人内皮细胞对正常人和再生障碍性贫血患者粒系造血的研究

以人脐静脉内皮细胞作铺层，进行体外CFU－GM半固体培养，观察内皮细胞对正常人和再生障碍性贫血患者粒系造血的影响。在有内皮细胞的各培养体系，CFU－GM的产率均显著高于无内皮细胞的对照体系。在无内皮细胞存在的体系中，需添加GM－CSF才能表现出对粒系造血的刺激作用。对15例初诊慢性再障患者骨髓粒系祖细胞的培养结果表明，无内皮细胞存在时，无论向培养体系中加何种细胞因子均无集落形成，而在有内皮细胞的双层培养中，部分患者骨髓培养中有CFU－GM集落形成。研究结果表明内皮细胞对正常人和再障患者粒系造血均有明显影响。     

肿瘤坏死因子,干扰素和免疫活性肽对癌瘤患者骨髓粒—巨造血祖细…

应用ＣＦＵ－ＧＭ软琼脂培养技术，在体外观察了不同浓度的ｒｈ－ＴＮＦ，ｒｈ－ＩＦＮα－２和ＩＡＰ对９例恶性淋巴瘤和１例睾丸癌患者的骨髓粒、巨造血祖细胞生长的影响。结果表明，在培养体系中，加入ｒｈ－ＴＮＦ５０和１００ｕ／ｍｌ，其ＣＦＵ－ＧＭ较空白对照均明显增多（Ｐ〈０．０５）。ｒｈ－ＩＦＮα－２１０００ｕ／ｍｌ可使ＣＦＵ－ＧＭ减少（Ｐ〈０．０５）。ＩＡＰ似乎对ＣＦＵ－ＧＭ无影响（Ｐ〉０．０５）。集落形     

肿瘤坏死因子、干扰素和免疫活性肽对癌瘤患者骨髓粒－巨造血祖细胞的影响

应用ＣＦＵ－ＧＭ软琼脂培养技术，在体外观察了不同浓度的ｒｈ－ＴＮＦ，ｒｈ－ＩＦＮα－２和ＩＡＰ对９例恶性淋巴瘤和１例睾丸癌患者的骨髓粒－巨造血祖细胞生长的影响。结果表明，在培养体系中，加入ｒｈ－ＴＮＦ５０和１００ｕ／ｍｌ，其ＣＦＵ－ＧＭ较空白对照均明显增多（Ｐ＜０．０５）。ｒｈ－ＩＦＮα－２１０００ｕ／ｍｌ可使ＣＦＵ－ＧＭ减少（Ｐ＜０．０５）。ＩＡＰ似乎对ＣＦＵ－ＧＭ无影响（Ｐ＞０．０５）。集落形态学显示，ｒｈ－ＴＮＦ和ＩＡＰ均能增加巨噬细胞为主形成的集落（Ｐ＜０．０１）。我们认为，该实验为骨髓移植及肿瘤患者在选择应用这些细胞因子上提供了一定的佐证。     

利用双层细胞培养技术对粒系造血的探讨

研究造血微环境及基质细胞对造血的影响，在生理和病理造血中均具有重要意义。我们采用以人脐静脉内皮细胞为铺层的双层细胞培养技术，对１０例正常骨髓细胞及１５例再生障碍性贫血（再障）患者粒系造血的情况进行了体外ＣＦＵ－ＧＭ半固体双层细胞培养的观察。１材料与方...     

脐血输注对急性白血病患者骨髓CFU－GM体外培养及血浆CSA、BPA的影响

以自身对比方法观察急性白血病化疗后骨髓衰竭患者输注脐血和外周血后，骨髓粒、单细胞系集落形成单位（ＣＦＵ－ＧＭ）、血浆集落刺激活性（ＣＳＡ）、爆式集落刺激活性（ＢＰＡ）及外周血白细胞计数（ＷＢＣ）的变化。结果脐血输注后可使患者ＣＦＵ－ＧＭ体外培养显著提高，ＣＳＡ、ＢＰＡ水平显著下降并趋于正常，可在输血量减少情况下，使ＷＢＣ显著增高。     

