Langerhans'' cells and T cells in human skin tumours: an immunohistological study

In this study normal skin and a range of skin tumours, both benign and malignant, have been examined using monoclonal antibodies to identify the distribution and morphology of Langerhans' cells and T cells, the distribution of T lymphocytes and their subsets have been analysed using monoclonal anti-T cell antibodies. The results indicated that Langerhans' cells can be reliably identified in both normal and malignant skin biopsies using monoclonal antibodies. A striking finding to emerge was that in benign skin lesions Langerhans' cells were increased, whereas in malignant tumours they were not only markedly depleted or absent but also grossly stunted and deformed in outline. The majority of lymphocytes surrounding these skin tumours were shown to be T cells with helper cells outnumbering suppressor cells by a ratio from 2 to 5:1. This study shows the usefulness of immunohistological techniques using monoclonal antibodies for examining the morphology and distribution of Langerhans' cells in skin pathology. In addition they are particularly appropriate for identifying their topographical relationships with other immunologically important cells such as T cells.     

The effects of adherent cells on measurement of the hyper-responsiveness of rheumatoid B lymphocytes to Epstein-Barr virus

Rheumatoid peripheral blood mononuclear cells show an increased responsiveness to superinfection with Epstein-Barr virus (EBV). We have investigated the role of adherent cells in this hyperresponsiveness using two different methods of adherent cell depletion. Depletion of adherent cells from both rheumatoid and normal mononuclear cells, using either dextran bead columns or plastic petri dishes, produced inconsistent changes in the response of autologous non-adherent cells to EBV. The addition of supernatants of cultured rheumatoid adherent cells also produced an inconsistent change in response although normal adherent cell supernatants increased the responsiveness of autologous non-adherent cells to EBV. The inconsistencies observed are discussed with respect to adherent cell subpopulations present in rheumatoid and normal peripheral blood mononuclear cell preparations when using recognized methods of adherent cell depletion.     

Allogeneic intrabone marrow-bone marrow transplantation plus donor lymphocyte infusion suppresses growth of colon cancer cells implanted in skin and liver of rats

We have recently found that allogeneic intrabone marrow-bone marrow transplantation (IBM-BMT) + donor lymphocyte infusion (DLI) using CD4(+) cell-depleted spleen cells (CD4(-) cells) can prevent graft-versus-host disease (GvHD) but suppress tumor growth (Meth A: fibrosarcoma) in mice. In the present study, we show that allogeneic IBM-BMT + DLI using CD4(-) cells also has suppressive effects on the growth of colon cancer cells implanted not only in the skin but also in the liver of rats. First, we examined the effects of allogeneic IBM-BMT + DLI on the subcutaneously inoculated ACL-15 (rat colon cancer cell line). Lethally irradiated Fischer rats (F344 rats) were transplanted with T-cell-depleted bone marrow cells (BMCs) from Brown Norway (BN) rats. Simultaneously, DLI was performed using whole spleen cells (whole cells), CD4(+) cell-depleted spleen cells (CD4(-) cells) or CD8(+) cell-depleted spleen cells (CD8(-) cells) of BN rats. Although allogeneic IBM-BMT + DLI suppressed tumor growth, a considerable number of rats treated with allogeneic IBM-BMT + DLI using whole cells or CD8(-) cells died due to GvHD. In contrast, allogeneic IBM-BMT + DLI using CD4(-) cells also suppressed tumor growth, but there was no GvHD. Based on these findings, we next examined the effects of allogeneic IBM-BMT + DLI using CD4(-) cells on the cancer cells implanted in the liver. Allogeneic IBM-BMT + DLI using CD4(-) cells via the portal vein significantly prolonged the survival. These results suggest that allogeneic IBM-BMT + DLI using CD4(-) cells could become a new strategy for the treatment of solid tumors.     

Adhesion of Subsets of Human Blood Mononuclear Cells to Endothelial Cells In Vitro, as Quantified by Flow Cytometry

Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.     

