系统性红斑狼疮患者骨髓造血干/祖细胞体外培养及其发病机制的研究

目的　比较正常人与系统性红班狼疮 (SLE)患者的造血干 /祖细胞生长情况及从造血干 /祖细胞水平探讨SLE的发病机制。方法　用半固体甲基纤维素集落培养法观察 16例活动期及其中 3例经治疗缓解的SLE患者的粒 巨噬细胞集落 (GM CFU)、红系集落 (E CFU)、巨核细胞集落 (MK CFU)。结果　活动期SLE患者的GM CFU、E CFU、MK CFU分别为 (182± 5 0 )、(5 6± 2 7)、(8± 5 ) ,均明显低于正常对照组 ,缓解期SLE患者的GM CFU、E CFU比活动期的明显增加 ,而MK CFU与活动期比较无明显变化。结论　活动期SLE患者的造血干 /祖细胞增生、分化功能异常 ,缓解后的SLE患者其巨核细胞系恢复迟于粒系、红系 ,SLE的发病可能与造血干 /祖细胞的异常有关     

羟基淀粉沉淀对免疫磁珠法分离脐血CD34^＋细胞及造血祖细胞增殖功能的影响

目的：应用羟基淀粉沉淀去除脐血红细胞和免疫磁株（MACS）阳性选择,建立富集,分离脐血CD34^ 细胞的简易实用方法,通过造血祖细胞集落培养分析其在细胞因子作用下的增殖潜能,观察羟基淀粉沉淀对造血细胞增殖功能的影响,方法：采用羟基淀粉沉淀和改进的MACS富集,分离脐血CD^34^ 细胞,以甲纤半固体造血细胞培养法观察其粒－单系细胞（CFU－GM）和红系爆式集落（BFU－E）的数量和特性。结果：脐血分离前,CD34^ 细胞纯度为1．5％－3％,MACS分离后其纯度可达85％,羟基淀粉沉淀和MACS分离可达91％,CD34^ 造血祖细胞培养有明显的增殖,采用IL－3,IL－6,SCF,GM－CSF,EPO等细胞因子组合扩增培养9－14d,CD34^ 细胞在液体培养中有明显扩增,MACS分离后CD34^ 细胞总数增加39倍,羟基淀粉沉淀和MACS分离可增加45倍。结论：应用羟基淀粉沉淀和改进的MACS系统可有效富集,分离肌血CD34^ 细胞,羟基粉沉淀对CD34^ 细胞的富集,分离无影响,脐血细胞分离后作造血祖细胞培养,可产生丰富的CFU－GM和BFU－E,说明羟基淀粉沉淀分离的CD34^ 细胞的增殖功能优于MACS分离CD34^ 细胞的增殖功能。     

白细胞介素-15对骨髓增生异常综合征骨髓造血干/祖细胞增殖的影响

目的：观察白细胞介素(IL-15)对体外培养的骨髓增生异常综合征(MDS)患者CD34^ 细胞增殖作用。方法：应用单克隆抗体免疫磁珠分离系统提取MDS患者CD34^ 细胞，以加IL-15组为实验组，不加IL-15组为对照组，进行液体和甲基纤维素半固体集落培养，计算培养后细胞数和CFU—E、BFU—E、CFU—GM、CFU—GEMM等集落数，并用MTT比色法检测IL-15对MDS患者CD34^ 细胞增殖的抑制作用，流式细胞术检测上述培养细胞周期的变异情况。结果：11例MDS对象平均CD34^ 细胞比例、回收率、纯度和富集倍数均达要求，MTT比色法检测IL-15对CD34^ 细胞的增殖作用呈最佳浓度效应，最佳浓度为20μg／L，细胞增殖抑制最低峰值时间为8d。用0μg／L IL-15(对照组)和20μg／L IL-15(实验组)作用MDS CD34^ 细胞，计数显示培养细胞最大增殖倍数和集落形成比率实验组均较对照组明显增加，IL-15作用后各细胞周期G1、S、G2期比例有明显改变，与对照组比较，差异有统计学意义。结论：IL-15对MDS CD34^ 细胞有促增殖效应，与其它造血生长因子具有协同作用，对MDS治疗可能有一定的应用前景。     