脐血输注对急性白血病患者骨髓CFU—GM体外培养及血浆CSA,BPA的影响

以自身对比方法观察急性白血病化疗后骨髓衰竭患者输注脐血和外周血后，骨髓粒、单细胞系集落形成单位（ＣＦＵ－ＧＭ）、血浆集落刺激活性（ＣＳＡ）、爆式集落刺激活性（ＢＰＡ）有外周血白细胞计数（ＷＢＣ）的变化。结果脐血输注后可使患者ＣＦＵ－ＧＭ体外增减显著提高，ＣＳＡ、ＢＰＡ水平显著下降并趋于正常，可在输血量减少情况下，使ＷＢＣ显著增高。     

APL经ATRA诱导后高白细胞征机制的进一步研究

目的：提示ＡＰＬ患者ＡＴＲＡ治疗后高白细胞征的机制。方法：外周血ＷＢＣ计为九及分类按常规进行，体内外粒－单系集落（ＣＦＵ－ＧＭ）按半固体琼脂法进行。ＡＴＲＡ体内实验中的骨髓与脾脏均用２００目风研磨制成单个细胞悬液后计ＷＢＣ数。结果：ＡＴＲＡ临床治疗ＡＰＬ患者可引起明显的ＷＢＣ、早幼粒及较成熟粒系升高。ＣＦＵ－ＧＭ呈现升 高趋势。而ＡＰＬ患者骨髓ＣＦＵ－Ｌ逐渐降低。正常小鼠的体内实验中，ＡＴＲＡ明显     

光卟啉-光照选择性杀灭白血病细胞株K562的研究

为研究光卟啉（ＹＨＰＤ）加光照对白血病细胞株Ｋ５６２的杀伤作用及对正常造血祖细胞的影响，通过细胞体外液体培养活细胞计数、体外半固体培养集落测定，以探讨该法用于体外净化自体移植骨髓中残留的白血病细胞的可能性。观察不同光敏剂剂量和光照时间的变化对杀伤白血病细胞作用的影响，结果表明，单用ＹＨＰＤ或单独日光荧光照射对Ｋ５６２细胞和正常人造血祖细胞均无明显杀伤作用。二者联合使用表现光敏效应。细胞存活率的对数与ＹＨＰＤ剂量、光照时间呈线性相关。ＹＨＰＤ加光照对Ｋ５６２细胞具有强大杀伤作用，对正常造血祖细胞影响较小，在能够杀伤白血病细胞大于５个对数级，其体外已不能形成集落的情况下，正常人骨髓－巨噬细胞集落形成单位（ＣＦＵ－ＧＭ）尚有１５．９７％，提示该法可望用于骨髓体外净化     

通关藤提取物体外对人正常免疫细胞及造血干细胞的影响

目的　观察通关藤提取物（消癌平注射液）对正常免疫细胞及造血干细胞功能的影响。方法　采用ＭＴＴ法检测通关藤提取物对正常人淋巴细胞以及ＣｏｎＡ、ＬＰＳ诱导的人淋巴细胞增殖活性的影响,中性红法检测巨噬细胞的吞噬功能,ＣＦＵ-ＧＭ半固体集落培养法检测造血干细胞的集落形成能力。结果　通关藤提取物体外对ＣｏｎＡ诱导的正常淋巴细胞和ＬＰＳ诱导的正常淋巴细胞有促增殖作用且呈剂量依赖性。通关藤提取物对正常巨噬细胞吞噬中性红染液的能力无明显影响。对正常骨髓／外周血造血干细胞ＣＦＵ-ＧＭ的形成没有显著影响。结论　通关藤提取物体外对正常免疫细胞和造血干细胞无明显细胞毒作用,并且有促进Ｔ、Ｂ细胞的增殖作用。     

重组人干细胞因子对人类急性髓系白血病干／祖细胞自我更新作用的实验研究

应用无血清液体一半固体二次培养法，观察了重组人干细胞因子（ｒｅｃｏｍｂｉｎａｎｔｈｕｍａｎｓｔｅｍｃｅｌｌｆａｃｔｏｒ，ｒｈＳＣＦ）对人类急性髓系白血病干／祖细胞（ＡＭＬ－ＣＦＵ）自我更新的影响。认为ｒｈＳＣＦ是一作用阶段早于白介素３（ＩＬ－３）和粒／巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）的造血生长因子（ＨＧＦ）。无论单一，还是与其它ＨＧＦ联合，ｒｈＳＣＦ对绝大部分ＡＭＬ患者，均有强大的维持其ＡＭＬ－ＣＦＵ自我更新的作用。但不同ＦＡＢ亚型及ＦＡＢ亚型相同的患者之间，这种维持作用差异较为显著。提示ＳＣＦ在ＡＭＬ病理过程中起重要作用。     

