胚胎干细胞和间充质干细胞分化为内皮细胞的研究现状

内皮细胞对于维持血管正常功能具有重要作用,然而其来源有限.因此,寻找内皮细胞的其他丰富来源一直为研究者们所关注.干细胞在体外培养具有能大量增殖、并在合适的条件下定向分化的特性.近年来,各种类型的干细胞诱导分化为内皮细胞的研究不断深入并各有优缺点.对胚胎干细胞和各种来源的间充质干细胞诱导分化为内皮细胞的研究现状作一综述.     

供体脾细胞输注诱导小鼠移植耐受及其机理的研究

目的以持续供体脾细胞输注的方法建立异基因嵌合体动物模型,并探讨移植耐受形成的机制。方法BALB/c（H-2     

Tolerogenic dendritic cells impede priming of naïve CD8+ T cells and deplete memory CD8+ T cells

Type 1 diabetes is a T‐cell‐mediated autoimmune disease in which autoreactive CD8⁺ T cells destroy the insulin‐producing pancreatic beta cells. Vitamin D3 and dexamethasone‐modulated dendritic cells (Combi‐DCs) loaded with islet antigens inducing islet‐specific regulatory CD4⁺ T cells may offer a tissue‐specific intervention therapy. The effect of Combi‐DCs on CD8⁺ T cells, however, remains unknown. To investigate the interaction of CD8⁺ T cells with Combi‐DCs presenting epitopes on HLA class I, naive, and memory CD8⁺ T cells were co‐cultured with DCs and proliferation and function of peptide‐specific T cells were analyzed. Antigen‐loaded Combi‐DCs were unable to prime naïve CD8⁺ T cells to proliferate, although a proportion of T cells converted to a memory phenotype. Moreover, expansion of CD8⁺ T cells that had been primed by mature monocyte‐derived DCs (moDCs) was curtailed by Combi‐DCs in co‐cultures. Combi‐DCs expanded memory T cells once, but CD8⁺ T‐cell numbers collapsed by subsequent re‐stimulation with Combi‐DCs. Our data point that (re)activation of CD8⁺ T cells by antigen‐pulsed Combi‐DCs does not promote, but rather deteriorates, CD8⁺ T‐cell immunity. Yet, Combi‐DCs pulsed with CD8⁺ T‐cell epitopes also act as targets of cytotoxicity, which is undesirable for survival of Combi‐DCs infused into patients in therapeutic immune intervention strategies.     

干细胞与肿瘤发生的关系

Stem ceils are a lineage with capacities of self- renewal and multipotential differentiation.They are exactly regulated by microenvironment where they live. With the understanding of stem ceils biology,researchers noticed that stem ceils have many similar biology characters with tumor cells, and thought that tu-mors were likely derived from stem ceils, and gave a concept that there were tumor stem ceils in tumor tissues.     

造血干细胞的可塑性及临床应用

干细胞是一类具有自我复制和多分化潜能的细胞 ,根据其发育阶段不同 ,分为胚胎干细胞和成体干细胞。胚胎干细胞具有多分化潜能 ;而成体干细胞的分化潜能则较为局限 ,只能定向分化为特化细胞。但新近的研究表明 ,成体干细胞在一定条件下可分化为与其所在组织不同的其他组织类型细胞 ,这种干细胞的可塑性被称为成体干细胞的横向分化(ｔｒａｎｓｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ)。其中造血干细胞 (ｈｅｍａｔｏｐｏｉｅｔｉｃｓｔｅｍｃｅｌｌｓ,ＨＳＣ)作为人们认识最深入 ,其临床应用也最为广泛的一类成体干细胞 ,目前已成为成体干细胞可塑性…     