骨髓增生异常综合征各系祖细胞的体外培养观察

本文对44例MDS患者的多向祖细胞(CFU—GEMM)、红系祖细胞(BFU—E)、前巨核祖细胞(BFU—MK)、后巨核祖细胞(CFU—MK)、粒系祖细胞(CFU—GM)进行了体外培养观察。结果表明部分MDS骨髓粒系祖细胞体外生长类似白血病生长型。其它各系祖细胞集落数量减少,且明显小于正常,说明祖细胞在增殖分化等方面有明显的缺陷。     

汞致单纯红细胞再生障碍性贫血的临床研究

目的：探讨重金属汞中毒致单纯红细胞再生障碍性贫血（PRCA）的发病机制、致毒量、体内代谢过程及治疗转归。方法：应用电感耦合氩等离子发射光谱法（ICAS）对血、尿汞含量进行动态检测。间接免疫荧光法、琼脂半固体单层培养法、甲基纤维素半固体培养法，骨髓基质细胞培养，淋巴细胞抑制造祖血细胞试验，观察了细胞亚群改变，粒单系祖细胞（CFU－GM）爆式红系祖细胞（BFU-E），基质细胞高峰集落数。患者外周血淋巴细胞对正常人骨髓细胞BFU－E影响。结果：致毒后血、尿汞含量超标值百余倍，T细胞亚群中CD细胞水平下降，CD细胞水平升高，CD／CD比率下降；骨髓祖细胞培养BFU－E生长受抑，其淋巴细胞对正常人骨髓BFU－E生长同时有抑制作用，对骨髓基质细胞的影响尚不能定论。结论：汞中毒对骨髓有一定影响，尤其红细胞系更敏感。伴随汞毒物质的驱除，上述指标渐趋恢复。     

系统性红斑狼疮患者T细胞活化及其对CD34+细胞的免疫调节

系统性红斑狼疮（SLE）T、B细胞的异常活化以及炎症因子异常导致了SLE患者的多系统损害。造血系统是SLE患者受累的器官之一，导致外周血细胞减少。免疫紊乱可影响骨髓造血和造血微环境。CD34^＋细胞是非均质性细胞群，其中含有造血干细胞和不同分化阶段的各系造血祖细胞，具有自我更新、多向分化的特性和重建造血和免疫系统的功能。骨髓中CD34^＋细胞的增殖、分化、凋亡的调节主要依赖免疫系统，用CD34^＋细胞移植治疗难治性SLE有一定的疗效。去除T细胞对移植后狼疮的复发有预防作用。提示T细胞活化在SLE发病中有重要作用。本文就活性T细胞对CD34^＋细胞的免疫调节进行综述。     

AA和MDS患者骨髓CD+34细胞表面GM-CSFR的表达及意义

目的 探讨再生障碍性贫血（AA）和骨髓增生异常综合征（MDS）患者骨髓CD34^＋细胞表面粒细胞-巨噬细胞集落刺激因子受体（GM—CSFR）的表达情况。方法 用流式细胞术（FCM）和单克隆抗体技术检测14例AA、23例MDS和8例非血液病患者骨髓CD34^＋细胞表面的GM—CSFR表达率。结果 MDS组骨髓CD34^＋细胞表面GM-CSFR的表达率明显高于对照组及AA组（P〈O．05），而AA组与对照组间差异无统计学意义（P〉0．05）。结论 AA患者造血干细胞没有质的缺陷；造血干细胞异常可能是MDS的主要发病机制之一。     

几种细胞因子对骨髓基质成纤维细胞集落形成的影响及意义

目的：观察细胞因子粒－巨噬细胞集落刺激因子（GM－CSF）、干细胞因子（SCF）、白细胞介素－3（IL－3）、肿瘤坏死因子（TNF）及其联合应用对骨髓基质成纤维细胞集落生成单位（CFU－F）形成的作用。方法：应用体外骨髓基质细胞粘壁培养的方法,分别加入不同的细胞因子,进行培养2周时的CFU－F计数和培养4周时贴壁细胞的增殖状态观察。结果：GM－CSF、SCF及TNF对骨髓基质细胞的增殖有明显的促进作用。结论：体外加入苛些细胞生长因子对骨髓基质细胞具有明显的扩增作用,提示基质细胞在体外适当细胞因子作用下扩增后进行回输,有可能成为促进造血损伤修复的新措施。     

