25例多发性骨髓瘤误诊病种分析

多发性骨髓瘤是淋巴-浆细胞系统恶性肿瘤,多发于40岁以上成年人.其临床特征为血清中出现单克隆球蛋白,溶骨性骨质破坏,骨髓中浆细胞异常增多.由于瘤细胞的恶性程度和浸润范围不一,合成分泌异常免疫球蛋白多寡不同,临床表现复杂多变,很容易误诊,误诊率60%以上[1],误诊病种达20余种.现将我院2000～2010年收治多发性骨髓瘤25例的误诊病种报告如下.     

多发性骨髓瘤分子水平研究的新进展

多发性骨髓瘤(Multiple Myeloma,MM)是骨髓浆细胞恶性增生性疾病,进年来,随着分子生物学技术的发展,人们可以直接分析骨髓瘤细胞DNA水平的变化,并在分子水平上的研究获得了一系列的成果,对于我们更好的了解骨髓瘤、改善骨髓瘤病症及治疗疾病具有极为重要的意义,本文就对多发性骨髓瘤在分子水平上的研究进展做一综述。     

无巨核细胞条件下促血小板生成素对骨髓基质细胞纤维形成的影响

目的：观察无巨核细胞存在的条件下促血小板生成素能否刺激骨髓基质细胞纤维形成。方法：用改良Dexter培养法进行体外不同浓度促血小板生成素（TPO）作用下的基质细胞培养,在培养过程中检测基质细胞相对增殖指数,纤维连接蛋白、层粘素和Ⅳ型胶原的表达,以及Ⅲ型前胶原蛋白的合成。结果：TPO可刺激基质细胞增殖,相对增殖指数随TPO浓度增加而增强,但不随作用时间延长而增强;纤维连接素、层粘素和Ⅳ型胶原在对照组与实验组均有阳性表达,但实验组强于对照组,但阳性强度不随培养时间的延长而增强;标记的Ⅲ型前胶原蛋白平均荧光强度实验组高于对照组,差异明显,但这种作用的强弱与TPO浓度相关性不强。结论：无巨核细胞存在的条件下,TPO可直接刺激骨髓基质细胞产生细胞外基质和胶原,促进其纤维形成。     

多发性骨髓瘤遗传学异常和克隆演变

多发性骨髓瘤(MM)是一种难以治愈的血液系统恶性肿瘤,从意义未明的单克隆丙种球蛋白血症(MGUS)进展到冒烟型骨髓瘤(SMM),再进一步进展至有症状MM甚至浆细胞性白血病(PCL),遗传学异质性和克隆演变在其中有着关键推动作用.近年来有关该转化进程的MM遗传学研究取得了很大的进展,该文将对其及临床意义进行综述,以期为科...     

小鼠骨髓内皮细胞条件培养液复合FL及TPO对HPP—CFC及CFU—GM增殖的影响

通过传代培养小鼠骨髓内皮细胞,收集无血清条件培养液（ECM）,并经超滤得到大于10kD的浓缩液,分别观察ECM和大于10kD的浓缩液复合flt3ligand(FL)及thrombopoietin(TPO)对体外培养HPP-CFC、CFU-GM的影响,结果表明：ECM或大于10kD的浓缩液对HPP-CFC、CFU-GM的生长均有支持作用;FL或/和TPO与ECM或大于10kD的浓缩液合用能加强对HPP-CFC、CFU-GM生长的刺激作用;FL加TPO与ECM或大于10kD的浓缩液合用对HPP-CFC、CFU-GM生长的刺激作用更加明显;选择FL和TPO特异性的引物,用RT-PCR技术未能检测到小鼠骨髓内皮细胞有FL和TPOmRNA的表达。     