角膜干细胞重建眼表后泪膜稳定性改变的试验研究

目的：研究角膜缘干细胞重建眼表术后泪膜生理功能改变，探讨利用角膜缘干细胞移植重建眼表的有效性和评价指标。方法：以健康雄性新西兰兔为实验对象，取左眼角膜缘干细胞体外培养，然后进行眼表重建，观察泪膜生理功能改变情况。结果：采用羊膜为载体培养角膜缘干细胞，移植修复眼表结构后，眼表细胞形态与烧伤前相似；泪膜破裂时间测试，修复后与烧伤后有显著差异（P<0.05），修复后与烧伤前无显著差异（P>0.05）。结论：利用角膜缘干细胞培养可能是重建眼表有效途径，修复后细胞结构形态和泪膜生理功能恢复良好；眼表细胞结构形态分析和泪膜破裂时间是良好的眼表重建疗效分析指标。     

Pathogenic T cells in MOBP-induced murine EAE are predominantly focused to recognition of MOBP21F and MOBP27P epitopic residues

Myelin-associated oligodendrocytic basic protein (MOBP) is a central nervous system (CNS)-specific myelin constituent important in stabilizing the unique multi-layered structure of the CNS-myelin sheath. Although autoimmunity against MOBP has been associated with multiple sclerosis pathogenesis, anti-MOBP pathogenic T cells have been poorly investigated as compared to T cells against other encephalitogenic myelin proteins. We have recently determined MOBP15-36 as the major encephalitogenic epitope of MOBP for SJL/J mice. In this study, epitope-specificity was dissected to the level of residues crucial for activation/control of MOBP-specific encephalitogenic T cells. Structural bioinformatic analysis of MOBP15-36/I-A(s) interaction and experimental data have determined MOBP21F and MOBP27P of I-A(s)-binding nonameric core epitope (MOBP20-28) as TCR-contact residues critical for functional activation of encephalitogenic MOBP-specific T cells. Accordingly, the non-encephalitogenic/nonstimulatory pMOBP16-21A27A-33 inhibited encephalitogenic MOBP-reactive T cells, shifted their cytokine secretion profile, and effectively suppressed pMOBP15-36-induced EAE. Moreover, pMOBP16-21A27A-33 fully reversed ongoing clinical EAE induced by whole MOBP as well as by pMOBP15-36. Data show that encephalitogenic MOBP-reactive T cells are predominantly focused to recognition of MOBP21F and MOBP27P, and suggest that both their selection and control are governed by MOBP21F and MOBP27P epitopic residues. Such focused epitopic recognition may affect profoundly on peripheral self-tolerance to MOBP and on potential altered peptide ligand-mediated therapy of MOBP-related autoimmune pathogenesis.     

The promoting effect of neutrophil-derived BAFF molecule on the proliferation and life span of CAL-27 oral squamous carcinoma cells

Tumor-associated neutrophils (TANs) are the major cellular component of the tumor microenvironment and have been shown to release of different bioactive molecules such as B-cell activating factor (BAFF). The data on the interactions between OSCC cells and neutrophils are limited and do not explain the actual role of the BAFF in the development of the OSCC. In the present study we examined the direct effect of neutrophils-derived BAFF on the OSCC cell line CAL-27 proliferation and apoptosis.PMNs of OSCC patients and healthy control were isolated from whole blood and separated by magnetic selection with monoclonal anti-human CD16 antibodies. CD-16 – positive neutrophils were incubated in the presence of TGF-β and/or LPS as well as flavonoids (luteolin and quercetin). CAL-27 cells were co-incubated with supernatants of neutrophils. BAFF expression in neutrophils, BAFF-R expression on CAL-27 cells and apoptosis of CAL-27 cells were assessed by flow cytometry. To determine the CAL-27 cells proliferation, the MTT test was used. Expression of select mitochondrial proteins in CAL-27 cells were measured by Western blot.Neutrophils from OSCC patients showed significantly higher expression of BAFF than those from the healthy controls. The results obtained revealed upregulation of the proliferation and downregulation of the apoptosis of the CAL-27 cells in the presence of the supernatants of TGF-β-treated neutrophils. Flavonoids reduced BAFF expression in neutrophils of patients with OSCC and control group. Lower intensity of apoptosis in CAL-27 cells was associated with the increased expression of anti-apoptotic Bcl-2, Mcl-1 and activated form of PI3K kinase (pPI3K) and simultaneously reduced expression of pro-apoptotic Bax protein in the presence of rhBAFF, as well as of supernatants of neutrophils derived from OSCC patients. In conclusion, the data presented confirm the previously suggested role of neutrophil-derived BAFF in OSCC development. The favorable effects of examined flavonoids on tumor-promoting BAFF expression in neutrophils suggest that they might be promising candidates as chemo-preventive agents in the therapy of patients with OSCC.     

