Homing-Associated Cell Adhesion Molecule (H-CAM/CD44) on Human CD34+ Hematopoietic Progenitor Cells

Human CD34⁺ hematopoietic progenitor cells (HPCs) express CD44 and can directly adhere to hyaluronate (HA) via CD44. Furthermore, CD44 may also be involved in the regulation of CD34⁺ HPC proliferation and development. The expression of CD44 molecules on CD34⁺ hematopoietic progenitor cells is significantly lower on bone marrow (BM) CD34⁺ cells compared with circulating CD34⁺ cells in cord blood and peripheral blood. Myeloid and erythroid progenitor cells are found predominantly in CD34⁺CD44^- cell fractions. More interestingly, CD34⁺CD44⁺ cells expressing B-lymphocyte-associated CD10 and CD19 would represent unique B-lymphocyte committed precursors in the BM, which might undergo apoptotic cell death in the early steps of B-cell differentiation.     

Growth Characteristics of Myelodysplastic CD34+ Cells

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders of hematopoiesis entailing hyperproliferative and ineffective hematopoiesis associated with morphologic evidence of marrow cell dysplasia resulting in refractory cytopenia(s), and an increased risk of transformation into acute myeloblastic leukemia (AML). The administration of colonystimulating factor(s) (CSFs) to patients with MDS increased blood neutrophil concentrations, in most patients, and it was anticipated to be of benefit to prevent infections. The progression to AML while being treated with CSFs has come under close scrutiny. In vitro studies are expected to produce more pertinent criteria for selection of patients who are likely to benefit, as well as the overall benefits of various therapies. For this purpose, in vitro colony assays are an excellent approach for investigation of the biologic characteristics of MDS progenitor cells. The stem cell phenotype CD34 is the one of the best markers of progenitor cells, and can be used for the purification of these cells to unify levels of maturation; a direct comparison of proliferative and differentiative capacity of MDS progenitor cells with normal CD34⁺ cells can thus be made. The properties of MDS CD34⁺ cells are described here in association with proliferation and differentiation, with special emphasis on the role of stem cell factor (a ligand for c-kit) in leukemic type growth of MDS CD34⁺ cells.     

Proportion of CD4+CD25+ regulatory T cell is increased in the patients with ovarian carcinoma

Objective: To study the frequency of the CD4⁺CD25⁺ regulatory T cells (Tregs) in the patients with ovarian carcinoma and its possible mechanism. Methods: The percentages of CD4⁺CD25⁺ Tregs in the peripheral blood lymphocytes (PBLs), tumor infiltrating lymphocytes (TILs) and tumor associated lymphocytes (TALs) from 13 patients with ovarian carcinoma and in the PBLs from 14 healthy women were determined by flow cytometry. The expression of CD69 on CD4⁺PBLs from the patients was detected. PBLs from healthy women were cultured in complete RPMI 1640 containing the supernatant from SKOV3 cell line with or without PHA (phytohemagglutinin) stimulation for 72 hours, then the percentage of CD4⁺CD25⁺ T cells was detected. Results: CD4⁺CD25⁺ Tregs in the PBLs from patients with ovarian carcinoma were significantly increased compared with those from the control. The percentage of CD4⁺CD25⁺ Tregs in TILs was higher than that in PBLs and TALs from the patients, but not significantly. The expression of CD69 on CD4⁺PBLs from the patients was negative. The percentages of CD4⁺CD25⁺ T cells in PBLs cultured with SKOV3 supernatant elevated significantly compared with those without supernatant whether PHA was added or not (P = 0.001 and 0.001, respectively). Conclusion: There is an increasing of the proportion of CD4⁺CD25⁺ Tregs in PBLs, TILs and TALs of the patients with ovarian carcinoma, which probably results from up-regulation of soluble factor secreted by ovarian carcinoma cells.     

Enhancement of Adenovirus-Mediated Gene Transfer to Human Bone Marrow Cells

Adenovirus infection of CD34⁺ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34⁺ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/β-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or β-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34⁺ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.     

Short-Term Effects of Early-Acting and Multilineage HematoPoietic Growth Factors on the Repair and Proliferation of Irradiated Pure Cord Blood (CB) CD34 Hematopoietic Progenitor Cells

Purpose: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation.

Methods and Materials: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34⁺ cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34⁺ cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose–response curves were fitted to the data.

