as a criterion of cell competence. When suspensions of C57BL bone marrow cells were incubated in thymus extract and injected into mice incapable of inducing a response in the graft-versus-host assay as a result of neonatal thymectomy, or adult thymectomy plus irradiation, or because of genetic similarity with the (C3H x C57BL)F₁ tissue used for challenge in the assay, competent cells were recovered from the spleens of the injected mice. The reactive cells were shown to be of bone marrow origin since immune reactivity was related to the genetic makeup of the bone marrow cells rather than that of the intermediate recipients. A thymic factor was involved in the process leading to immune reactivity by these cells, as bone marrow cells incubated in xenogeneic or syngeneic thymic extracts induced a graft-versus-host response after passage through nonresponsive mice, whereas incubation of bone marrow cells in xenogeneic lymph node or spleen extracts or in culture medium only did not lead to subsequent reactivity. Participation of peripheral lymphoid tissue seemed essential in this process since bone marrow cells tested directly after exposure to thymic extract failed to induce a graft-versus-host response. C57BL bone marrow cells exposed to thymus extract and cultured together with fragments of (C3H x C57BL)F₁ spleen tissue in vitro were competent to induce a graft-versus-host response; thus, these components would seem to be sufficient as well as necessary for the immunodifferentiation process leading to graft-versus-host activity. It is concluded that one step in the process by which bone marrow cells acquire competence vis-a-vis the graft-versus-host response depends upon a thymic agent that is noncellular and extractable, and that another stage in this process is under the influence of components found within the peripheral lymphoid tissue environment. It is suggested that differentiation of precursor cells to competence could occur by progressive development of the cells in separate compartments of the lymphoid system.     

THE AUTOPLASTIC TRANSPLANTATION OF TISSUES INTO THE BONE MARROW CAVITY : I. THE THYMUS.

When the thymus is autoplastically transplanted into the marrow cavity of the tibia of a guinea pig, the transplant degenerates, regenerates, and finally atrophies. The degeneration and regeneration of the thymus when so transplanted are similar to the homologous changes occurring when it is autoplastically transplanted into the abdominal wall. For comparative study the material used in previously published work on transplantation into the abdominal wall was reviewed. A difference in the final result of transplantation of thymus in these two situations is observed. In the abdominal wall the thymus regenerates and persists apparently indefinitely. Abdominal wall thymus transplants more than a year old have been observed. In the bone marrow cavity, the regeneration, which is slower and less abundant, is followed by an atrophy of the gland, and finally its complete disappearance. This atrophy is, we believe, the result of the encasement of the thymus in a bony capsule. That the genetic character of the transplanted tissue has much to do with the success of the transplantation in the marrow, is evident from the fact that some other tissues regenerate without encapsulation, even though they may finally disappear. The blood clot about the thymus which fills the rest of the curetted cavity, is quickly vascularized and replaced by regenerated cellular bone marrow and newly formed bony trabeculæ. The difference in the fate of the thymus when transplanted in the abdominal wall and in the marrow, is probably due to difference in the reciprocal relations of the thymus to the tissue in those situations. From experience gained in the transplantation of other tissues into the marrow cavity, we have learned that encapsulation of the transplant does not occur with all tissues. The reaction of the marrow to transplanted thymus is similar to its reaction to any tissue foreign to it.