人骨髓基质细胞的培养及鉴定

目的:探讨骨髓基质细胞的分离、体外培养方法,为基因治疗提供载体细胞.方法:采用密度梯度离心法结合贴壁筛选法分离人骨髓进行体外培养扩增,用倒置显微镜观察细胞形态学特征并用流式细胞仪鉴定其CD90表达.结果:分离培养的骨髓基质细胞似成纤维状,原代细胞呈集落生长,并可表达CD90,传代后细胞形态较一致,且随着传代次数的增加CD90表达越高,第3代可达94.31%.结论:骨髓基质细胞可通过体外培养纯化获得,随着传代次数增加,纯度越高,该方法是一种较理想的骨髓基质细胞培养方法.     

脐血基质细胞与骨髓基质细胞造血支持功能的比较

目的：探讨脐血基质细胞造血支持功能并与骨髓基质细胞进行相关比较.方法：选择脐带血10份，骨髓标本10份，采用体外培养的方法，观察了脐血基质细胞的体外生长特性，以及其对造血的支持功能，并与骨髓基质细胞进行了相关比较.结果：①脐血基质细胞的形态以圆形和椭圆形为主，脐血基质细胞贴壁时间晚于骨髓基质细胞.②骨髓基质细胞层上悬浮细胞产率在第1、2、3，周均高于脐血细胞层上细胞产率，且两者都高于无贴壁细胞层，均P＜0.05，差异有统计学意义.③骨髓基质细胞层和脐血基质细胞层上的粒单系祖细胞集落（CFU-GM）产率都高于无贴壁细胞层，脐血基质细胞层上的CFU-GM产率低于骨髓基质细胞层，均P＜0.05，差异有统计学意义.结论：脐血基质细胞有支持造血干/祖细胞生长的作用，但其造血支持功能不如骨髓基质细胞。     

白血病干细胞和骨髓基质细胞共培养的方法学探讨

目的 探讨白血病干细胞和骨髓基质细胞共培养的可行方法及扩增白血病干细胞的特点.方法 体外用两种方法共培养急性髓系白血病（AML）患者的骨髓单个核细胞（MNCs）：一种方法是将MNCs中的贴壁基质细胞单独培养,第2代或第3代基质细胞用丝裂霉素处理后做饲养层,再同悬浮造血细胞共培养;另一种方法是将MNCs持续培养即原代共培养.观察MNCs形态学特征、长期存活情况及增殖特性,用流式细胞仪检测分析细胞表面标志.结果 两种方法共培养的细胞部分可以长期存活,在共培养中有卵石样区域细胞及悬浮细胞团的形成.原代共培养的细胞所形成的卵石样区域数目多,排列规律.结论 两种方法都可以起到扩增白血病干细胞的作用.同经过丝裂霉素处理之后的饲养层相比,没有经过处理的骨髓基质细胞在共培养时能够更有效地扩增白血病干细胞.     

急性白血病化疗前后基质细胞上连接蛋白43表达及功能改变的研究

目的:研究急性白血病骨髓基质细胞在化疗前后基质细胞上连接蛋白43(connexin43,Cx43)表达及由其介导的细胞通讯功能的变化。方法:体外培养初发急性白血病及其化疗后完全缓解的骨髓基质细胞,采用细胞免疫化学方法及计算机灰度检测观察二者之间Cx43表达的变化,采用细胞划痕染料传输技术比较二者之间通讯功能的差异。结果:化疗后达到完全缓解的急性白血病骨髓基质细胞的Cx43的表达增加,细胞间通讯功能较化疗前明显增强。结论:化疗后完全缓解的急性白血病骨髓基质细胞Cx43的表达及由其介导的细胞间通讯功能较化疗前明显增强。     

稳定表达分泌型脑源性神经营养因子的神经胶质细胞构建

目的制备可以持续表达和分泌脑源性神经营养因子(brain derived neurophic factor,BDNF)的神经胶质细胞,以此作为滋养层细胞培养体系,促进原代神经元接种的存活率。方法采用慢病毒介导的BDNF表达载体感染神经胶质细胞并进行连续传代,通过Western印迹和ELISA方法检测并获得稳定表达和分泌BDNF的滋养细胞层,将体外分离获得的原代神经元细胞接种于该细胞层,通过MAP2细胞免疫染色检测神经元的形态和存活数量。结果 BDNF过表达慢病毒感染神经胶质细胞后可以在培养基中稳定表达和分泌BDNF,以此为支持滋养细胞进行原代神经元的培养可以显著提高神经元的存活率。结论本实验成功构建了稳定表达和分泌BDNF的神经胶质细胞,该滋养细胞显著提高了原代神经元接种后的存活率,能够满足以高纯度和高密度原代神经元为细胞模型的神经药理学实验的需要。     

