人脐带间充质干细胞作为维持人胚胎干细胞生长饲养层细胞的研究

目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。     

CD44+CD24-/low乳腺癌干细胞的流式分选及其肿瘤干细胞特性的初步鉴定

目的:通过表面标志分选法富集乳腺癌干细胞,并初步鉴定其肿瘤干细胞特性。方法:采用流式细胞分选术从人乳腺癌细胞系MCF-7中分选CD44+CD24-/low乳腺癌干细胞,并进行干细胞比例分析;用免疫荧光法检测、比较分选获得的细胞和对照细胞的干性和分化标记物Oct-4、SOX-2、CK-18和α-SMA的表达状态。结果:分选获得的CD44+CD24-/low乳腺癌干细胞阳性比例达90%以上;免疫荧光检测结果显示,CD44+CD24-/low细胞亚群比non-CD44+CD24-/low细胞亚群高表达干细胞转录因子Oct-4、SOX-2,低表达分化因子CK-18、α-SMA;体外实验表明,CD44+CD24-/low细胞亚群具有更强的成球生长能力,并具有双向分化潜能。结论:CD44+CD24-/low表面标记物分选的方法可以富集高纯度的乳腺癌干细胞,且呈现干性因子Oct-4和SOX-2高表达。     

三阴乳腺癌CD44~+/CD24~(-/LOW)细胞与CXCR4表达的临床意义

目的探讨CD44+/CD24-/low表型在三阴乳腺癌中的临床病理意义以及与CXCR4表达的相关性。方法根据ER、PR、Her-2表达情况将118例乳腺癌分为三阴及非三阴组,应用免疫组织化学检测其CD44、CD24及CXCR4表达情况,分析CD44+/CD24-/low表型及CXCR4在三阴乳腺癌中表达情况及其与临床病理的关系。结果 34例三阴乳腺癌肿瘤细胞CD44+/CD24-/low表型阳性率为55.9%,CD44+/CD24-/low阳性与一般临床特征不相关,但与远处转移或复发密切相关。CXCR4的表达与CD44+/CD24-/low阳性细胞成正相关。结论三阴乳腺癌的复发和远处转移可能与CD44+/CD24-/low较高的表达率有关,并且可能是由CXCR4介导的。     

CD44+CD24-Λow乳腺癌干细胞的流式分选及其肿瘤干细胞特性的初步鉴定

目的:通过表面标志分选法富集乳腺癌干细胞,并初步鉴定其肿瘤干细胞特性.方法:采用流式细胞分选术从人乳腺癌细胞系MCF-7中分选CD44+CD24-Λow乳腺癌干细胞,并进行干细胞比例分析;用免疫荧光法检测、比较分选获得的细胞和对照细胞的干性和分化标记物Oct-4、SOX-2、CK-18和α-SMA的表达状态.结果:分选获得的CD44+CD24-Λow乳腺癌干细胞阳性比例达90％以上;免疫荧光检测结果显示,CD44+CD24-Λow细胞亚群比non-CD44+CD24-Λow细胞亚群高表达干细胞转录因子Oct-4、SOX-2,低表达分化因子CK-18、α-SMA;体外实验表明,CD44+CD24-Λow细胞亚群具有更强的成球生长能力,并具有双向分化潜能.结论:CD44+CD24-Λow表面标记物分选的方法可以富集高纯度的乳腺癌干细胞,且呈现干性因子Oct-4和SOX-2高表达.     

胃癌中CD44v6 表达及与微血管密度和生物学行为的关系

目的:探讨CD44v6在胃癌中的表达及其与微血管密度(microvessel density,MVD)和生物学行为的关系。方法:采用免疫组化法检测80例胃癌组织CD44v6、CD34的表达,以CD34标记肿瘤微血管,并在显微镜下计数微血管密度(MVD)。结果:CD44v6在胃癌组织中的表达与肿瘤浸润深度、临床分期、淋巴结转移相关(P<0.05),CD44v6强阳性表达组中MVD明显高于CD44v6阴性表达组(P<0.05)。结论:CD44v6的表达和MVD计数是反映胃癌生物学特性的良好的指标,对判断胃癌的浸润转移具有一定的临床意义。     