联合应用生长因子促骨髓间充质干细胞向韧带样细胞分化

背景：利用生长因子诱导骨髓间充质干细胞定向分化成韧带样细胞并促其分泌胶原蛋白是构建组织工程化韧带的关键。生长因子碱性成纤维细胞生长因子和转化生长因子β1在细胞定向分化过程中都起着十分重要的作用。 目的：采用转化生长因子β1和碱性成纤维细胞生长因子诱导体外培养的兔骨髓间质干细胞,转化为韧带样细胞,并研究此种韧带样细胞的生物特性。 方法：自幼兔四肢骨抽取骨髓分离纯化骨髓间充质干细胞并培养、增殖。采用10 μg/L转化生长因子β1和25 μg/L碱性成纤维细胞生长因子对骨髓间质干细胞进行诱导分化,分为空白组、转化生长因子β1组、碱性成纤维细胞生长因子组、转化生长因子β1+碱性成纤维细胞生长因子组。观察生长因子对骨髓间质干细胞生长、形态的影响,使用MTT法绘制细胞生长曲线,使用天狼腥红染色法定量对比骨髓间质干细胞分泌胶原蛋白量。把转化生长因子β1+碱性成纤维细胞生长因子联合诱导的骨髓间质干细胞使用BrdU荧光染色标记,然后移植到脱细胞真皮基质上,观察细胞增殖分布情况,并与空白组对照。 结果与结论：转化生长因子β1+碱性成纤维细胞生长因子联合组,细胞形态优于空白组及单一因子组,细胞增殖率、胶原分泌量也较高。脱细胞真皮基质上,转化生长因子β1+碱性成纤维细胞生长因子组细胞增殖分布情况明显优于空白组。提示联合使用生长因子转化生长因子β1和碱性成纤维细胞生长因子刺激兔骨髓间质干细胞,能够促使兔骨髓间质干细胞定向转化为韧带样细胞,对组织工程前交叉韧带的构建具有积极意义。     

脂肪干细胞与Bio-oss支架构建组织工程骨治疗种植体周围骨缺损

背景：目前治疗种植体周围炎的方法主要有手术治疗、激光治疗、超声洁治加局部用药单独或联合治疗等,但并未完全达到理想的治疗效果。 目的：探讨由骨形态发生蛋白修饰的脂肪干细胞与Bio-oss支架构建组织工程骨治疗种植体周围炎性骨缺损的可行性。 方法：应用计算机检索1988年1月至2011年12月Pubmed数据库相关文章,检索词为“adipose-derived stem cells、tissue engineering”,并限定文章语言种类为英文。同时计算机检索2005年1月至2011年12月CNKI数据库相关文章,检索词为“脂肪干细胞、种植体周围炎”,并限定文章语言种类为中文。共检索到文献56篇。 结果与结论：利用组织工程原理与方法再生组织的特点和优势,将脂肪干细胞作为理想的种子细胞,骨形态发生蛋白2作为理想的细胞因子与Bio-oss生物材料混合,构成三维环境,Bio-oss晶体内广泛交织的空隙结构,有利于脂肪干细胞的迁移。骨形态发生蛋白2与脂肪干细胞复合,促进控制其分化向骨形成中心募集,并分化为骨系细胞,通过钙盐沉积形成新骨,达到治疗种植体周围骨缺损的目的。     

Improved detection of rare CALLA-positive cells in peripheral blood using multiparameter flow cytometry

A major limitation to the detection of rare cell types in the peripheral blood using monoclonal antibodies is nonspecific binding of the antibody reagent to normal cells. Detection of rare common acute lymphoblastic leukemia antigen (CALLA)-positive cells in peripheral blood is significantly improved by using multiple flow cytometric parameters to exclude a variety of mature blood cells which may nonspecifically bind the antibody reagent. Monocytes and granulocytes are excluded by gating out cells with high 90 degrees light scatter. By gating on red fluorescence, a variety of mature cell types binding to phycoerythrin (PE)-conjugated Leu 3, Leu 2, and M3 monoclonal antibodies are also excluded. CALLA-positive lymphoblasts from 6 consecutive patients were not excluded on the basis of these parameters. Gating on log 90 degrees light scatter and log red fluorescence in this fashion reduced the incidence of nonspecific binding to peripheral blood mononuclear cells of a fluorescein-conjugated irrelevant monoclonal antibody by 98% from 308 cells per million to 5 cells per million. One CALLA-positive lymphoblast per 100,000 peripheral blood mononuclear cells could be detected in mixture experiments using this method. The normal range of CALLA-positive cells in adults is less than 16 cells per million peripheral blood mononuclear cells. This low background of CALLA-positive peripheral blood cells may permit the detection of early leukemic relapse in acute lymphoblastic leukemia by analysis of the peripheral blood. This methodology can be applied to the detection of any rare cell type by using phycoerythrin-conjugated antibodies to markers that the cell type does not possess.     