低温冻存对骨髓基质细胞生物学特性的影响

目的：探讨低温冻存对骨髓细胞和贴壁基质细胞生物学特性的影响。方法：取新鲜骨髓和经Dexter法培养14d的骨髓贴壁基质细胞(称“基质细胞”),经-196℃液氮冻存(前者称“冻存骨髓”,后者称“冻存基质细胞”)2周,复温,再用Dexter法培养这些细胞,检测细胞增殖、细胞形态、细胞化学染色、细胞表面抗原及基质细胞支持另一骨髓造血细胞形成的鹅卵石造血区(CAFC),长期培养起始细胞(LTCIC)的变化,比较冻存对骨髓细胞和基质细胞生物学特性的影响。结果：生长特性：冻存骨髓比新鲜骨髓、冻存基质细胞比新鲜基质细胞培养后融合成片的时间延迟,细胞增殖数比也有减低。细胞成分：冻存骨髓比新鲜骨髓形成的成纤维细胞、内皮细胞比率下降,而巨噬细胞和脂肪细胞比率升高,冻存基质细胞上述现象更明显：冻存后含凋亡小体的细胞在骨髓细胞和基质细胞内均有增加。细胞表面抗原：冻存骨髓、冻存基质细胞CD14、HLA-DR抗原表达百分率比新鲜骨髓、新鲜基质细胞高,CD45、CD33反之。支持造血：冻存前后骨髓和基质细胞支持形成的CAFC和LTC-IC,生长良好,无显著差异。结论：骨髓细胞和经培养生成的贴壁基质细胞,经冻存和复温,生物学特性有一定变化,但仍可以保留良好的支持造血重建功能。     

脐血造血细胞的体外扩增：1.生长规律的研究

考察了在添加细胞因子和未添加细胞因子培养条件下的造血细胞群体的生长和代谢,研究了长期培养条件下造血祖细胞生长规律。在静态培养条件下,脐血造血细胞群体的比生长速率为0.34d^-1,倍增时间为2d。培养后期,造血细胞消耗了大部分葡萄糖,乳酸浓度可达40 mmol/L。在造血细胞长期培养条件下,CFU-GM产出最高峰在培养第2周与第3周之间。BFU-E产出最高峰在培养第1周。每天换50%培养液,造血细胞总数扩增了14倍,CFU-GM扩增了13倍,BFU-E扩增了5倍。     

多发性骨髓瘤中NF-κB作用机制研究

多发性骨髓瘤(multiple myeloma,MM)是浆细胞异常增生的恶性肿瘤,其发病机制比较复杂。研究表明,活化NF-κB具有多种生物学功能,它既能调节细胞因子;影响细胞周期,又与血管发生有关,并且与多发性骨髓瘤治疗密切相关。该文就NF-κB在多发性骨髓瘤中的作用机制做一综述。     

免疫固定电泳检测对浆细胞肿瘤相关肾损害的诊断价值

目的:探讨免疫固定电泳(immunofixation electrophoresis,IFE)检测对浆细胞肿瘤相关肾损害的诊断价值。方法:回顾性分析我院肾病科665例住院患者的免疫固定电泳分型结果及检出M蛋白带的患者骨髓检查结果。结果:665例肾病科患者中检出44例单克隆免疫球蛋白带,检出率6.6%,其中IgG型24例(κ型6例,λ型18例),IgA型10例(κ型6例,λ型4例),IgM型2例(均为κ型),单纯轻链κ型2例,单纯轻链λ型6例;此44例患者经骨髓检查,38例确诊为多发性骨髓瘤(multiple myeloma,MM),2例确诊为巨球蛋白血症(waldenstrom macroglobulinemia,WM),骨髓涂片中骨髓瘤细胞比例均≥10%;骨髓瘤细胞10%的4例,诊断为意义未明单克隆免疫球蛋白血症(monoclonal gammopathy of un-determined significance,MGUS)。结论:免疫固定电泳检测可对浆细胞肿瘤相关肾损害提供重要的诊断和分型依据,值得临床应用。     

混合及是血清在骨髓长期培养中的效应

目的揭示脐血清在骨髓长期培养中的效应,为脐血清的应用提供基础。方法以Dexter培养法,观察混合脐血清(MCBS)、组合细胞因子(CK)在长期骨髓培养中对骨髓单个核细胞(BMMNC)形成鹅卵石造血区(CAFC)、长期培养起始细胞(LTC-IC)、有核细胞(NCC)的生长。结果10例人骨髓,106BMMNC培养5周后,CAFC、LTC-IC分别为37.1±12．4／(ml.well),40．9±10．6/(ml.well),NCC由接种时的106/(ml.well)增至（1．63±0．17）106/(ml.well),加入10％MCBS则可使三者得到明显扩增,但不及组合CK;10％MCBS还能明显提高组合CK对三者的扩增;20％MCBS不能取代骨髓长期培养中的血清和组合CK对三者的扩增。结论MCBS中含有类似GM-CSF、SCF、IL-3、IL-6、EPO等一类能使CAFC、LTC-IC、NCC得到明显扩增的“活性物质”。     