Results: The radiobiological parameters (D₀ and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34⁺ cells compared to optimal two-factor combinations. The D₀ values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56–1.15, 0.41–2.24, and 0.56–1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n = 1 for all tested GF(s). DNA repair capacity of CD34⁺ cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34⁺ cells was similar.

Conclusion: Our data indicate that increased survival of irradiated CB CD34⁺ cells after short-term GF treatment is mediated through proliferative GF effects on the surviving fraction but not through improved DNA repair capacity.     

Multipotent and Committed CD34+ Cells in Bone Marrow Transplantation

In order to study the role of CD34⁺ cells in hematological recovery following bone marrow transplantation (BMT), bone marrow cells stained with HPCA-1 (CD34) and MY-9 (CD33) monoclonal antibodies were analyzed by using a fluorescence-activated cell sorter on or about days 14 and 28, as well as at later times, following BMT in 6 recipients. Single cell cultures of CD34⁺ cells were also performed to evaluate their in vitro hematopoietic function. CD34⁺ cells were detectable in bone marrow cells on day 14. More than 80% of CD34⁺ cells co-expressed the CD33 antigen, and macrophage (Mac) colony-forming cells predominated among total colony-forming cells of CD34⁺ cells. In normal bone marrow cells, CD34⁺, CD33⁺ cells amounted to about 40% of CD34⁺ cells, and the incidences of erythroid bursts, granulocyte/macrophage (GM) colonies, and Mac colonies were similar to each other. After more than 10 weeks, CD34⁺, CD33⁻ cells gradually recovered, as erythroid burst colony-forming cells increased following GM colony-forming cells. This phenomenon was well-correlated with the time course of peripheral blood cell recovery. CD34⁺, CD33⁺ cells as committed progenitors and CD34⁺, CD33⁻ cells as multipotent stem cells have distinctive biological behaviors in BMT.     

Ultrastructural Features of CD34+ Hematopoietic Progenitor Cells from Bone Marrow, Peripheral Blood and Umbilical Cord Blood

Hematopoietic progenitor cells from different sources have been widely characterized, but their ultrastructural morphology has never been described in detail. In this study, imunomag-netically separated CD34⁺ cells from normal bone marrow (BM), mobilized peripheral blood (PBSC) and human umbilical cord blood (CB) were studied by transmission electron microscopy (TEM) using a cytochemical method which reveals endogenous myelo-peroxidase (MPO) activity. This technique is particularly suited for detecting early signs of the myeloid commitment. The CD34⁺ cells from PBSC were morphologically very homogeneous and 94.7 ± 4.5% of these cells were MPO^-: these ultrastructural features are generally considered typical of immature cells. The CD34⁺ BM cells were instead more heterogeneous, with 24.6 ± 7.4% showing intense MPO activity. The ultrastructural characteristics of CB cells fell between those observed in PBSC and BM, but there was a high percentage of morphologically immature cells with no evidence of MPO activity (about 83%). The number of apoptotic cells within samples from different sources was also examined both by TEM and flow cytometry. The percentage of apoptotic cells was 0.7% in PBSC, 2.3% in BM, 2.9% in CB from vaginal delivery and 11.6% in CB from cesarean section. These observations confirm the relative phenotypic immaturity of CB in comparison with BM cells; they also suggest that CB collected after cesarean section may be associated with reduced stem cells viability.     

CD44和CD24标记的宫颈癌细胞亚群特性

目的 考察Siha细胞系中的CD44^+/CD24⁺能否富集宫颈癌干细胞。方法 用流式细胞仪分选出CD44⁺/CD24⁺Siha细胞,用无血清悬浮培养观察成球能力、裸鼠移植瘤实验观察成瘤能力、透射电子显微镜观察辐射前后两组细胞形态变化,并通过Transwell侵袭实验比较细胞侵袭能力的差异。结果 耐放疗细胞中CD44^+/CD24⁺Siha细胞比例明显高于其在亲代Siha细胞中的比例;无血清培养CD44⁺/CD24⁺Siha细胞组可以形成致密且体积较大的细胞球,CD44^+/CD24⁺Siha细胞组致瘤时间早,成瘤率高;辐射后CD44⁺/CD24⁺Siha细胞较亲代Siha细胞更抗凋亡;CD44⁺/CD24⁺Siha细胞组的迁移细胞数明显高于亲代Siha细胞组,所有数据均具有统计学意义。结论 CD44⁺/CD24⁺Siha具备部分干细胞特性,CD44⁺/CD24⁺可能成为宫颈癌干细胞特异性表面标志物。     