非淋巴细胞白血病原代细胞诱导成树突状细胞的研究

目的：用白血病细胞诱导树突状细胞,为白血病的免疫治疗提供新途径。方法：分离人的非淋巴细胞白血病原代细胞,体外流体培养体系中加入ＧＭ－ＣＳＦ、ＴＮＦ－α及ＩＬ－４,培养１２ｄ后,与培养前的细胞在形态、ＣＤ１ａ、ＨＬＡ－ＤＲ及ＣＤ８０的表达情况进行比较。结果：经１２ｄ诱导,细胞形态表现典型的树突状细胞的特征;ＣＤ１ａ、ＨＬＡ－ＤＲ及ＣＤ８０的表达明显增高。结论：可将非淋巴细胞白血病原代细胞诱导成树突状     

急性白血病化疗前后基质细胞上连接蛋白43表达及功能改变的研究

目的研究急性白血病骨髓基质细胞在化疗前后基质细胞上连接蛋白43(connexin43, Cx43)表达及由其介导的细胞通讯功能的变化.方法体外培养初发急性白血病及其化疗后完全缓解的骨髓基质细胞,采用细胞免疫化学方法及计算机灰度检测观察二者之间Cx43表达的变化,采用细胞划痕染料传输技术比较二者之间通讯功能的差异.结果化疗后达到完全缓解的急性白血病骨髓基质细胞的Cx43的表达增加,细胞间通讯功能较化疗前明显增强.结论化疗后完全缓解的急性白血病骨髓基质细胞Cx43的表达及由其介导的细胞间通讯功能较化疗前明显增强.     

大鼠骨髓基质细胞体外培养转化为神经元细胞的实验研究

目的探讨体外原代培养骨髓基质细胞（BMSC）向神经元细胞转化情况.方法取成年Wistar大鼠BMSC,无血清和有血清培养基进行细胞克隆,获取骨髓间质干细胞（MSCs）,应用碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）进行细胞扩增及诱导分化.结果分离纯化后的BMSC经原代培养形成细胞克隆团,经传代后其数量明显增多,其分化细胞形态多样,包括神经元细胞、星形胶质细胞和少突胶质细胞,仍具有神经细胞所特有的性质.结论BMSC具有较强的自我更新能力和多分化潜能,在合适的环境条件下,可诱导分化出神经元细胞和神经胶质细胞,是较为理想的种子细胞.     

胎盘滋养层细胞的感染和凋亡与HBV的宫内传播机制

目的:通过HBV感染体外培养的人绒毛膜滋养层细胞,探讨HBV宫内感染发生的机制.方法:对人早孕绒毛膜滋养层细胞进行原代培养和对人滋养层细胞系JEG-3进行传代培养,将HBV感染血清与原代及传代培养细胞共同孵育8-48h,倒置显微镜观察细胞形态及细胞间连接,细胞免疫荧光和免疫组织化学染色方法检测滋养层细胞中HBsAg和HBcAg的表达,荧光定量PCR方法检测被感染的滋养层细胞中的HBV DNA,TUNEL方法检测滋养层细胞的凋亡.结果:与HBV阳性血清共同孵育对滋养层细胞的形态和细胞间连接无明显影响;细胞免疫荧光和免疫组织化学染色结果显示,滋养层细胞与HBV感染血清共同孵育(8,24,48h)后,滋养层细胞可以出现HBsAg和HBcAg的阳性表达,24 h时HBsAg阳性细胞数量最多(8h:18.0%±3.67%;24h:30.6%±2.88%;48 h:24.8%±4.21%);荧光定量PCR方法检测到被感染的滋养层细胞中HBV DNA的存在;TUNEL结果显示,与HBV感染血清共同孵育可导致滋养层凋亡细胞数量逐渐增加(24h:18.6%±3.05%;48h:26.8%±2.86%:P<0.01).结论:滋养层细胞的感染可能是HBV通过胎盘屏障的途径之一;HBV感染可以诱导滋养层细胞产生凋亡,这可能是胎盘屏障阻止HBV宫内感染的一种保护性机制.     