Difference in susceptibility to morphological changes in the nucleus to aneugens between p53-competent and p53-abrogated lymphoblastoid cell lines (TK6 and NH32 cells) in the in vitro micronucleus assay

We previously reported that the proportion of large-size micronuclei (MN) can be a reliable parameter to discriminate aneugens from clastogens in the in vitro MN assay using Chinese hamster lung cells. The frequencies of polynuclear (PN) and mitotic (M) cells are also supposed to be useful parameters for the same purpose since they are known to be increased by aneugens. In the present study, we investigated whether morphological observations of the cell nucleus can be applied for the in vitro MN assay using the p53-competent human lymphoblastoid cell line, TK6 cells. Our present MN assay with six clastogens and six aneugens revealed that the frequencies of large-size MN or PN cells cannot distinguish aneugens from clastogens, while the frequencies of M cells can distinguish them, suggesting that the M-cell frequency is a recommended parameter to determine a mode of action for MN induction in the in vitro MN assay using TK6 cells. Our further investigation using p53-null mutant NH32 cells showed that the frequencies of large-size MN or PN cells induced by aneugen treatments were higher than those in TK6 cells but not by clastogen treatments. These findings suggest that p53 abrogation promotes the susceptibility for morphological changes in the nucleus to aneugens and that morphological observation of the cell nucleus including size-classifying MN counting could distinguish aneugens from clastogens in the MN assay using NH32 cells.     

Production of human T cell growth factor

We provide here a protocol for production of T cell growth factor (TCGF) using cells isolated from defibrinated blood. Whether combined in allogeneic pools or tested as single donors, these cells consistently yield high activity TCGF, following PHA stimulation. Protocols using cells isolated from heparinized blood have often included addition of indomethacin or removal of adherent cells, or both, to overcome the problem of frequent nonproducing cultures. Our failure to find nonproducing cultures using this source of cells suggests that inhibitory cells or factors are removed by the blood clot formed during defibrination.

A distincteconomic advantage is gained by using these cells, not only because of the reliability of obtaining active supernatants, but also because the same blood can be used for preparation of a serum pool for cell cultures and the erythrocytes are useful for absorption of contaminating PHA present in the TCGF-containing media. Not only can defibrinated blood leukocytes be stimulated by PHA to release TCGF, but when cultured in allogeneic mixtures containing no PHA, they also release active TCGF.     

Separation of Lipase-Positive Cells from Suspensions of Pancreas Cells in an Isokinetic Gradient of Ficoll in Tissue Culture Medium

Lipase-positive cells were separated from suspensions of pancreas cells both by velocity sedimentation and by isopycnic sedimentation. Lipase-positive cells were 51.0 ± 9.6% of the disaggregated pancreas cells in the starting sample suspension. The purest gradient fractions from experiments using velocity sedimentation for cell separation contained a mean of 98.8 ± 0.6% lipase-positive cells. Cell separation using isopycnic centrifugation was less effective than cell separation using velocity sedimentation and resulted in a mean purity of only 89.8 ± 6.8% lipase-positive cells. Lipase positivity was assessed using the Gomori technic with the conditions for fixation slightly modified to be suitable for disaggregated cells.     

小鼠附睾脂肪干细胞的分离培养及鉴定

背景：脂肪干细胞作为一种新的成体干细胞,具有来源丰富、取材容易、增殖能力强等优点,受到人们越来越多地关注。 目的：体外分离培养小鼠附睾脂肪组织中的脂肪干细胞,并鉴定其生物学特性。 方法：无菌切取小鼠附睾处脂肪组织,采用胶原酶进行消化,利用1次消化多次收集与差速贴壁法分离纯化脂肪干细胞。倒置显微镜及透射电子显微镜观察脂肪干细胞的形态。绘制脂肪干细胞的生长曲线。流式细胞术检测脂肪干细胞的免疫表型。加入细胞诱导剂对脂肪干细胞进行成骨及成脂肪的诱导分化。扫描电子显微镜观察脂肪干细胞与胶原支架材料的相容性。 结果与结论：倒置显微镜下可见脂肪干细胞呈长梭形或成纤维细胞样,密集排列成漩涡状或成束状交织,可在体外稳定传至第9代。透射电子显微镜下可见脂肪干细胞表面有丰富的微绒毛结构,细胞核体积大,细胞质内可见线粒体、粗面内质网等细胞器。脂肪干细胞表达CD44、CD29,不表达CD34。经成脂肪诱导剂诱导后,多数脂肪干细胞胞质内可见小脂滴,油红O染色可将小脂滴染成红色。经成骨诱导剂诱导后,在脂肪干细胞生长密集区域内的细胞结构模糊、细胞间界限不清楚。经茜素红染色后,可见较多大小不一的折光率较强颗粒状结构沉积。扫描电子显微镜观察到脂肪干细胞呈铺展状生长于胶原支架材料上。结果表明该方法分离出的脂肪干细胞能在体外扩增并稳定传代,在一定诱导条件下,能够分化为成骨细胞和脂肪细胞并且与胶原支架具有良好的相容性。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Detection of monocyte/macrophage cell populations in effusions: a comparative study using flow cytometric immunophenotyping and immunocytochemistry