Proliferation and Differentiation of Myelodysplastic CD34+ Cells

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders of hematopoiesis involving hyperproliferative and ineffective hematopoiesis associated with morphologic evidence of marrow cell dysplasia resulting in refractory cytopenia(s), and an increased risk of transformation into acute myeloblastic leukemia (AML). The administration of colony-stimulating factor(s) (CSFs) to patients with MDS increased blood neutrophil concentrations, in most patients, and was also expected to be beneficial and to prevent infections. However, the progression to AML during the treatment with CSFs was suspected in some patients. Therefore, extensive in vitro studies were expected to lead to the establishment of criteria for selection of patients who are likely to benefit from CSF's as well as to establish the overall value of the different types of CSFs therapy. For this purpose, in vitro colony assays provide an excellent tool for investigating the biologic characteristics of MDS progenitor cells. However, conditions of the culture must be such that each progenitor can express its full potential for proliferation and differentiation.

Because of the above, MDS progenitor cells cannot be used because they carry an impairment in proliferation and differentiation. To address this problem, one needs to know how many cells are being handled and the maximum numbers of colonies and clusters expected. CD34, a stem cell phe-notype, is at present one of the best markers of progenitor cells, and can be used for purposes of purification. Using a defined number of CD34⁺ cells, it was feasible to make direct investigations on MDS progenitor cells. In this review the properties of MDS progenitor cells are described, in association with proliferation and differentiation, with special emphasis on the phenotypic subpopula-tions of MDS CD34⁺ cells.     

Characterization of CD34+ Human Hemopoietic Progenitor Cells from the Peripheral Blood: Enzyme-, Carbohydrate- and Immunocytochemistry, Morphometry, and Ultrastructure

Following an immunomagnetic isolation and enrichment procedure, CD34⁺ cells were harvested from the peripheral blood of about 50 healthy donors. A battery of cytochemical staining reactions, monoclonal and carbohydrate-specific antibodies, proliferation markers and lectins was applied on smears and sections from paraffin-embedded pellets. Additionally, a morphometric analysis and ultrastruc-tural investigation was carried out. More than 95% of the total yield of progenitor cells expressed CD34 and CD43 (MT1) and of these about 90% CD45 (LCA) and 25% CD45RA (MT2). The CD34VCD45RA population was thought to represent very primitive, probably not lineage-restricted stem cells. On the other hand, reactivity with ANAE, CD11c, CD15, CD20, Ret40f, KiMlP, and CD61 (ranging between 1 to 20%) was considered to indicate a transition into more differentiated elements of hemopoiesis. The failure to detect any staining with proliferation markers (Ki-67/MIBl, PCNA, KiS1) was in keeping with a quiescent status. Carbohydrate antigens revealed a pattern which underlines the fact that the CD34 and CD43 antigens belong to the family of heavily O-glycosylated sialomucins. Blood group antigens which are located at the peripheral regions of mucin-oligosaccharides (H type 2, Lewis^x, Lewis^a) could be demonstrated, but no t. A, B, Sialyl-Lewis^x and Lewis^y. Morphometric analysis revealed that CD34⁺ progenitors were larger than small lymphocytes. Electron microscopy showed a relatively primitive cytoplasmic organization and numerous tiny magnetic beads clustered at the plasma membrane.     

供者单采外周血中幼稚粒细胞检测的研究

目的 :探讨异基因外周血干细胞移植 ( Allo-PBSCT)供者经细胞集落刺激因子动员后 ,采集的外周血干细胞 ( PBSC)移植物中的幼稚粒细胞与单个核细胞 ( MNC)及 CD+ 3 4 细胞数之间相关性和移植的剂量标准。方法 :对 6例 Allo-PBSCT供者用 G-CSF或 G-CSF+GM-CSF进行动员 ,于动员前及动员后 ,对外周血及收集的MNC收集物中幼稚粒细胞及 CD+ 3 4 细胞进行检测计数 ,并计算出输给患者每公斤体重的 MNC、幼稚粒细胞及 CD+ 3 4细胞数。结果 :动员后外周血中的幼稚粒细胞与 CD+ 3 4 细胞数同步增加 ,外周血 MNC中幼稚粒细胞数与 CD+ 3 4 细胞数之间有较好的相关性。 6例患者全部植活恢复造血功能 ,并为染色体核型或血型转为供者型所证实。结论 :Allo-PBSCT供者经 G-CSF或 G-CSF+GM-CSF动员之后 ,外周血 MNC中幼稚粒细胞与 CD+ 3 4 细胞数有较好的相关性。因此 ,MNC与幼稚粒细胞数结合可作为 Allo-PBSCT的剂量标准 ,该法检测方便快速 ,稳定性好 ,便于推广     