Induction and long-term maintenance of Thy-1 positive T lymphocytes: Derivation from continuous bone marrow cultures

In the present study we investigated the presence of T-lymphocyte progenitors in the long-term murine bone marrow culture system described by Dexter: mature Thy-1 antigen-bearing T lymphocytes are lost in these cultures after a few days. By culturing nonadherent cells from such cultures in the presence of a supernatant of concanavalin A-stimulated spleen cells, a source of T-cell growth factor, we found that Thy-1 positive blast cells proliferated together with a second population of Thy-1 negative cells. These two populations of cells have been maintained in long-term in vitro cultures by passaging the cells in fresh conditioned medium at regular intervals. Moreover, we have been able to establish pure cultures of the Thy-1-bearing blast cells after separating them from the non-T cells using their adherence property to plastic surfaces. Long-term cultures of T lymphocytes can thus be established from long-term marrow cultures as well as from the spleen, thymus or fresh bone marrow.     

Morphologic analysis of long-term bone marrow cultures that support B-lymphopoiesis or myelopoiesis

Summary A comparative morphological analysis of the Whitlock-Witte long-term B-cell culture and the predominantly myeloid Dexter long-term bone marrow culture demonstrates that similarities and differences exist between the two systems. Cells from the long-term B-cell cultures have a characteristic lymphoid morphology, whereas those from the Dexter cultures are predominantly granulocytes and macrophages along with a few undifferentiated blast cells. A multilayered stromal cell layer is a common feature of both systems. Scanning electron micrographs show the cells in this layer to be large, irregularly shaped and flattened. The data further indicate that there are unique features in the relationship between developing B cells and stromal cells in the long-term B-cell cultures. Large, mononuclear cells are present that have numerous membrane infoldings within which numerous lymphoid cells lie. The relationship of these cells to macrophages and epithelial/reticular cells is discussed.Supported by the National Institutes of Health Grants AI2125601 (KD) and HD13704 (WHF)     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

In vitro study of bone marrow from leukemia patients and patients without hematologic disease

The bone-marrow of 26 patients not affected with hematological diseases and 10 patients with untreated leukemia was investigated according to Dexter in long-term cultures. Survival time and cell content of those long-term cultures started with normal bone marrow were not influenced significantly, if reinoculation was made with autologeneic or allogeneic bone marrow. Even without repeated inoculation, leukemic cells grew for a longer time in long-term cultures than normal bone marrow cells. As far as the outcome of the disease is concerned, no conclusion can be drawn from the duration of cultivating leukemic cells growth.     

Promotion of hematopoietic stem cell differentiation in vitro by a soluble mediator,allogeneic effect factor

This study was designed to investigate the effects of allogeneic effect factor (AEF), a soluble mediator derived from short-term mixed lymphocyte cultures (MLC) of in vitro alloantigen-primed T cells, on cultures of murine bone marrow cells. Cultures established under suboptimal conditions namely, in the absence of a pre-established adherent cell layer as required in conventional Dextertype cultures–declined and lost their stem cell activity rapidly. In contrast, supplementation of these cultures, at initiation and thereafter, with AEF, but not with T cell growth factor (TCGF), induced cell growth and proliferation for several weeks. Such AEF-supplemented cultures exhibited cellular heterogeneity and stem cell activity for significantly longer periods than the control cultures. Even in conventional Dexter cultures, established under optimal conditions, AEF had a beneficial effect on cellular growth and proliferation and myeloid progenitor cell (CFU-C) activity. Furthermore, cells capable of synergizing with suboptimal numbers of mature T cells in con A-induced mitogenic responses, shown by others to be pre-T cells, were detected in the AEF-supplemented cultures for several weeks.     