The objective of the present study was to compare the efficiency of immunophenotyping using flow cytometry (FCM) and immunocytochemistry (ICC) in the detection of macrophages in serous effusions. Cytoblock sections from 90 effusions were stained for the monocyte/macrophage marker CD14, using ICC. Fresh-frozen samples of all cases were analyzed for CD14 expression, using FCM. Epithelial, lymphoid, and mesothelial cell populations were identified using antibodies against Ber-EP4, CD45, and N-cadherin, respectively. Results were compared with clinical parameters and morphological diagnosis. Thirty-nine specimens were cytologically diagnosed as malignant, containing tumor cells of nonhematologic origin, whereas 46 were interpreted as benign. Two additional specimens were diagnosed as indeterminate or suspicious for malignancy, and 3 specimens contained lymphoma cells. CD14-positive cells were detected in 85/90 (94%) of effusions using FCM, and in all 90 specimens using ICC. The percentage of CD14-positive cells was highly variable, but in some specimens was as high as 76% using FCM and 85% using ICC. A good association was observed between the two methods in the detection of CD14-positive cells (P < 0.001). The presence of macrophages in effusions showed an association with female gender, using both FCM (P = 0.002) and ICC (P = 0.011), but none with effusion site, patient age, clinical and cytological diagnosis, or presence of Ber-EP4-positive cells (P > 0.05). The presence of Ber-EP4-positive cells showed a strong association with the cytological diagnosis of malignancy (P < 0.001). In conclusion, macrophages are a significant cell population in effusions, of both benign and malignant etiology, due to both their size and their possible confusion with cancer cells. Both FCM and ICC aid in the recognition of these cells, and thus provide an effective tool for the identification of different cell populations in effusions.     

Immunofluorescence technique using HeLa cells expressing recombinant nucleoprotein for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.     

Let-7维持乳腺癌干细胞的特性及机制的研究

目的:研究let-7在乳腺癌干细胞中的表达,探索let-7影响乳腺癌干细胞的特性及机制。方法:采用SP分选法,分选乳腺癌细胞系MCF-7中的SP及NSP细胞亚群;运用实时定量PCR法检测MCF-7细胞系中SP、NSP亚群let-7a/b/c的表达;通过Western blot法检测Ras、ERK蛋白在MCF-7细胞系中SP、NSP亚群中的表达,探索let-7维持乳腺癌干细胞特性的机制。结果:SP侧群细胞占MCF-7细胞的3.3%,加入维拉帕米抑制干细胞外排荧光染料Hoechst33342的功能后,SP侧群细胞占乳腺癌MCF-7细胞的比例下降至0.4%。Let-7miRNA在SP细胞中的表达低于NSP细胞,其中let-7b/c在SP及NSP亚群中表达差异最大;p-Ras、p-ERK在SP细胞中的表达高于NSP细胞,t-Ras及t-ERK的表达无明显差异。结论:SP法是一种可有效分离干细胞的方法,乳腺癌细胞系MCF-7中存在肿瘤干细胞亚群;let-7在乳腺癌干细胞中的表达低于普通肿瘤细胞,而p-Ras、p-ERK在乳腺癌干细胞中的表达高于普通肿瘤细胞,let-7的低表达失去了对Ras mRNA的抑制,使Ras信号转导通路激活,进而磷酸化的p-Ras和p-ERK表达升高,维持乳腺癌干细胞的功能。     

Increased smooth muscle actin expression from bone marrow stromal cells under retinoic acid treatment: an attempt for autologous blood vessel tissue engineering

Vascular replacement in vital organs is sometimes necessary for human life for example because of atherosclerosis. Blood vessel tissue engineering is applied for autologous transplantations to avoid graft rejections. Stem cells are used for blood vessel tissue engineering because they are the origin of smooth muscle cells, endothelial cells and fibroblasts. This paper shows that bone marrow stromal cells (BMSCs) can be induced to differentiate into the early stage of smooth muscle cells by using 0.01 microM retinoic acid. The differentiation of BMSCs to smooth muscle cells was detected by the expression of smooth muscle alpha actin (SM alpha-actin), the earliest smooth muscle cell marker. The SM alpha-actin marker expression was demonstrated using indirect immunofluorescence technique and Western blot analysis. The induction of BMSC to form early stages of smooth muscle cells in this study is appropriate for blood vessel tissue engineering because the early stage smooth muscle cells may be stimulated to develop vascular walls with endothelial cells using a co-culture system.     