人巨细胞病毒对粒-单及红系祖细胞体外增殖的影响

目的：探讨人巨细胞病毒(HCMV)对脐血粒—单系祖细胞(CFU—GM)、红系爆式祖细胞(BFU—E)及红系祖细胞(CFU—E)体外增殖的抑制作用。方法：采用造血祖细胞体外半固体培养技术，研究HCMV—ADl69株对脐血CFU—GM、BFU—E及CFU—E集落生长的影响；用聚合酶链反应(PCR)技术检测集落细胞中HCMV—ADl69 DNA。结果：①HCMV—ADl69对CFU—GM、BFU—E及CFU—E均有抑制作用。②其抑制作用与HCMV浓度有关，高浓度(1：10，1：100)感染组与对照组比较集落产率明显下降(P＜0.01)，集落维持的时间明显缩短(P＜0.01)，而低浓度组(1：1000)无此作用。③经PCR检测发现病毒感染组CFU—GM、BFU—E及CFU—E集落细胞中有HCMV—ADl69 DNA存在。结论：①HCMV—ADl69可抑制CFU—GM、BFU—E及CFU—E集落的生长。②造血祖细胞是HCMV—ADl69的宿主细胞之一，HCMV—ADl69能直接感染造血祖细胞。HCMV感染患儿出现的粒细胞减少和贫血可能与此有关。     

脐血造血细胞中AC133抗原表达及其功能特征研究

目的 探讨AC133抗原在正常人造血细胞中表达的意义。方法 采用双色直接免疫荧光标记法,使用流式细胞仪测定脐血中造血细胞的免疫表型。通过祖细胞集落形成单位（CFC）和长期培养启动细胞（LTC-IC）的测定对脐血中AC133^ ,CD34^ 和AC133^-CD34^ 细胞的造血潜能进行了研究。结果 脐血单个核细胞（MNCs)中AC133^ 细胞比例为0.67%,AC133^ 细胞中93.4%表达CD34抗原,94.2%为PKH26阳性,虽然三群细胞产生的CFC总数差异无显著性。但AC133^ 细胞中50%以上为粒-单集落形成单位,CD34^ ,AC133^-CD34^ 细胞中60%和80%以上为红系集落形成单位,AC133^ 细胞产生的LTC-IC显著高于CD34^ 和AC133^-CD34^ 细胞。体外培养7d,AC133^ 细胞组细胞总数和CFC分别扩增28.2和7.1倍,6.8%细胞仍保持PKH26阳性。结论 脐血中早期造血干/祖细胞富集在AC133^ 细胞群中,AC133^ 细胞在体外具有较强的增殖能力,既能产生大量的早期定向祖细胞,又能保持一定数量的早期干/祖细胞,AC133抗原可作为临床分选造血干/祖细胞的有效标志。     

消化系恶性肿瘤病人LAK细胞和NK细胞功能与表型的变化

通过观察20例正常人和24例消化系恶性肿瘤病人外周血自然杀伤细胞(NK)和淋巴因子激活的杀伤细胞(LAK)的活性变化,以及加用重组白细胞介素2(rIL-2)刺激前后T淋巴细胞表型变化。结果发现肿瘤病人的NK细胞活性明显下降,但经rIL-2激活后LAK细胞活性得到明显提高,其溶解率接近正常水平。肿瘤病人的总T淋巴细胞(CD_(3+))和辅助/诱导T淋巴细胞(CD_(4+))水平低于正常,但抑制/杀伤淋巴细胞(CD_(8+))水平正常。辅助/诱导淋巴细胞与抑制/杀伤淋巴细胞之比为1.18,低于正常水平(1.55)。经加入rIL-2培养后,CD_(3+)和CD_(8+)淋巴细胞的比率明显升高并达正常水平。而在正常人此变化不明显,且加用rIL-2培养与不加者无显著差异。IL-2受体的表达正常人与肿瘤病人无异。结果显示胃肠道恶性肿瘤病人的免疫机制受到抑制,但能被IL-2提高至正常水平。     