Contribution of sex steroids and prolactin to the modulation of T and B cells during autoimmunity

In this review we discuss how sex steroids and prolactin affect regulation and responsiveness of B and T cells. Sex hormones exert profound effects on several physiological processes of non- reproductive tissues. In the immune system, several studies with experimental models for SLE have shown a noticeable pro-inflammatory role for ERα, contributing to disease development reflected in proteinuria and renal pathology. On the other hand, ERβ appears to have an anti- inflammatory and immunosuppressive effect. Estrogen/ERα signaling induced an increase of Th17 cells in lymph nodes as well as the expression of its correspondent chemokine receptor CCR6 during collagen induced arthritis acute phase. High levels of anti- DNA antibodies and increased mortality was observed when given high E and prolactin doses to NZB/NZW mice, as compared with mice receiving low E and prolactin doses, or high E and low prolactin doses. Intracellular progesterone receptors have been detected in TCD4⁺ cells but in contrast as observed with ERs, it suppresses T cell dependent responses. Progestagen administration on female NZB/NZW mice decreased anti DNA IgG, improved survival, decreased glomerulonephritis and proteinuria.     

In vivo activation of Treg cells with a CD28 superagonist prevents and ameliorates chronic destructive arthritis in mice

Although regulatory T (Treg) cells are necessary to prevent autoimmune diseases, including arthritis, whether Treg cells can ameliorate established inflammatory disease is controversial. Using the glucose‐6‐phosphate isomerase (G6PI)‐induced arthritis model in mice, we aimed to determine the therapeutic efficacy of increasing Treg cell number and function during chronic destructive arthritis. Chronic destructive arthritis was induced by transient depletion of Treg cells prior to immunization with G6PI. At different time points after disease induction, mice were treated with a CD28 superagonistic antibody (CD28SA). CD28SA treatment during the induction phase of arthritis ameliorated the acute signs of arthritis and completely prevented the development of chronic destructive arthritis. CD28SA treatment of mice with fully developed arthritis induced a significant reduction in clinical and histological signs of arthritis. When given during the chronic destructive phase of arthritis, 56 days after disease induction, CD28SA treatment resulted in a modest reduction of clinical signs of arthritis and a reduction in histopathological signs of joint inflammation. Our data show that increasing the number and activation of Treg cells by a CD28SA is therapeutically effective in experimental arthritis.     

骨髓间质干细胞输注对外周血干细胞移植造血恢复的影响

目的:观察骨髓间质干细胞 (MSCs)输注对外周血干细胞移植 (PBSCT)后造血恢复的影响。方法:去脾小鼠经粒细胞集落刺激因子动员后收获外周血单个核细胞 (PBMC)和扩增培养的骨髓间质干细胞 (MSCs),移植给经放 /化疗预处理的BALB/c小鼠,数量分别为10⁶PBMC(PBSCT组)、10⁴MSCs和10⁶ PBMC(实验1组)、10⁶ MSCs和10⁶ PBMC(实验 2组),观察受体鼠 4周的生存率、骨髓有核细胞 (BMNC)、粒细胞巨噬细胞集落形成单位 (CFU-GM)、成纤维细胞集落形成单位 (CFU-F)、外周血白细胞 (WBC)计数等指标。结果:实验 2组的生存率、BMNC、CFU-GM、CFU-F显著高于PBSCT组,WBC计数恢复较PBSCT组快 (P <0.05);实验1组和PBSCT组比较,WBC计算恢复快,CFU-F产率高 (P <0.05)。结论:骨髓间质干细胞输注有促进外周血干细胞移植造血恢复的作用。     