5-氮胞苷对培养兔骨髓基质细胞心房利钠肽表达的影响

目的研究5-氮胞苷(5-azacytidine,5-aza)对体外培养兔骨髓基质细胞中心肌特异性心房利钠肽(atrialnatriureticpolypeptide,ANP)表达的影响,探讨骨髓基质细胞向心肌样细胞分化的条件。方法体外分离培养兔骨髓基质细胞用不同浓度5-aza诱导培养,以RT-PCR方法测定诱导前后ANP的表达。结果正常培养兔骨髓基质细胞不表达ANP。经5-aza诱导并继续培养24h后,骨髓基质细胞表达ANP,在一定时间、浓度范围内ANP含量逐渐增加。结论5-aza诱导体外培养兔骨髓基质细胞表达心肌特异性ANP,与诱导时间及浓度呈正相关,提示可诱导兔骨髓基质细胞向心肌样细胞分化。     

Growth pattern and clinical correlation of subcutaneously inoculated human primary acute leukemias in severe combined immunodeficiency mice

We examined the ability of patient-derived human leukemic blasts to generate leukemic growth and dissemination in severe combined immunodeficiency (SCID) mice by subcutaneous inoculation without conditioning treatment or administration of growth-promoting cytokines. Additionally, we correlated the growth pattern with the clinical outcome of patients from whom the leukemic cells were derived. The leukemias displayed three distinct growth patterns, ie, either aggressive, indolent, or no tumor growth. Leukemic cells from 6 of 13 patients with acute myeloid leukemia (AML), 4 of 7 T-cell acute lymphoblastic leukemia (T-ALL), and 11 of 16 patients with B-lineage ALL grew as subcutaneous tumors, with a significant number subsequently disseminating into distant organs in SCID mice. Patients whose leukemic blasts displayed an aggressive growth and dissemination pattern in SCID mice had a relatively poor clinical outcome, whereas patients with AML and T- or B-lineage ALL whose leukemic blasts grew indolently or whose cells failed to induce growth had a more favorable clinical course. Our study has shown that the subcutaneous inoculation of patient-derived human leukemic cells in SCID mice can engraft and grow as subcutaneous tumors with subsequent dissemination to distant organs in a manner analogous to their pattern of growth in humans. Additionally, these data suggest a clinical correlation to the growth and dissemination of some leukemic subtypes that may represent not only an additional prognosticator for patient outcome, but also a vehicle for the study of the biologic behavior of human leukemias and the development of novel therapeutic strategies.     

Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic leukemia

To identify new markers of minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL), gene expression of leukemic cells obtained from 4 patients with newly diagnosed ALL was compared with that of normal CD19(+)CD10(+) B-cell progenitors obtained from 2 healthy donors. By cDNA array analysis, 334 of 4132 genes studied were expressed 1.5- to 5.8-fold higher in leukemic cells relative to both normal samples; 238 of these genes were also overexpressed in the leukemic cell line RS4;11. Nine genes were selected among the 274 overexpressed in at least 2 leukemic samples, and expression of the encoded proteins was measured by flow cytometry. Two proteins (caldesmon and myeloid nuclear differentiation antigen) were only weakly expressed in leukemic cells despite strong hybridization signals in the array. By contrast, 7 proteins (CD58, creatine kinase B, ninjurin1, Ref1, calpastatin, HDJ-2, and annexin VI) were expressed in B-lineage ALL cells at higher levels than in normal CD19(+)CD10(+) B-cell progenitors (P <.05 in all comparisons). CD58 was chosen for further analysis because of its abundant and prevalent overexpression. An anti-CD58 antibody identified residual leukemic cells (0.01% to 1.13%; median, 0.03%) in 9 of 104 bone marrow samples from children with ALL in clinical remission. MRD estimates by CD58 staining correlated well with those of polymerase chain reaction amplification of immunoglobulin genes. These results indicate that studies of gene expression with cDNA arrays can aid the discovery of leukemia markers. (Blood. 2001;97:2115-2120)     

Adhesion-dependent survival of normal and leukemic human B lymphoblasts on bone marrow stromal cells