猫4种不同来源间充质干细胞的生物学特性比较

为比较猫的脂肪、骨髓、羊膜、脐带等器官组织4种不同器官来源间充质干细胞(Mesenchymal stem cells,MSC)的生物学特性,本研究通过全血培养法分离培养骨髓来源的间充质干细胞,并采用消化法分离培养脂肪、脐带和胎盘来源的间充质干细胞,比较它们的细胞形态、生长曲线、成脂成骨分化能力以及细胞表面标志物等生物学特性。结果显示,分离得到的4种不同来源的MSC均呈贴壁生长,细胞形态呈梭形、多棱形;均具有成脂成骨分化能力;脂肪源和骨髓源MSC的细胞增殖速度较快,细胞倍增时间较短,而羊膜与脐带源的MSC增殖能力较差,细胞倍增时间较长,4种细胞中脂肪来源MSC的增长活性最好,细胞传至第9代的时候依然保持良好的增殖活性,提示AD-MSC的临床应用可能比较好;4种MSC细胞表面均高表达CD105、CD90和CD44等标志物,低表达CD34。     

血小板第四因子对脐血CD34+细胞趋化作用的研究

目的 :研究PF4及其小肽PF417 70对新鲜脐血CD34+细胞的趋化作用及对粘附分子表达的影响。方法 :采用免疫磁珠法 (MACS)分选CD34+细胞 ,利用Transwell穿孔板测定PF4对CD34+细胞的趋化作用 ;流式细胞仪检测免疫荧光标记的粘附分子及CXCR4的表达。结果 :①PF4对脐血CD34+细胞有趋化作用 ,PF4组的趋化百分比为 15 7.43%± 5 0 .0 6 %(P <0 .0 5 ) ,PF417 70组为 187.0 2 %± 10 .6 9%(P <0 .0 5 )。②PF4作用于CD34+细胞时 ,CD49d和CXCR 4表达增加 ,对其它粘附分子CD31,CD44 ,CD11a ,CD6 2 p ,CD6 2E的表达没有影响。 结论 :PF4对脐血CD34+细胞有趋化作用 ,促进整合素CD49d及CXCR4的表达 ,PF4有助于脐血干细胞的归巢。     

cxcr2基因修饰小鼠骨髓间充质干细胞的构建与鉴定

目的构建小鼠CXC型趋化因子受体2(CXCR2)基因cxcr2过表达的骨髓间充质干细胞(Bone marrow mes-enchymal stem cell,BMSC)并进行鉴定。方法全骨髓贴壁法分离培养小鼠BMSC,采用流式细胞术检测干细胞抗原1(stem cell antigen-1,SCA-1)、CD44、CD43、CD45、IA/IE表达率,并诱导成骨分化。以含有小鼠cxcr2的质粒为模版进行PCR扩增,将获得的cxcr2克隆到慢病毒载体,命名为p Lenti-cxcr2-GZ;将其与慢病毒包装质粒共转染HEK-293T细胞,收获慢病毒后,通过离心法感染BMSC,经过1μg/mL zeocin压力选择建立了稳定表达CXCR2的小鼠BMSC(CXCR2-BMSC)。采用流式细胞术和RT-PCR分别检测其CXCR2蛋白和m RNA表达水平,Transwell趋化实验检测其迁移能力。结果 90%以上的第3代BMSC表达CD44、SCA-1,几乎不表达IA/IE、CD34、CD45,且成功诱导成骨分化。菌液PCR、质粒双酶切后,琼脂糖凝胶电泳鉴定结果得到特异、大小正确的条带及测序鉴定正确,表明成功构建了p Lenti-cxcr2-GZ表达质粒。流式细胞术和RT-PCR结果显示,CXCR2-BMSC的CXCR2蛋白和m RNA表达水平均明显高于对照组BMSC,差异有统计学意义(P<0.001)。Transwell结果显示,CXCR2-BMSC迁移能力高于对照组BMSC,差异有统计学意义(P<0.01)。结论利用慢病毒系统成功构建了稳定表达CXCR2的BM-SC,cxcr2基因修饰BMSC后可明显增加BMSC的迁移能力。     

山羊胚胎干细胞的分离与培养

对关中奶山羊配种后6～7天的桑椹胚和囊胚,分别采用全胚培养法、酶消化法和免疫外科法进行处理.将处理后的胚胎培养于小鼠胎儿成纤维细胞(MEF)饲养层上,分离培养山羊胚胎干细胞(Embryonic stem cell,ESC).对分离传代的山羊ESCs分别进行免疫组化染色,RT-PCR检测和体外诱导分化试验.结果表明.全胚培养法易于胚胎贴壁形成原代集落,采用全胚培养法获得的ESCs有一株目前已传至18代.山羊ESCs Nanong、Oct4、SSEA-3免疫组化染色呈阳性,SSEA-1免疫组化染色呈弱阳性,SSEA-4免疫组化染色呈阴性,RT-PCR检测显示其表达Nanog、Oct4、端粒酶、CD117.山羊ESCs经DMSO体外诱导可以向心肌细胞分化.这些试验均表明该细胞具有ESCs的生物学特性.     