Robust measurement of telomere length in single cells

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.Telomeres are the ribonucleoprotein structures that cap and protect linear chromosome ends from genomic instability and tumorigenesis (1, 2). Intriguingly, telomere shortening protects against tumorigenesis by limiting cell growth (3, 4), but also can impair tissue regenerative capability and cell viability (5, 6).Thus far, most assays of telomere length measure average telomere length from aggregates of many cells derived from dissected tissues, cultured cells, or blood (7). Telomere restriction fragment (TRF) determination (1, 8), a Southern blot-based technique, remains the “gold standard” for determining absolute telomere length, but requires a large amount of starting material (0.5–5 µg DNA) and several days for processing. Moreover, the requirements for gel electrophoresis and hybridization limit the scalability of this assay. Recently, a quantitative PCR (qPCR)-based method for telomere length measurement was developed, providing the convenience and scalability of PCR (9). Although the DNA requirement (35 ng) for qPCR is significantly less than TRF, it still relies on populations of cells to derive sufficient amount of DNA.Quantitative FISH (Q-FISH) allows sensitive visualization of relative telomere length from individual cells and individual telomeres, but this method requires many cells or metaphase arrested cells, which precludes its application to many sample types, including postmitotic cells, senescent cells, and other nondividing cells, and when only one actual cell is required to test. In addition, preparing chromosome spreads requires significant technical skill, and only proliferating cells within a population reach metaphase stage, so this analysis potentially biases the estimates of telomere length for a given cell population (10–12). High-throughput Q-FISH, flow FISH, and single telomere length analysis can be used for telomere measurement of dividing, nondividing, and senescent cells, but these methods also require large cell populations (13–15).The ability to measure telomere length in single cells rather than relying upon average telomere length in cell populations or the entire tissue enables the study of biological heterogeneity on a cell-by-cell basis, an issue of fundamental importance for studies of aging, development, carcinogenesis, and many other diseases. Here, we demonstrate an accurate determination of telomere length in individual cells, with the resolution and scalability of the qPCR telomere length assay.The basis of qPCR is that within a given cell, the ratio of the copy number of telomere repeats to the copy number of a multicopy reference gene is fixed (3), and this method, because of its simplicity, has been widely used to investigate a variety of telomere shortening-associated diseases (7), even sensitive enough to identify mild telomere dysfunction resulting from chronological life stress (16, 17). We adapted qPCR to measure telomere length in individual cells by using a preamplification step that specifically targets both the telomere and multicopy genes, followed by a qPCR assay to obtain telomere to reference gene (T/R) ratio. A single-cell telomere (SCT) length measurement method (SCT-pqPCR) runs robustly, and shows an identical T/R ratio for two sister blastomeres from two-cell–stage mouse embryos. The average result from SCT-qPCR with multiple single cells is linearly correlated to Q-FISH, TRF, and conventional qPCR assays designed for a large number of cells. The heterogeneity of telomere length among several populations of cells by SCT-pqPCR run on multiple single cells is consistent with—and sometimes superior to—results obtained by Q-FISH. Application of SCT-pqPCR to study telomere length during early embryo development, aging, and cancer demonstrate the value of this single-cell telomere length assay method.     

Glycolipid presentation to natural killer T cells differs in an organ-dependent fashion

It has been shown that dendritic cells (DCs) are able to present glycolipids to natural killer (NK) T cells in vivo. However, the essential role of DCs, as well as the role of other cells in glycolipid presentation, is unknown. Here, we show that DCs are the crucial antigen-presenting cells (APCs) for splenic NK T cells, whereas Kupffer cells are the key APCs for hepatic NK T cells. Both cell types stimulate cytokine production by NK T cells within 2 h of glycolipid administration, but only DCs are involved in the systemic, downstream responses to glycolipid administration. More specifically, CD8alpha+ DCs produce IL-12 in response to glycolipid presentation, which stimulates secondary IFN-gamma production by NK cells in different organs. Different APCs participate in glycolipid presentation to NK T cells in vivo but differ in their involvement in the overall glycolipid response.     