High frequency of autoantibody-secreting cells and long-lived plasma cells within inflamed kidneys of NZB/W F1 lupus mice

Autoantibodies to double-stranded (ds) DNA represent a serological hallmark of systemic lupus erythematosus (SLE) and may critically contribute to the pathogenesis of lupus nephritis. Self-reactive antibodies might be partially produced by long-lived plasma cells (PCs), which mainly reside within the bone marrow and spleen. In contrast to short-lived PCs, long-lived PCs are extremely resistant to therapy and may sustain refractory disease courses. Recently, antibody-secreting cells were found within the inflamed kidneys of New Zealand black/white (NZB/W) F1 lupus mice as well as of patients with SLE. To analyze the longevity of the IgG-producing cells present in nephritic kidneys of NZB/W F1 mice we performed in vivo BrdU-labeling. We identified a higher frequency of long-lived than short-lived renal PCs, indicating that survival niches for long-lived PCs also exist within inflamed kidneys. Using ELISPOT assays, we found that on average 31% of renal IgG-producing cells reacted with dsDNA and 24% with nucleolin. Moreover, the frequencies of IgG-secreting cells specific for the autoantigens dsDNA and nucleolin were higher in the kidneys compared with those in the spleen and bone marrow.     

人骨髓源干细胞向胰岛素分泌细胞分化(英文)

目的：探讨人骨髓源干细胞向具有功能的胰岛素分泌细胞分化的可能性。方法：从人骨髓分离间充质干细胞。采用表皮生长因子、β-巯基乙醇和高糖培养基诱导其向胰岛素分泌细胞分化。经诱导后,用RT-PCR检测胰岛β细胞相关基因的表达,并采用免疫细胞化学染色检测胞浆胰岛素的表达。此外,诱导后细胞分泌的胰岛素定量及胰岛素释放实验将采用化学发光法进行检测。将经诱导后的细胞移植到糖尿病小鼠的右侧肾被膜下。在移植后16 d持续检测小鼠的血糖水平,最后对右侧肾脏进行免疫组化检测。结果：经诱导后,细胞能表达胰岛β细胞相关基因;免疫细胞化学染色也能检测到胞浆有胰岛素的表达;而且这些细胞能对糖刺激有所反应。细胞被移植到糖尿病小鼠的肾被膜下,能起降血糖作用。其肾脏的免疫组化显示：肾被膜下有胰岛素阳性细胞。结论：人骨髓源干细胞具有向胰岛素分泌细胞分化的潜能,这将为糖尿病细胞治疗提供丰富的细胞来源。     

TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells into the primary repertoire

Toll-like receptors (TLR) have been shown to play an essential role in the generation of autoantibodies in mouse models of autoimmunity, but the timing and context of these effects are poorly understood. One hypothesis is that TLR ligands assist in the positive selection of self-reactive B cells into the primary repertoire and, in this way, distinguish between immunogenic and tolerogenic forms of self-antigen. To explore this idea we generated hen egg lysozyme-specific immunoglobulin (Ig(HEL)) and isotype class-switching anti-HEL mice deficient in MyD88, TLR4 or TLR9 signalling and studied B cell development and autoantibody secretion in the presence or absence of an intracellular form of self-antigen HEL that positively selects B1 cells. Our findings show that TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells in the primary B cell repertoire, nor is MyD88 required for the generation of isotype-switched antibodies in the absence of antigen. These results suggest that the significant effects of TLR on autoimmunity occur in the established repertoire and not during B cell development.     