Characteristics of Granulocyte-Macrophage Colony Formation in Chronic Myelomonocytic Leukemia: A Comparative Study with Other Myelodysplastic and Myeloproliferative Disorders

We studied granulocyte-macrophage (GM) colony formation in chronic myelomonocytic leukemia (CMML, 6 cases), as compared with that in myelodysplastic syndromes (MDS, 6 cases) and myeloproliferative disorders (MPD, 12 cases). GM colony formation of bone marrow cells by colony-stimulating factor (CSF) was normal in CMML and MPD patients, but was decreased in MDS patients. Circulating granulocyte-macrophage progenitors (CFU-GM) were detected in CMML and MPD patients, but not in MDS patients. GM colony formation without CSF was observed in CMML patients, but not in MDS or MPD patients. These endogenous colonies decreased markedly after adherent cell (AdC) depletion, but AdC did not form endogenous colonies in sufficient numbers to explain their marked decrease after AdC depletion. In CMML patients, the numbers of circulating CFU-GM and endogenous colonies correlated with leukocyte and monocyte counts, respectively. The cellular composition of GM colonies was normal in MDS and MPD patients, whereas granulocytic colonies predominated in all CMML patients but one. The CSF-producing capacity of peripheral blood cells was also studied and was found to be increased in CMML patients. This capacity was markedly decreased by AdC depletion; and AdC could produce CSF only in CMML patients. CSF produced by CMML patients supported granulocytic colonies to a greater extent than CSF produced by MDS or MPD patients. These results suggest that enhanced granulopoiesis in CMML patients is closely associated with the possible hyperproduction of granulocytic CSF by their adherent monocytes.     

Characteristics of granulocyte-macrophage colony formation in chronic myelomonocytic leukemia: a comparative study with other myelodysplastic and myeloproliferative disorders

We studied granulocyte-macrophage (GM) colony formation in chronic myelomonocytic leukemia (CMML, 6 cases), as compared with that in myelodysplastic syndromes (MDS, 6 cases) and myeloproliferative disorders (MPD, 12 cases). GM colony formation of bone marrow cells by colony-stimulating factor (CSF) was normal in CMML and MPD patients, but was decreased in MDS patients. Circulating granulocyte-macrophage progenitors (CFU-GM) were detected in CMML and MPD patients, but not in MDS patients. GM colony formation without CSF was observed in CMML patients, but not in MDS or MPD patients. These endogenous colonies decreased markedly after adherent cell (AdC) depletion, but AdC did not form endogenous colonies in sufficient numbers to explain their marked decrease after AdC depletion. In CMML patients, the numbers of circulating CFU-GM and endogenous colonies correlated with leukocyte and monocyte counts, respectively. The cellular composition of GM colonies was normal in MDS and MPD patients, whereas granulocytic colonies predominated in all CMML patients but one. The CSF-producing capacity of peripheral blood cells was also studied and was found to be increased in CMML patients. This capacity was markedly decreased by AdC depletion; and AdC could produce CSF only in CMML patients. CSF produced by CMML patients supported granulocytic colonies to a greater extent than CSF produced by MDS or MPD patients. These results suggest that enhanced granulopoiesis in CMML patients is closely associated with the possible hyperproduction of granulocytic CSF by their adherent monocytes.     