A role of CXC chemokine ligand 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its receptor CXCR4 in fetal and adult T cell development in vivo

The functions of a chemokine CXC chemokine ligand (CXCL) 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its physiologic receptor CXCR4 in T cell development are controversial. In this study, we have genetically further characterized their roles in fetal and adult T cell development using mutant and chimeric mice. In CXCL12(-/-) or CXCR4(-/-) embryos on a C57BL/6 background, accumulation of T cell progenitors in the outer mesenchymal layer of the thymus anlage during initial colonization of the fetal thymus was comparable with that seen in wild-type embryos. However, the expansion of CD3(-)CD4(-)CD8(-) triple-negative T cell precursors at the CD44(-)CD25(+) and CD44(-)CD25(-) stages, and CD4(+)CD8(+) double-positive thymocytes was affected during embryogenesis in these mutants. In radiation chimeras competitively repopulated with CXCR4(-/-) fetal liver cells, the reduction in donor-derived thymocytes compared with wild-type chimeras was much more severe than the reduction in donor-derived myeloid lineage cells in bone marrow. Triple negative CD44(+)CD25(+) T cell precursors exhibited survival response to CXCL12 in the presence of stem cell factor as well as migratory response to CXCL12. Thus, it may be that CXCL12 and CXCR4 are involved in the expansion of T cell precursors in both fetal and adult thymus in vivo. Finally, enforced expression of bcl-2 did not rescue impaired T cell development in CXCR4(-/-) embryos or impaired reconstitution of CXCR4(-/-) thymocytes in competitively repopulated mice, suggesting that defects in T cell development caused by CXCR4 mutation are not caused by reduced expression of bcl-2.     

CXCR3 is induced early on the pathway of CD4+ T cell differentiation and bridges central and peripheral functions

Chemokine receptors on T cells are frequently categorized as functioning either in immune system homeostasis within lymphoid organs, or in peripheral inflammation. CXCR3 is in the latter category and is reported to be expressed selectively on Th1 cells. We found that CXCR3 was expressed in vivo on newly activated tonsillar CD4(+) T cells. Using CD4(+) T cells from cord blood, we found that CXCR3 was induced by cellular activation in vitro independently of the cytokine milieu, although on resting cells, expression was maintained preferentially on those that had been activated in type 1 conditions. In inflamed tonsils, CXCR3(+)CD4(+) T cells were localized around and within germinal centers. The inference that CXCR3 has a role in germinal center reactions was supported by the finding that the CXCR3 ligand CXC chemokine ligand 9 was expressed in a pattern demarcating a subset of germinal centers both in tonsil and in lymph nodes from an HIV-infected individual. We next investigated the role of CXCR3 on peripheral effector/memory CD4(+) T cells by comparing its pattern of expression with that of CCR5, another Th1-cell associated chemokine receptor. Analysis of cells directly from peripheral blood and after activation in vitro suggested that CXCR3 expression preceded that of CCR5, supporting a model of sequential induction of chemokine receptors during CD4(+) T cell differentiation. Taken together, our data show that CXCR3 can be expressed at all stages of CD4(+) T cell activation and differentiation, bridging central function in lymphoid organs and effector function in peripheral tissues.     

Expression of stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4, on CD34+ human bone marrow cells is a phenotypic alteration for committed lymphoid progenitors.

We found that the stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4 (CXCR4), is expressed on human CD34+ bone marrow (BM) cells. Stringently FACS-sorted CD34+CXCR4+ BM cells completely lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential (myeloid progenitors), but give rise to B and T lymphoid progenitors, whereas CD34+CXCR4- BM cells can generate colonies formed by myeloid progenitors and can also develop into these lymphoid progenitors. Therefore, expression of CXCR4 on CD34+ BM cells can allow lymphoid progenitors to be discriminated from myeloid progenitors. Because CD34+CXCR4+ cells are differentiated from CD34+CXCR4- cells, multipotential progenitors located in the BM are likely to be negative for CXCR4 expression. CXCR4 seems to be expressed earlier than the IL-7R and terminal deoxynucleotidyl transferase during early lymphohemopoiesis. These results suggest that the expression of CXCR4 on CD34+ BM cells is one of the phenotypic alterations for committed lymphoid progenitors.     