小鼠骨髓多能成体祖细胞的分离、培养及体外诱导分化为胰岛素分泌细胞的研究

目的：探讨培养骨髓多能成体祖细胞的条件和生物学特性及其向胰岛素分泌细胞分化的可能性。 方法： 采用文献报道的条件培养液培养小鼠骨髓多能成体祖细胞，观察其细胞形态、生长情况、细胞表面标志及mRNA水平，检测其oct-4基因表达，并以含胰高糖素多肽-1的无血清培养液诱导分化细胞，基因水平检测与胰岛素分泌细胞有关的基因表达。 结果： 小鼠骨髓多能成体祖细胞类圆形，细胞核大，胞质少；细胞增殖能力尚可；细胞表面CD13+、CD44-、CD45-、MHCⅡ-；有oct-4基因表达；诱导分化后，可见与胰岛素分泌细胞有关的基因表达。 结论： 成功培养小鼠骨髓多能成体祖细胞，具有与文献报道类似的生物学特征；有被诱导分化为胰岛素分泌细胞的可能性。     

人胚胎成纤维细胞对人胚胎干细胞生长的作用

目的：比较人和小鼠胚胎成纤维细胞对人胚胎干细胞生长的作用,为胚胎干细胞定向诱导各系统细胞应用于临床,消除异种蛋白污染打下基础。方法：分别采用人胚胎成纤维细胞和小鼠胚胎成纤维细胞为饲养层细胞,支持人受精卵的培养,观察其增殖和分化情况。结果：人和小鼠胚胎成纤维细胞分别加入白血病抑制因子（hLIF）均能很好支持人胚胎干细胞生长增殖,并保持未分化状态。结论：完全可以使用人胚胎成纤维细胞支持人胚胎干细胞增殖,消除异种蛋白污染的可能性,为胚胎干细胞定向诱导分化发育应用于临床打下坚实基础。     

Application of central immunologic concepts to cancer: Helping T cells and B cells become intolerant of tumors

CD4‐mediated T‐cell help in the activation of CD8⁺ T cells and B cells, through linked‐recognition of antigenic determinants, is a long‐standing concept foundational to our understanding of immunity (presence of help) versus tolerance (lack of help). Surprisingly, this function of CD4⁺ T cells has not been extensively examined as a means to overcome immune tolerance of the self‐antigens made by tumor cells. Hesitation to employ this powerful mechanism may be due to the potential to cause unwanted autoimmune pathology. In this issue of the European Journal of Immunology, Snook et al. [Eur. J. Immunol. 2014. 44: 1956–1966] identify a state of split tolerance, showing that CD4⁺ T cells specific for a number of tumor‐associated self‐antigens are robustly tolerant, while their CD8⁺ T‐cell and B‐cell counterparts are far less tolerant. Furthermore, the authors demonstrate that provision of linked foreign helper epitopes, such as influenza hemagglutinin, substantially enhances both CD8⁺ T‐cell and B‐cell responses to tumor self‐antigens without causing any overt autoimmune pathology. These findings provide a strong rationale to employ foreign helper epitopes in cancer vaccines and highlight the need to fully explore therapeutic strategies that are based on well‐established immunologic concepts.     

猴表皮干细胞的分离和培养

目的： 探索猴表皮干细胞分离纯化和培养方法。 方法: 将猴皮肤标本剪成皮条,加入0.25％胰酶浸泡过夜；然后去除角质层，刮上皮面获取表皮细胞进行培养。取第2-4代的细胞,经消化后，用Ⅳ型胶原黏附法获得为快吸附的表皮细胞。对快吸附的表皮细胞行流式细胞仪、免疫组化和RT-PCR检测。 结果: 快吸附细胞从mRNA转录水平及蛋白表达水平均呈现表皮干细胞的特性：β1整合素、K15和α6整合素阳性，而CD71和K1/K10阴性。 结论: 所采用的分离纯化方法和培养条件适合猴表皮干细胞的分离纯化及生长。