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: I, Diverse Patterns of Expression in Mature (Post-Thymic) T-Cell Proliferations

By simultaneous two- and three-colour flow cytometry, this study analysed the expression of membrane CD45RA (2H4) and CD45RO (UCHL1) determinants by normal thymocytes (n = 5) and peripheral blood lymphocyte subpopulations (CD4⁺, n = 21; CD8⁺, n = 12; CD8^dim+, n = 12) and compared these patterns with those of T-cells from representative CD4⁺CD8^- (n = 8), CD4⁺CD8⁺ (n = 2), CD4^-CD8⁺ (n = 10) and CD4^-CD8^- (n = 1) proliferations. These comprised cases of prolymphocytic leukaemia (T-PLL, n = 5), adult T-cell leukaemia-lymphoma (ATLL, n = 2), Sezary Syndrome (SS, n = 4), chronic lymphocytic leukaemia (T-CLL, n = 4), and lymphoproliferative disease of granular lymphocytes (LDGL, n = 5). Normal thymocyte fractions, of which a mean of 85% cells co-expressed membrane CD4 and CD8, were predominantly (mean 89%) 2H4^-UCHL1⁺ with the remaining cells consisting of 2H4^intUCHL1⁺ and 2H4⁺UCHL1^- components. Further analysis showed that virtually all CDla⁺ thymocytes were UCHL1⁺ whereas the CD1a^- fraction comprised similar proportions of both UCHL1^- and UCHL1⁺ subpopulations. Similarly, normal blood CD4⁺, CD8⁺ and CD8^dim+ lymphocytes showed reciprocal CD45RA/CD45RO expression and could be phenotypically grouped into 2H4⁺UCHL1^- 2H4^intUCHL1⁺ and 2H4^-UCHL1⁺ subpopulations. Mean proportions of 48% and 68%, for CD4⁺ and CD8⁺ lymphocytes respectively, showed a composite 2H4⁺UCHL1^- phenotype, whereas the percentage of NK-associated CD8^dim+ cells with this phenotypic pattern was considerably higher (mean, 85%). Normal lymphocyte subpopulations lacking both determinants (2H4^-UCHL1^-) were only rarely noted. Comparing normal patterns of CD45RA/CD45RO expression with those of the T-cell proliferations revealed diverse and abnormal patterns of staining for 3/6 of the CD4⁺CD8^- SS and ATLL, and for 5/5 of the T-PLL (CD4⁺CD8^-, n = 2; CD4⁺CD8⁺, n = 2; and CD4^-CD8⁺, n = 1) cases studied. In contrast, the nine cases of CD4^-CD8⁺ T-CLL and LDGL all showed CD45RA/CD45RO staining patterns similar to that of normal CD8⁺/CD8^dim+ blood lymphocytes (i.e. a predominance of 2H4⁺UCHL1^- cells). Although the variant CD45RA/CD45RO pattern types of the CD4⁺ proliferations did not appear to be related to either the diagnostic category or other phenotypic characteristics, the high proportion of abnormal patterns within this case group suggests that recognition of these abnormalities may be potentially relevant to the differentiation of benign and malignant CD4⁺ proliferations and, in addition, may be of aetiological importance with respect to the diverse acquired defects in immunity commonly seen in patients with such disorders.     