Cutting edge: profile of chemokine receptor expression on human plasma cells accounts for their efficient recruitment to target tissues

We systematically examined the repertoire of chemokine receptors expressed by human plasma cells. Fresh bone marrow plasma cells and myeloma cells consistently expressed CXCR4, CXCR6, CCR10, and CCR3. Accordingly, plasma cells responded to their respective ligands in chemotaxis and very late Ag-4-dependent cell adhesion to fibronectin. Immobilized CXC chemokine ligand (CXCL)16, a novel transmembrane-type chemokine and CXCR6 ligand, also directly induced adhesion of plasma cells without requiring G(alpha i) signaling or divalent cations. Furthermore, we revealed consistent expression of CXCL12 (CXCR4 ligand), CXCL16 (CXCR6 ligand), and CC chemokine ligand 28 (CCR10 and CCR3 ligand) in tissues enriched with plasma cells including bone marrow, and constitutive expression of CXCL12, CXCL16, and CC chemokine ligand 28 by cultured human bone marrow stromal cells. Collectively, plasma cells are likely to be recruited to bone marrow and other target tissues via CXCR4, CXCR6, CCR10, and CCR3. CXCR6 may also contribute to tissue localization of plasma cells through its direct binding to membrane-anchored CXCL16.     

Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2

The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.     

Evolving complexity of MIF signaling

Macrophage migration inhibitory factor (MIF) is a cytokine expressed in various cell types, including hematopoietic, epithelial, endothelial, mesenchymal and neuronal cells. Altered MIF expression has been associated with a multitude of diseases ranging from inflammatory disorders like sepsis, lupus and rheumatoid arthritis to organ pathologies such as heart failure, myocardial infarction, acute kidney injury, organ fibrosis and a number of malignancies. The implication of MIF in these diseases was supported by numerous animal studies. MIF acts in an autocrine and paracrine manner via binding and activating the receptors CD74/CD44, CXCR2, CXCR4 and CXCR7. Upon receptor binding, several downstream signaling pathways were shown to be activated in vivo, including ERK1/2, AMPK and AKT. Expression of MIF receptors is not uniform in various cells, resulting in differential responses to MIF across various tissues and pathologies. Within cells, MIF can directly bind and interact with intracellular proteins, such as the constitutive photomorphogenic-9 (COP9) signalosome subunit 5 (CSN5), p53 or thioredoxin-interacting protein (TXNIP). D-dopachrome tautomerase (D-DT or MIF-2) was recognized to be a structural and functional homolog of MIF, which could exert overlapping effects, raising further the complexity of canonical MIF signaling pathways. Here, we provide an overview of the expression and regulation of MIF, D-DT and their receptors. We also discuss the downstream signaling pathways regulated by MIF/D-DT and their pathological roles in different tissue, particularly in the heart and the kidney.     

Pertussis toxin-sensitive signal controls the trafficking of thymocytes across the corticomedullary junction in the thymus

We investigated a role of chemokines in thymocyte trafficking. Genes encoding stromal cell-derived factor-1 and its receptor CXCR4 were detected in the cortex by in situ hybridization. Early immigrant cells did not express CXCR4, whereas their descendant CD44+CD25+CD4-CD8- cells did. CXCR4 expression was down-modulated when CD4+CD8+ double-positive cells became CD4+CD8- or CD4-CD8+ single-positive (SP) cells. Positively selected CD69+CD3intermediate cells gained CCR4, of which ligand, thymus activation-regulated chemokine, was expressed in the medulla. At the next developmental stage, CD69-CD3high cells lost CCR4 but gained CCR7. These results suggest that thymocytes use different chemokines along with their development. Blockade of chemokine receptor-mediated signaling by pertussis toxin perturbed the normal distribution of SP cells and resulted in the accumulation of SP cells in the cortex. Thus, a pertussis toxin-sensitive event controls the trafficking of SP cells across the corticomedullary junction.     