Evaluation of an exposure system using cells grown on collagen gels for detecting highly volatile mutagens in the CHO/HGPRT mutation assay

Chinese hamster ovary (CHO) cells were grown on hydrated collagen gels, the overlaying medium removed leaving the cells at an air/collagen interface, and the cells exposed to a dynamic flow of ethylene oxide. Increases in CHO cell mutant frequency and decreases in cell viability were observed. To establish if the exposure system could be simplified, cells were exposed in sealed bottles (static system) to ethylene oxide. No substantial changes in cytotoxicity, mutant frequency, or effective concentration were noted when comparing static versus dynamic exposure systems. The general usefulness of the exposure system using cells grown on collagen gels was evaluated in a static system using propylene oxide and 1,2-dichloroethane, both of which were found to be mutagenic and cytotoxic. Comparatively, the exposure of cells by the collagen gel method was as effective in detecting genotoxic damage as were conventional methods (cells covered with medium) using cells grown on glass substrates. The exposure of CHO cells on collagen gels to highly volatile mutagens was simple and inexpensive, and may be generally useful in the detection of gaseous or volatile mutagens.     

不同培养方法对人脐带间充质干细胞的影响

BACKGROUND:Umbilical cord stem cells mainly derive from full-term infants, and common culture methods include tissue-attached method and trypsin-digestion mehod. However, effects of different culture methods on the separation of umbilical cord mesenchymal stem cells remain many disputes. OBJECTIVE:To observe the effects of different culture methods on umbilical cord mesenchymal stem cells. METHODS:Umbilical cords of 30 healthy full-term and caesarean delivery infants were selected, and cultured using tissue-attached method or trypsin-digestion method to isloate and culture human umbilical cord mesenchymal stem cells. Meanwhile, cell growth was measured by flow cytometry. RESULTS AND CONCLUSION:The fusiform-shaped cells began to separate from the umbilical cord tissue that was primary cultured using tissue-attached method, and 10 days later, the cell fusion reached 80%; after the umbilical cord was cultured using collagenase-trypsin digestion for 5 days, a small amount of adherent cells with different shapes appeared, and the fiber-like cells reached 80% of confluence until 2-week culture. There was no significant difference in the growth of umbilical cord mesenchymal stem cells cultured by different culture methods (P > 0.05). Moreover, cells cultured by two methods were all positive for CD13, CD29, CD44 and CD105. These results demonstrate that human umbilical cord mesenchymal stem cells exhibit a high success rate in primary culture using tissue-attached method, which is superior to the trypsin-digestion method.     

Characterization of an idiotype-binding helper cell required for the formation of timothy grass pollen antigen-B-specific IgE

Experiments were conducted using an excess of biosynthetically labeled timothy grass pollen antigen-B-specific (AgB-specific) T helper factor (THF) with enriched T cells, enriched B cells, or normal spleen cells to determine the site of the THF action. These studies indicated that the labeled THF bound preferentially to T cells, and analysis of the cell surface characteristics suggested that these T cells were Lyt 123+ cells. A panning technique using partially purified AgB-specific THF-coated Petri dishes depleted a population of idiotype-binding T cells that appears to be required for the formation of AgB-specific IgE antibody.     

TUNEL法与MTT法比较分析检测肿瘤细胞药敏

本文用细胞凋亡法 (TUNEL法 )和MTT法在体外对白血病细胞株细胞、肝癌腹水中肿瘤细胞及实体瘤中分离出的肿瘤细胞对 8种化疗药物的敏感性进行了检测。对白血病细胞株 (HL 6 0与Molt4) ,TUNEL法和MTT法所测得的结果高度一致 ;对肝癌腹水中肿瘤细胞的药敏结果表明两法所得结果基本一致 ,而对实体瘤中分离出的肿瘤细胞同时用两法检测药敏的实验结果有较大差异 ,统计分析表明TUNEL法优于MTT法。提示在细胞高度均一时 (如白血病细胞株细胞 )TUNEL法和MTT法均可准确反映药敏情况 ,而随着所分离的肿瘤细胞纯度降低 ,MTT法难以准确反映药敏实验结果。表明TUNEL法由于可直接在荧光显微镜下观察发生凋亡的肿瘤细胞 ,因此可减少混杂的组织细胞对结果的干扰。