CD44+/CD24+表型的宫颈癌Siha细胞对顺铂的耐药及机制研究

背景与目的：肿瘤干细胞的存在是肿瘤细胞抗拒化疗的原因之一。该研究探讨CD44⁺/CD24⁺宫颈癌Siha细胞对顺铂的耐药性及相关机制。方法：体外培养Siha细胞,流式荧光激活细胞分选仪分选CD44⁺/CD24⁺Siha细胞,噻唑蓝(thiazolyl blue,MTT)法检测不同浓度顺铂对细胞的体外抑制情况,采用流式细胞仪检测10 μg/mL顺铂作用于CD44⁺/CD24⁺ Siha细胞24、48和72 h时的细胞凋亡率,实时定量聚合酶链式反应(quantitutive real-time polymerase chain reaction,qRT-PCR)和蛋白［质］印迹法(Western blot)检测CD44⁺/CD24⁺ Siha细胞中Oct-4、ABCG2、Bcl-2的表达,同时设立亲代Siha细胞作为对照。结果：不同浓度顺铂(0.1、1、5、10、15和20 μg/mL)对CD44⁺/CD24⁺ Siha细胞的增殖抑制作用较亲代Siha细胞小［(88.4±1.5)% vs (92.9±1.5)%,(79.9±1.0)% vs (84.7±1.1)%,(69. 8±0.8)% vs (75.1±2.9)%,(59.0±0.7)% vs (65.8±2.7)%,(49.6±0.9)% vs(52.1±0.5)%,(45.1±0.7)% vs (48.8±1.0)%,P<0.05］;与亲代细胞相比,当10 μg/mL顺铂作用于CD44⁺/CD24⁺Siha细胞时,发现在24、48和72 h时的细胞凋亡率均较小［(3.05±0.16)% vs (5.17±0.27)%,(17.94±2.02)%vs (32.60±4.28)%和(40.14±3.01)% vs (56.62±5.32)%,P<0.05］。qRT-PCR和Western blot实验均显示CD44⁺/CD24⁺ Siha细胞高表达Oct-4、ABCG2和Bcl-2,与亲代Siha细胞比较差异有统计学意义(P=0.015)。结论：CD44⁺/CD24⁺ Siha细胞可以抵抗顺铂诱导的细胞凋亡,具有化疗抵抗性,并且高表达肿瘤干细胞表面标志物如Oct-4和ABCG2,该结果在宫颈癌肿瘤干细胞的有效分选以及肿瘤靶向治疗方面有深远意义。     

结直肠癌及腺瘤患者CD4+、CD8+和CD28+T淋巴细胞的亚群变化及临床意义

背景与目的：结直肠癌（colorectal cancer,CRC）严重影响患者生存。探讨肿瘤微环境（tumor microenvironment,TME）T细胞亚群在CRC和腺瘤中的表达及意义。方法：用免疫组织化学法和流式细胞术对51例健康人（对照组）、46例结直肠腺瘤（腺瘤组）、100例CRC（癌症组）和15例CRC术后（癌术后组）患者进行T细胞亚群检测。结果：① 对照组、腺瘤组及癌症组3组中CD4⁺T细胞的阳性率分别是90.00%、43.75%及32.65%,CD8⁺T淋巴细胞的阳性率分别是30.00%、56.25%及75.51%,CD28⁺T淋巴细胞的阳性率分别是42.86%、30.00%及20.00%。② 对照组、腺瘤组及癌症组3组中CD4⁺、CD4⁺/CD8⁺、CD28⁺、CD8⁺CD28⁺和CD8^－CD28⁺逐渐降低,CD8⁺、CD8⁺CD28^－逐渐增加（P＜0.05）;癌术前术后T细胞亚群差异有统计学意义（P＜0.05）。结论：① CRC微环境T细胞亚群中CD4⁺、CD4⁺/CD8⁺、CD28⁺、CD8⁺CD28⁺和CD8^－CD28⁺呈递减趋势,CD8⁺、CD8⁺CD28^－呈递增趋势,且在癌前病变腺瘤中已逐步出现上述趋势变化。② CRC患者行肿瘤切除术后,其T细胞亚群有所恢复,故在一定程度上,CRC中T细胞亚群的变化可以早期预测结直肠疾病的发展。     

Role of glycosylphosphatidylinositol-specific phospholipase D in the homing of umbilical cord blood, mobilized peripheral blood and bone marrow-derived hematopoietic stem/progenitor cells