A CXCR4-dependent chemorepellent signal contributes to the emigration of mature single-positive CD4 cells from the fetal thymus

Developing thymocytes undergo maturation while migrating through the thymus and ultimately emigrate from the organ to populate peripheral lymphoid tissues. The process of thymic emigration is controlled in part via receptor-ligand interactions between the chemokine stromal-derived factor (SDF)-1, and its cognate receptor CXCR4, and sphingosine 1-phosphate (S1P) and its receptor S1PR. The precise mechanism by which S1P/S1PR and CXCR4/SDF-1 contribute to thymic emigration remains unclear. We proposed that S1P-dependent and -independent mechanisms might coexist and involve both S1P-induced chemoattraction and SDF-1-mediated chemorepulsion or fugetaxis of mature thymocytes. We examined thymocyte emigration in thymi from CXCR4-deficient C57BL/6 embryos in a modified assay, which allows the collection of CD62L(high) and CD69(low) recent thymic emigrants. We demonstrated that single-positive (SP) CD4 thymocytes, with the characteristics of recent thymic emigrants, failed to move away from CXCR4-deficient fetal thymus in vitro. We found that the defect in SP CD4 cell emigration that occurred in the absence of CXCR4 signaling was only partially overcome by the addition of the extrathymic chemoattractant S1P and was not associated with abnormalities in thymocyte maturation and proliferative capacity or integrin expression. Blockade of the CXCR4 receptor in normal thymocytes by AMD3100 led to the retention of mature T cells in the thymus in vitro and in vivo. The addition of extrathymic SDF-1 inhibited emigration of wild-type SP cells out of the thymus by nullifying the chemokine gradient. SDF-1 was also shown to elicit a CXCR4-dependent chemorepellent response from fetal SP thymocytes. These novel findings support the thesis that the CXCR4-mediated chemorepellent activity of intrathymic SDF-1 contributes to SP thymocyte egress from the fetal thymus.     

Differential role of CD133 and CXCR4 in renal cell carcinoma

The chemokine receptor CXCR4 and CD133, putative stem cell markers, were previously described in renal cancer (RCC). To evaluate the biological and prognostic role of CD133 and CXCR4 in RCC the expression was evaluated through qPCR and immunoblotting in human renal cancer cell lines (786-O, A498, ACHN, CAKI-1, SN12C, TK10, UO31) and patients biopsies. Renal cancer cells and surgical biopsies expressed functional CXCR4 while CD133 was not detectable. CXCR4 and CD133 expression was then evaluated in 240 renal cancer patients through immunohistochemistry. CXCR4 and CD133 were low in 19.1% and 59.6%; intermediate in 20% and 17.9%; high in 60.8% and 22.5% of the cases, respectively. CXCR4 was overexpressed in tumours (p= 0.02), while CD133 was over expressed in healthy tissues (p= 0.04). Disease free survival Kaplan Meier plots suggest that prognosis is unfavourable for patients whose primary tumours express CXCR4 (p= 0.0199) but nor CD133 (p= 0.151) neither the concomitant CXCR4-CD133 (p=0.848) high expression affected prognosis. Analysis of prognostic factors suggests that age, clinical presentation, AJCC stage and CXCR4 had a significant prognostic value at the univariate analysis. The CXCR4 predictive ability was confirmed at the multivariate analysis while no prognostic role was identified for CD133. Thus concomitant CD133 and CXCR4 evaluation is not worth in RCC patient while the CXCR4 prognostic role encourage CXCR4 antagonists as promising therapeutic option.     

MIF,CD74 and other partners in kidney disease: Tales of a promiscuous couple

Macrophage migration inhibitory factor (MIF) is increased in kidney and urine during kidney disease. MIF binds to and activates CD74 and chemokine receptors CXCR2 and CXCR4. CD74 is a protein trafficking regulator and a cell membrane receptor for MIF, D-dopachrome tautomerase (D-DT/MIF-2) and bacterial proteins. MIF signaling through CD74 requires CD44. CD74, CD44 and CXCR4 are upregulated in renal cells in diseased kidneys and MIF activation of CD74 in kidney cells promotes an inflammatory response. MIF or CXCR2 targeting protects from experimental kidney injury, CD44 deficiency modulates kidney injury and CXCR4 activation promotes glomerular injury. However, the contribution of MIF or MIF-2 to these actions of MIF receptors has not been explored. The safety and efficacy of strategies targeting MIF, CD74, CD44 and CXCR4 are under study in humans.