We investigated the requirement for intimate contact between bone marrow stroma and B lymphoblasts from normal donors and children with leukemia. By scanning electron microscopy, both normal and leukemic cells seeded onto stroma were surrounded by folds of stromal cells or were linked to the stroma by fine tendrils and uropods. Separation of normal B progenitors from stroma by use of microporous membranes led to significantly lower cell recoveries compared with results when contact was unimpeded. For instance, 22.5% +/- 1.8% (mean +/- SEM) of CD19+, CD34+ cells (most immature subset) were recovered after a 7-day culture directly on stroma, compared with 5.2% +/- 0.7% after growth on membranes (P < .001 by Student's t test). In 6 of 11 cases of B-lineage acute lymphoblastic leukemia, separation of progenitors from stroma resulted in apoptosis and a greater than 60% reduction in cell recovery. In the remaining 5 cases, however, this effect was much less pronounced, with reductions in cell recoveries ranging from 48.5% to less than 1% (median, 39.0%) of control values. Inhibition of very late antigen-4, a surface molecule critical for adhesion of B lymphoblasts to stroma, was associated with a greater loss of normal CD34+ B progenitors compared with that for equivalent leukemic cells. These results establish direct contact with stroma as a survival requirement of normal B lymphoblasts and show marked heterogeneity in stromal dependency among B-lineage leukemic cells.     

Temporal association of CD40 antigen expression with discrete stages of human B-cell ontogeny and the efficacy of anti-CD40 immunotoxins against clonogenic B-lineage acute lymphoblastic leukemia as well as B-lineage non-Hodgkin's lymphoma cells.

Detailed immunophenotypic analyses of immunologically classified leukemias and lymphomas showed that CD40 displays an exquisite B-lineage specificity within the human lymphopoietic system. Notably, 82% of B-lineage chronic lymphocytic leukemias (CLLs), 82% of B-lineage hairy cell leukemias (HCLs), 86% of B-lineage non-Hodgkin's lymphomas (NHLs), and 29% of B-lineage acute lymphoblastic leukemias (ALLs) were CD40+. Quantitative analyses of the correlated expression of CD40 and other B-lineage differentiation antigens on fetal lymphoid precursor cells by multiparameter two-color/three-color flow cytometry, combined with analyses of sequential antigen expression on fluorescence-activated cell fluorescence activated cell sorter (FACS) isolated immunologically distinct fetal B-cell precursor subpopulations during in vitro proliferation and differentiation, provided evidence that the acquisition of CD40 antigen in human B-cell ontogeny occurs subsequent to the expression of CD10 and CD19 antigens but before the surface expression of CD20, CD21, CD22, CD24, and surface immunoglobulin M (sIgM). Some leukemic pro-B cells from ALL patients as well as normal pro-B cell clones from fetal livers displaying germline Ig heavy chain genes were CD40+, indicating that the acquisition of CD40 antigen likely precedes the rearrangement of Ig heavy chain genes. CD40+ FACS-sorted malignant cells from B-lineage ALL as well as B-lineage NHL patients were capable of in vitro clonogenic growth, indicating the CD40 antigen is expressed on clonogenic leukemia and lymphoma cells. This hypothesis was confirmed by the ability of an anti-CD40 immunotoxin that we used as an antigen-specific cytotoxic probe to effectively kill clonogenic B-lineage ALL and NHL cells.     

Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia

In children with acute lymphoblastic leukemia (ALL), response to treatment is assessed by bone marrow aspiration. We investigated whether minimal residual disease (MRD) can be effectively monitored in peripheral blood. We used flow cytometric techniques capable of detecting 1 leukemic cell among 10 000 or more normal cells to compare MRD measurements in 718 pairs of bone marrow and peripheral blood samples collected from 226 children during treatment for newly diagnosed ALL. MRD was detected in marrow and blood in 72 pairs and in marrow but not in blood in 67 pairs; it was undetectable in the remaining 579 pairs. Remarkably, findings in marrow and blood were completely concordant in the 150 paired samples from patients with T-lineage ALL: for each of the 35 positive marrow samples, the corresponding blood sample was positive. In B-lineage ALL, however, only 37 of 104 positive marrow samples had a corresponding positive blood sample. Notably, peripheral blood MRD in these patients was associated with a very high risk for disease recurrence. The 4-year cumulative incidence of relapse in patients with B-lineage ALL was 80.0% +/- 24.9% for those who had peripheral blood MRD at the end of remission induction therapy but only 13.3% +/- 9.1% for those with MRD confined to the marrow (P =.007). These results indicate that peripheral blood may be used to monitor MRD in patients with T-lineage ALL and that peripheral blood MRD may provide strong prognostic information in patients with B-lineage ALL.     