Recent studies suggested that glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) correlated with tumor malignancy and prognosis of certain tumors. As hematopoietic stem/progenitor cells (HS/PC) homing was similar to tumor invasion and metastasis in some mechanisms, which arose our intrests in whether GPI-PLD contribution to the homing of HS/PC. In this study, CD34⁺ cells from umbilical cord blood (UCB), mobilized peripheral blood (MPB), and bone marrow (BM) were assayed for their differences in adhesion, migration, respectively. The expression of GPI-anchored proteins (CD48, CD90) on the cells were analyzed by flow cytometry. Semi-quantitive RT-PCR was used to detect GPI-PLD expression in the three different CD34⁺ cells. The results showed that GPI-PLD had no effect on the adhesion of CD34⁺ cells. While, spontaneous and SDF-1 induced migration of UCB and MPB, but not BM CD34⁺ cells were decreased after 1,10-phenanthroline (an inhibitor of GPI-PLD) pretreatment. Furthermore, we found little difference in GPI-anchored adhesion molecules (CD48, CD90) expression between untreated and pretreated CD34⁺ cells. GPI-PLD mRNA was low expressed in MPB and undetected in UCB and BM CD34⁺ cells. Our results suggested that GPI-PLD probably had no contribution to HS/PC homing, which may due to its low or no expression in UCB, BM and MPB CD34⁺ cells.     

不同乳腺癌干细胞亚群的自我更新和致瘤能力差异

目的 分析原代培养乳腺癌细胞中不同乳腺癌干细胞亚群的自我更新、致瘤能力差异。方法 采用流式细胞术和免疫荧光实验,从乳腺癌原代培养细胞中分选、鉴定CD44⁺CD24^-/low、ALDH1⁺和ALDH1⁺CD44⁺CD24^-/low细胞亚群,分析各分选细胞占总肿瘤细胞的比例;通过克隆形成实验观察各组细胞的自我更新能力;通过裸鼠致瘤实验观察各组细胞的致瘤能力。结果 CD44+CD24^-/low细胞占总细胞比例的7.2%,ALDH1⁺细胞所占比例为4.6%,ALDH1⁺CD44⁺CD24^-/low细胞所占比例为1.5%。三组细胞自我更新和致瘤能力由强至弱依次为ALDH1⁺CD44⁺CD24^-/low、ALDH1⁺、CD44⁺CD24^-/low细胞,各组细胞间比较差异均存在统计学意义（P<0.05）。 结论 乳腺癌组织中CD44+CD24-/low、ALDH1⁺和ALDH1⁺CD44⁺CD24^-/low三组乳腺癌干细胞亚群之间的自我更新和致瘤能力有明显差异,ALDH1⁺CD44⁺CD24^-/low亚群细胞具有最强的自我更新和致瘤能力,提示ALDH1⁺CD44⁺CD24^-/low可能是更具有特异性的乳腺癌干细胞标志物。     

Use of Flow Cytometric CD34 Analysis to Quantify Hematopoietic Progenitor Cells

This review summarizes our experiments on flow cytometric analysis of CD34 positive mono-nuclear cells (MNC) and on colony formation of myeloid hematopoietic progenitor cells in the clonogenic assay. We examined MNC isolated by density centrifugation of bone marrow, cord blood and peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth factors or from patients treated with G-CSF or GM-CSF. We attempted to correlate the results obtained by CD34 analysis with the cloning efficiency determined after a 14 day culture period in the methylcellulose-based clonogenic assay. The highest cloning efficacy (60%-100%) was observed in cord blood, however, a good correlation was found in both untreated and GM-CSF treated peripheral blood samples in which a mean of 50% and 20% of the number of CD34 positive MNC gave rise to myeloid colonies. In bone marrow, the cloning efficacy was generally lower and ranged between 5% and 15%. The lowest values were observed in G-CSF treated peripheral blood in which colonies were grown from only l%-9% of the CD34⁺ MNC. Due to the variable numbers of CD34⁺ lymphoid and/or more committed myeloid precursors which form either no colonies or only clusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysis of cord blood MNC and untreated or GM-CSF treated peripheral blood MNC provides reliable results with respect to the content of myeloid progenitors. Analysis of bone marrow MNC and G-CSF treated peripheral blood MNC requires two colour staining using CD34 and CD45RA. Exclusion of the CD34⁺/CD45RA⁺⁺ cell fraction will improve the correlation and give results similar to those obtained with untreated blood MNC.     