EFFECTS OF BETA MERCAPTOETHANOL ON THE PROLIFERATION AND DIFFERENTIATION OF HUMAN OSTEOPROGENITOR CELLS

Antioxidants are known to influence metabolism and promote cell survival in a number of cell culture systems. However, their effects on the modulation of bone cell differentiationin vitroare not clearly defined. In the present studies we have investigated the effects of β-mercaptoethanol (βME) and ascorbate alone and in combination on human osteoprogenitors derived from bone marrow fibroblasts. In primary marrow cultures, βME stimulated colony formation (2-fold), alkaline phosphatase activity (3.5-fold) and, increased DNA synthesis (8-fold) after 21 days. Cell proliferation was increased significantly by βME during the first 4 days of a 10-day culture period, indicating stimulation of marrow osteoprogenitor proliferation. Ascorbate did not significantly augment the effects of βME in primary cultures or long-term cultures of passaged bone marrow fibroblasts. These findings indicate a potential beneficial role for βME addition for the optimal maintenance of colony formation, cell proliferation and differentiation of marrow osteoprogenitor cells in primary human bone marrow fibroblast cultures.     

Studies on normal human bone marrow cultures in controlled environment thin-film culture systems

Summary Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first 2 weeks while an increasing number of monocytes and macrophages appeared in the media of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO₂ concentrations, i.e. 2%, 5%, 10%; similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera. Supported by U.S.P.H.S. Grant CA-5834 from the National Cancer Institute. Department of Medicine A. Department of Cell Physiology Department of Immunology and Immunochemistry.     

狗骨髓长期培养的贴壁层细胞及条件培养液对粒系祖细胞的影响

本文报告狗骨髓长期培养的初步研究结果。采用马血清与狗血清混合加入培养体系中,可建立起比较稳定的贴壁细胞层,可以维持19周以上。但是,这种贴壁层不能有效地维持造血干细胞的增殖与分化,上清液中血细胞与GM-CFU_C的数目,在第二次接种骨髓后2—3周即迅速下降。狗骨髓培养的条件液中存在着抑制GM-CFU_C生长的物质,但它对小鼠骨髓GM-CFU_C和CFU-S无影响。如以贴壁细胞作为底层,用双层琼脂平皿培养法与正常狗骨髓共同培养,发现贴壁细胞不仅没有抑制反而有加强CSF刺激GM-CFU_C生长的作用。因此对狗骨髓长期培养体系不能长期支持造血干细胞生长和繁殖的可能原因尚须作进一步探索。     

In vitro differentiation of B lymphocytes from primitive hemopoietic precursors present in long-term bone marrow cultures

B lymphocytes are not produced in the Dexter long-term bone marrow cultures, but a primitive B cell precursor is present. The findings presented in this study demonstrate that this precursor can be induced to produce B lymphocytes by transferring the cultures to the Whitlock conditions for the long-term growth of B cells in vitro. Two weeks after the transfer of cultures maintained at 33 degrees C in medium supplemented with horse serum and steroids to low concentrations of fetal calf serum at 37 degrees C, marked effects can be observed. The pattern of cell growth changes from one in which the hemopoietic cells are clustered in tight foci containing several hundred cells to smaller ones in which the cells are not as densely packed. Fat cells in the adherent layer disappear and the supporting stroma becomes more uniform in appearance. This change in the culture format is accompanied by a decrease in the number of nonadherent cells and a shift from myelopoiesis to lymphopoiesis. The numbers of granulocyte-macrophage progenitors decline weekly after the change in culture conditions and are not detected by the third week. B cell colony-forming units appear by 3 wk. Cells that express the 14.8 cell surface antigen are induced by 1 wk after the change in culture conditions, followed by the appearance of surface IgM-bearing cells 2 wk later. This shift to lymphopoiesis can be confirmed morphologically. Granulocytes and macrophages disappear from the cultures by 4 wk, at which time almost all of the cells have a characteristic lymphocyte morphology. Upon switching these cultures back to the original Dexter conditions, only low levels of transient myelopoiesis can be reinitiated.     

小型猪骨髓间充质干细胞的分离和培养

目的建立小型猪骨髓间充质干细胞（mesenchymal stem cells,MSCs）的体外分离和培养方法。方法穿刺小型猪髂后上嵴抽取骨髓,经密度梯度法离心得到骨髓单个核细胞,接种后形成单层贴壁细胞。用形态学方法鉴定培养的MSCs。结果经培养存活的MSCs原代和一代呈纺锤型、多边型或星型。至二代起呈均一纺锤型,似成纤维细胞样,长宽比例约为（2～3）？1。体外培养的原代MSCs8～10d达到融合,传代后仍具有较强的增殖能力。结论小型猪MSCs可在体外长期、稳定培养,其分离、培养体系的建立为基础研究和组织工程技术提供了一个有价值的动物模型。