Processivity of peptidoglycan synthesis provides a built-in mechanism for the robustness of straight-rod cell morphology

The propagation of cell shape across generations is remarkably robust in most bacteria. Even when deformations are acquired, growing cells progressively recover their original shape once the deforming factors are eliminated. For instance, straight-rod-shaped bacteria grow curved when confined to circular microchambers, but straighten in a growth-dependent fashion when released. Bacterial cell shape is maintained by the peptidoglycan (PG) cell wall, a giant macromolecule of glycan strands that are synthesized by processive enzymes and cross-linked by peptide chains. Changes in cell geometry require modifying the PG and therefore depend directly on the molecular-scale properties of PG structure and synthesis. Using a mathematical model we quantify the straightening of curved Caulobacter crescentus cells after disruption of the cell-curving crescentin structure. We observe that cells straighten at a rate that is about half (57%) the cell growth rate. Next we show that in the absence of other effects there exists a mathematical relationship between the rate of cell straightening and the processivity of PG synthesis—the number of subunits incorporated before termination of synthesis. From the measured rate of cell straightening this relationship predicts processivity values that are in good agreement with our estimates from published data. Finally, we consider the possible role of three other mechanisms in cell straightening. We conclude that regardless of the involvement of other factors, intrinsic properties of PG processivity provide a robust mechanism for cell straightening that is hardwired to the cell wall synthesis machinery.     

Regenerative medicine for the treatment of heart disease

Heart failure is a major cause of mortality worldwide with a steady increase in prevalence. There is currently no available cure beyond orthotopic heart transplantation, which for a number of reasons is an option only for a small fraction of all patients. Considerable hope has therefore been placed on the possibility of treating a failing heart by replacing lost cardiomyocytes, either through transplantation of various types of stem cells or by boosting endogenous regenerative mechanisms in the heart. Here, we review the current status of stem and progenitor cell‐based therapies for heart disease. We discuss the pros and cons of different stem and progenitor cell types that can be considered for transplantation and describe recent advances in the understanding of how cardiomyocytes normally differentiate and how these cells can be generated from more immature cells ex vivo. Finally, we consider the possibility of activation of endogenous stem and progenitor cells to treat heart failure.     

Sickle cell vasoocclusion: heterotypic, multicellular aggregations driven by leukocyte adhesion

Homozygous expression of sickle beta-globin alters the function of blood cells and the endothelium, producing a wide spectrum of clinical manifestations. Intravital microscopy studies in sickle cell mice suggest that vasoocclusion is a complex, sequential, multistep phenomenon involving (1) endothelial activation by sickle erythrocyte (SSRBC), (2) leukocyte (WBC) adhesion to the endothelium, and (3) the direct interaction between SSRBCs and adherent WBCs, which leads to reduced blood flow and tissue ischemia. Each of these steps represents a potentially useful therapeutic target. The identification of molecular determinants mediating vasoocclusion will provide new strategies for the prevention and treatment of this debilitating illness.     

特应性皮炎的免疫学发病机制

特应性皮炎是慢性复发性炎症性皮肤疾病，发病机制复杂，其中变态反应因素在发病机制中扮演着重要角色。目前认为Th1／Th2平衡失调是特应性皮炎重要的发病机制。本文围绕这一机制综述T细胞、树突状细胞、角质形成细胞及IgE在特应性皮炎发病机制中的作用。     

Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry

Left-right (LR) asymmetry (handedness, chirality) is a well-conserved biological property of critical importance to normal development. Changes in orientation of the LR axis due to genetic or environmental factors can lead to malformations and disease. While the LR asymmetry of organs and whole organisms has been extensively studied, little is known about the LR asymmetry at cellular and multicellular levels. Here we show that the cultivation of cell populations on micropatterns with defined boundaries reveals intrinsic cell chirality that can be readily determined by image analysis of cell alignment and directional motion. By patterning 11 different types of cells on ring-shaped micropatterns of various sizes, we found that each cell type exhibited definite LR asymmetry (p value down to 10(-185)) that was different between normal and cancer cells of the same type, and not dependent on surface chemistry, protein coating, or the orientation of the gravitational field. Interestingly, drugs interfering with actin but not microtubule function reversed the LR asymmetry in some cell types. Our results show that micropatterned cell populations exhibit phenotype-specific LR asymmetry that is dependent on the functionality of the actin cytoskeleton. We propose that micropatterning could potentially be used as an effective in vitro tool to study the initiation of LR asymmetry in cell populations, to diagnose disease, and to study factors involved with birth defects in laterality.