In vitro proliferative and differentiating characteristics of CD133(+) and CD34(+) cord blood cells in the presence of thrombopoietin (TPO) or erythropoietin (EPO). Potential implications for hematopoietic cell transplantation

We investigated the characteristics of cord blood (CD) CD133⁺ and CD34⁺ cells, by flow cytometry, clonogenic assays and assessment of the replating ability (area under the curve (AUC)) following 7-day liquid culture in the presence of early acting growth factors and either thrombopoietin (TPO) or erythropoietin (EPO). The CD34⁺ population showed a more effective proliferation in all parameters tested and TPO proved to be more effective than EPO. On the contrary, the CD133⁺ cell fraction retained and expanded more immature elements in a modest but consistent manner with either TPO or EPO. We conclude that CD133⁺ and CD34⁺ expanded cord blood cells could potentially be used in combination to overcome the shortcomings of cord blood transplantation in older children and adults.     

过继性免疫治疗对非小细胞肺癌患者外周血T细胞亚群的影响

目的 研究细胞因子诱导的杀伤（cytokine-induced killer,CIK）细胞治疗对非小细胞肺癌（non-small cell lung cancer,NSCLC）患者外周血T细胞亚群及一般情况的影响。方法 采集非小细胞肺癌患者外周血,常规方法体外扩增CIK细胞,分三次回输患者体内,流式细胞仪检测非小细胞肺癌患者治疗前与治疗3次后T细胞亚群的变化,分析其与健康对照组相比外周血T细胞亚群的差异,并观察CIK细胞治疗前后患者一般情况变化。结果 与对照组相比,NSCLC患者外周血中CD3⁺ T 细胞比例、CD3⁺CD4⁺T细胞比例、CD4⁺/CD8⁺ 比值显著下降（P<0.05）,CD3⁺CD8⁺ T细胞比例显著升高（P<0.05）。CIK细胞治疗后与治疗前,CD3⁺ T 细胞比例、CD3⁺CD4⁺ T细胞比例、CD4⁺/CD8⁺ T细胞比值显著升高（P<0.05）,CD3⁺CD8⁺ T细胞比例显著降低（P<0.05）。CIK细胞治疗后与对照组相比,CD3⁺ T 细胞比例、CD4⁺/CD8⁺T细胞比值显著下降（P<0.05）。CD3⁺CD4⁺ T细胞比例、CD3⁺CD8⁺ T细胞比例升高,但差异无统计学意义（P>0.05）。CIK细胞治疗前Tregs较对照组显著升高（P<0.05）。CIK细胞治疗后与治疗前相比,Tregs比例显著下降（P<0.05）。结论 CIK细胞输注治疗可以增强NSCLC患者的免疫功能,并改善患者的一般情况,提高生活质量,且未见明显不良反应。     

Flow Cytometric Analysis of Protein Phosphorylation in the Hematopoetic System

Cellular growth and differentiation in blood cells are regulated by the phosphorylation status of growth factor receptors and downstream proteins. Protein kinases and phosphatases balance the homeostasis of protein phosphorylation. Various diseases are associated with alterations in these tightly regulated processes. Aberrations have been proved to be of diagnostic value and might enhance the pathophysiological insight into the origin of the disease. However, quantitation of protein phosphorylation is currently not feasible in a clinical situation.

We developed a flow cytometric methodology which enables for direct investigation of protein phosphorylation in cell populations defined by multi-color flow cytometry. This assay does not only overcome drawbacks of traditional methodologies (e.g. Western blotting) but also allows quantitative analyses even in rare cell populations.

We accurately examined phosphorylation levels in different cell populations of hematological interest and especially analyzed CD34⁺ hematopoetic progenitor cells. CD34⁺ cells in bone marrow and in cord blood contained similar, low levels of phosphotyrosine. Circulating pheripheral blood system cells PBSC in patients exposed to G-CSF for stem cell mobilization exhibited significantly increased levels of phosphotyrosine. In vitro exposure of CD34⁺ progenitors to growth factors (G-CSF, IL-3, SCF) raised the levels of tyrosine phosphorylation in bone marrow and cord blood. Effects were dose and time dependent. Interestingly, in vivo stimulated CD34⁺ PBSC could not be further stimulated in vitro.

In conclusion, we present a new powerful methodology for analysis of protein phosphorylation in hematological specimens. The method does not only allow for accurate detection of phosphorylation levels in vivo, but also enables for quantitative analysis of growth factor receptor stimulation in vitro and in vivo.