Leukemic cell growth in SCID mice as a predictor of relapse in high- risk B-lineage acute lymphoblastic leukemia

Mice with severe combined immunodeficiency (SCID) provide a model system to examine the in vivo homing, engraftment, and growth patterns of normal and malignant human hematopoietic cells. The relation between leukemic cell growth in this model and the treatment outcome in patients from whom cells were derived has not been established. Leukemic cells from 42 children with newly diagnosed high-risk B- lineage acute lymphoblastic leukemia were inoculated intravenously into CB.17 SCID mice. Mice were killed at 12 weeks or when they became moribund as a result of disseminated leukemia. All mice were necropsied and subjected to a series of laboratory studies to assess their burden of human leukemic cells. Twenty-three patients whose leukemic cells caused histopathologically detectable leukemia in SCID mice had a significantly higher relapse rate than the 19 patients whose leukemic cells did not (estimated 5-year event-free survival: 29.5% v 94.7%; 95% confidence intervals, 11.2% to 50.7% v 68.1% to 99.2%; P < .0001 by log- rank test). The occurrence of overt leukemia in SCID mice was was a highly significant predictor of patient relapse. The estimated instantaneous risk of relapse for patients whose leukemic cells caused overt leukemia in SCID mice was 21.5-fold greater than that for the remaining patients. Thus, growth of human leukemic cells in SCID mice is a strong and independent predictor of relapse in patients with newly diagnosed high-risk B-lineage acute lymphoblastic leukemia.     

Terminal differentiation of human promyelocytic leukemic cells in primary culture in response to retinoic acid

The recent finding that retinoic acid induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line, HL-60, prompted an investigation of the sensitivity to this inducer of human myelocytic leukemia cells in primary suspension culture. Of the 21 leukemic specimens, only cells from the two patients with acute promyelocytic leukemia differentiated in response to retinoic acid. After an incubation period of 5--7 days in 1 microM retinoic acid, the cells from these two patients showed extensive morphological and functional maturation. Thus, because it appears that retinoic acid specifically induces granulocytic differentiation of leukemic promyelocytes, this compound may have therapeutic utility in the treatment of acute promyelocytic leukemia.     

Methodological approach to minimal residual disease detection by flow cytometry in adult B-lineage acute lymphoblastic leukemia

A flow cytometric approach to minimal residual disease (MRD) monitoring useful in childhood B-lineage acute lymphoblastic leukemia (ALL) is discussed here in the context of ALL in adults. Of 64 leukemia samples analyzed, 95.3% had at least one abnormal phenotype (57.3% had two or more) as compared to physiologic B-cell precursors in adult bone marrow. The method was sensitive enough to detect one leukemic cell among 10,000 normal cells in 16/19 experiments (84.2%). Blast phenotypes were stable in culture and at relapse, and were useful for MRD monitoring in patients. Marker combinations for childhood ALL are also applicable to adult cases.     

Detection of leukemic cells in the CD34(+)CD38(-) bone marrow progenitor population in children with acute lymphoblastic leukemia

Successful autologous hematopoietic stem cell (HSC) transplantation in childhood acute lymphoblastic leukemia (ALL) requires the ability to either selectively kill the leukemia cells or separate normal from leukemic HSC. Based on previous studies showing that more than 95% of childhood B-lineage ALL express CD38, this study evaluated whether normal CD34(+)CD38(-) progenitors from children with B-lineage ALL could be isolated by flow cytometry. CD34(+) cells from bone marrow samples from 10 children with B-lineage ALL were isolated at day 28 of treatment, when clinical remission had been attained. The CD34(+) progenitor cells were flow cytometrically sorted into CD34(+)CD38(+) and CD34(+)CD38(-) populations. The absolute numbers of CD34(+)CD38(-) cells that could be isolated ranged from 401 to 6245. The cells were then analyzed for the presence of clonotypic rearrangements of the T-cell receptor (TCR) Vdelta2-Ddelta3 locus. Only patients whose diagnostic marrow had an informative TCR Vdelta2-Ddelta3 rearrangement were included in this study. Detection thresholds were typically 10(-4) to 10(-5) leukemic cells in normal marrow. In 6 of 10 samples analyzed, the sorted CD34(+)CD38(-) cells had no detectable Vdelta2-Ddelta3 rearrangements. In 4 cases, the clonotypic leukemic Vdelta2-Ddelta3 rearrangement was detected in the CD34(+)CD38(-) population, indicating that the putative normal HSC population also contained leukemic cells. The data indicate that although most childhood ALL cells express CD34 and CD38, leukemic cells are also frequently present in the CD34(+)CD38(-) population. Therefore, strategies to isolate and transplant normal HSC from children with ALL will require a more stringent definition of the normal HSC than the CD34(+)CD38(-) phenotype. (Blood. 2001;97:3925-3930)