In-vitro granulopoiesis in adult acute lymphoblastic leukemia at various phases of the disease

We used the in-vitro agar culture technique to monitor the granulopoiesis in 68 adult patients with ALL during the course of their disease. Bone marrow cells were cultured from 42 patients at diagnosis, 26 patients in relapse, 36 patients in early remission and 31 patients in full remission. The results of culture growth were characterized by sparse or no growth at diagnosis. No inhibition of normal CFU-C by leukemic cells was demonstrated by co-culture experiments. In relapsed marrows with blasts exceeding 60%, the culture results were identical to those at first presentation. The colonies grown in ALL cultures showed normal morphology with a normal granulocytic and monocytic differentiation. The colony-forming potential gradually increased following induction therapy, but there was no relationship between the CFU-C number and the percentage of blasts. The impaired granulopoiesis usually recovered once a remission was obtained and remained normal throughout the remission period. In some instances, cultures were performed within a short period prior to relapse or carried out more than one occasion during stable remission, wide fluctuations in CFU-C incidences were observed. Our study indicates that the CFU-C assay in ALL is useful for monitoring the in-vitro granulopoietic activity at various phases of the disease, but is of limited value in predicting the response to treatment as well as in determining the remission-relapse status.     

Hematopoietic Progenitors and Synergism of Interferon-γ and Stem Cell Factor

Interferon-γ (IFN-γ), an immunoregulatory cytokine produced by activated T cells and natural killer cells in response to viral infection or other stimuli, is generally recognized as a suppressor of hematopoiesis. IFN-γ inhibited in vitro colony formation by granulocyte-macrophage (GM), erythroid and multipotential progenitors. This cytokine exerted direct suppression on the proliferation process, but not on the commitment, of GM progenitors. The antiproliferative effects of IFN-γ may, in part, result from the prolongation of the doubling time of GM progenitors. Clinically, IFN-γ may play an important role in the pathogenesis of pancytopenia in aplastic anemia and in the hemophagocytic syndrome. However, as well as showing inhibitory effects, IFN-γ increased the number of pure and mixed megakaryocyte colonies formed by post-5-fluorouracil treated bone marrow cells and, moreover, the addition of IFN-γ to culture containing stem cell factor resulted in a synergistic effect on the development of both primitive hematopoietic progenitors and mature populations. These findings suggest that IFN-γ has bifunctional activity in hematopoiesis.     

Acute erythremic myelosis (DiGuglielmo) following atypical aplastic anemia

A patient presented with acute erythromyelosis (DiGuglielmo) which was developed after 3 yr of aplastic anemia. Aplastic anemia differed from the classical form, since erythroid cells and megakaryocytes were relatively preserved in the bone marrow. Treatment with androgens induced the increase of hematocrit and reticulocyte as well as general improvement. The sudden appearance of hemorrhagic syndrome due to thrombocytopenia was associated with aggravation of anemia and granulocytopenia. In the bone marrow, giant multinuclear proerythroblasts with bizarre nuclear morphology and PAS positivity with coarse granules was found. Serum erythropoietin (Ep) level was high. Bone marrow cells culture in vitro revealed two types of erythroid colonies: typical and giant multinuclear cells, both benzidine-positive. The number of colonies was irrespective to the Ep dose. "Autonomous" Ep independent growth of these colonies was also demonstrated. The number of colonies was more than 3 times higher per number of cells seeded when compared to normals, which indicated malignant proliferation and Ep independent growth. Treatment with 6-mercaptopurine and transfusions was without effect and the patient died after 15 days with signs of cerebral bleeding.     

人类脐血与正常骨髓CFU-F特性的比较研究

用液体培养法对１４份人类脐血和１０份正常骨髓ＣＦＵ－Ｆ的生长特性、形态学、细胞化学及超微结构等进行了比较研究，结果除生长特性及集落数不同外，两者无明显差异。说明脐血含有基质细胞。在脐血ＣＦＵ－Ｆ培养中，还发现一种来源、性质不明的细胞，提示脐血具有不同于骨髓之处     

Growth kinetics and blast-colony forming cell binding capacity of aplastic anaemic stromal cells.

The kinetics of bone marrow cell growth and a special function of stromal cells (the capability of binding blast colony forming cells) were studied in patients with aplastic anaemia (AA). All 10 patients studied showed faster growth of bone marrow stromal cells. The time for a confluent stromal layer formation was 24.5 days for AA bone marrow as opposed to 33.0 days for normal bone marrow. This faster growth rate could also be observed if normal bone marrow cells, depleted of plastic non-adherent fraction, were plated, suggesting that at least one of the reasons for altered stromal cell growth kinetics in AA is the changes in the ratio of plastic adherent/non-adherent cells. Functionally, i.e. in supporting the growth of normal bone marrow blast colonies, AA stromal layers did not differ from that of normal stromal layers, independently of the clinical state of the disease (AA or SAA; in one patient before or after ATG treatment; in two patients after successful allogenic bone marrow transplantation). Moreover, in some AA patients this blast colony forming cell binding function of AA stromal layers could also be detected in samples cultured without hydrocortisone (i.e. in the absence of fat cells), suggesting that AA stroma also differs qualitatively from normal stroma without inducing a defective microenvironment for stem cell homing.     

Growth kinetics and blast-colony forming cell binding capacity of aplastic anaemic stromal cells

The kinetics of bone marrow cell growth and a special function of stromal cells (the capability of binding blast colony forming cells) were studied in patients with aplastic anaemia (AA). All 10 patients studied showed faster growth of bone marrow stromal cells. The time for a confluent stromal layer formation was 24.5 days for AA bone marrow as opposed to 33.0 days for normal bone marrow. This faster growth rate could also be observed if normal bone marrow cells, depleted of plastic non-adherent fraction, were plated, suggesting that at least one of the reasons for altered stromal cell growth kinetics in AA is the changes in the ratio of plastic adherent/non-adherent cells. Functionally, i.e. in supporting the growth of normal bone marrow blast colonies, AA stromal layers did not differ from that of normal stromal layers, independently of the clinical state of the disease (AA or SAA; in one patient before or after ATG treatment; in two patients after successful allogenic bone marrow transplantation). Moreover, in some AA patients this blast colony forming cell binding function of AA stromal layers could also be detected in samples cultured without hydrocortisone (i.e. in the absence of fat cells), suggesting that AA stroma also differs qualitatively from normal stroma without inducing a defective microenvironment for stem cell homing.     

The influence of thyroid hormones on colony growth of peripheral CFU-GM from normal and leukemic subjects

In order to clarify the alterations of granuloblastic cells in chronic and acute myeloid leukemia, the colony growth behavior of cultured CFU-GM from the peripheral blood of normal and leukemic subjects was examined in basal conditions and after adding to the medium T3 or T4 and/or thioproline and/or flurbiprofen. These drugs had in previous investigations proved their ability to modify cellular receptors and the uptake of thyroid hormones. The study was carried out in semisolid (double agar layer) and liquid medium, utilizing the techniques described previously. Both thyroid hormones enhanced the colony growth from normal peripheral blood CFU-GM and the response was more evident with T4 than T3. The effect on leukemic CFU-GM (from CML and AML) was less clear, probably due to the presence in leukemic cells of a defect of cellular uptake and to the utilization of T3 and T4. Indeed, on addition to the culture medium of thioproline, which modifies membrane permeability, and of fluorbiprofen, which inhibits PGE synthesis, the colony number and growth from leukemic CFU increased considerably in accordance with the results of our previous studies on these substances showing that they are able to modify cellular receptors for thyroid and several other hormones.     

Clonal growth of haemopoietic progenitor cells from myelodysplastic marrow in response to recombinant haemopoietins.

The growth factor requirements of granulocyte-macrophage (GM) and erythroid marrow progenitor cells from 12 myelodysplastic (MDS) patients have been analysed. GM progenitors from two of six patients who grew normal numbers of colonies in response to conditioned medium + erythropoietin (5637CM + Epo) showed defective responses to either GMCSF and/or IL-3. Of all the recombinant factors tested (IL-3, IL-1, GCSF, GMCSF, MCSF), GMCSF was the strongest stimulator of myeloid clonal growth, inducing normal numbers of GM colonies from marrow of six patients (two of whom were neutropenic). Erythroid colonies were low in 5637CM + Epo-supplemented cultures of marrow from all but one patient and remained poor in the presence of any of the haemopoietins. tested. Supraoptimal doses (for normal marrow) of these haemopoietins improved colony growth in only one patient (GM colonies in response to IL-3). Combinations of factors were also largely ineffective at raising myeloid or erythroid colony numbers. These data indicate that the defective response of MDS progenitor cells to growth factors is not amenable to experimental manipulation of recombinant factor levels or combinations. Clonal assays might suggest a role for GMCSF therapy in a subpopulation of neutropenic MDS patients but their potential now needs to be evaluated in association with clinical trials.