B7基因转染联合LAK细胞治疗肝癌的体外研究

目的：探讨体外诱导的LAK细胞对B₇基因转染的肝癌细胞的杀伤作用。方法：构建含人B_7-1基因cDNA的真核表达载体pRCCMV-B_7-1,脂质体介导转染法将pRCCMV-B_7-1转入人肝癌细胞系SMMC7721,G418筛选转染肝癌细胞（B₇⁺SMMC7721）。RT-PCR、流式细胞仪鉴定目的基因表达。MTT比色试验体外检测LAK细胞对B_7-1转染肝癌细胞的杀伤活性。结果：B₇⁺SMMC7721细胞稳定表达B₇基因。LAK细胞对B₇⁺SMMC7721细胞的杀伤活性明显高于野生型SMMC7721者,结果有统计学意义。结论：B₇基因体外转染人肝癌细胞后可表达有活性的B₇分子,LAK细胞对表达B_7-1的肿瘤呈现增强的杀伤作用。     

卡介菌多糖核酸对肺癌细胞粘附及骨架结构的影响

目的　研究卡介苗多糖核酸 (BCG PSN)对肺癌细胞上粘附分子配体 (slex)表达及肺癌细胞质内骨架的表现特征的影响。方法　采用流式细胞仪及透射电镜观察BCG PSN对高转移人肺巨细胞癌 (PG)细胞和低转移人肺腺癌 (PAa)细胞上slex 表达的影响及胞质内骨架结构的变化。结果　①流式细胞仪分析提示PAa细胞slex 表达高 ( 66.8% ) ,PG细胞表达低 ( 5 .72 % )。经BCG PSN处理后 ,PAa细胞slex 表达呈浓度依赖下降。②超微结构观察 :PAa细胞微丝稀疏 ,PG细胞微管微丝减缺。BCG PSN处理后 ,PG细胞微丝较前丰富、完整 ,树样分布 ,PAa细胞仅见少量的微丝。结论　细胞内微管、微丝等细胞骨架成分的改变与癌细胞运动迁徙能力有关 ,BCG PSN对肺癌细胞粘附性的抑制环节不是细胞骨架介导的粘附强化 ,可能是粘附分子表达下降所致的粘附强化启动过程。     

芳维A酸乙酯对HL-60细胞CD11b、CD15表达的影响

目的观察芳维A酸乙酯(arotinoid ethylester, AE)在HL-60细胞分化成熟过程中对其CD11b、CD15表达的影响,初步探讨AE治疗脓疱型银屑病的可能机制.方法 HL-60细胞以二甲基亚砜(dimethylsulfoxide, DMSO)、全反式维A酸(all-trans-retinoic acid, ATRA)、AE诱导培养,5 d后用流式细胞仪检测HL-60细胞表面CD11b和CD15的表达.结果 HL-60细胞经DMSO、ATRA、AE诱导后CD11b和CD15荧光标记率均比诱导前提高;ATRA组HL-60细胞CD11b和CD15荧光强度显著低于DMSO组,AE组HL-60细胞CD11b和CD15荧光强度显著低于ATRA组.结论 AE可明显抑制HL-60细胞表达CD11b、CD15;AE可能通过抑制参与炎症反应的中性粒细胞表达CD11b、CD15而降低中性粒细胞参与炎症反应的过程.     

Effect of siRNA mediated suppression of signaling lymphocyte activation molecule on replication of peste des petits ruminants virus in vitro

Signaling lymphocyte activation molecule (SLAM) expression was inhibited in B95a cell line using siRNA and the effect of SLAM inhibition on peste des petits ruminants virus (PPRV) replication and infectivity titre was studied. SLAM suppression was assessed using real-time PCR and flow cytometry to confirm suppression at the m-RNA and protein levels, respectively. Three chemically synthesized siRNAs were transfected individually using oligofectamine into B95a cell line. This resulted in SLAM suppression from 48 to 454-folds, in comparison to the untransfected B95a cell line. When the SLAM suppressed B95a cell line was infected with PPRV, replication was reduced by 12-143-folds and virus titre was reduced from log10 1.09 to 2.28. siRNA 3 showed the most potent inhibition of SLAM expression both at m-RNA and protein levels. This also caused the maximum reduction of virus replication and virus titre. A 100-fold reduction in PPRV titres was seen in anti-SLAM antibody neutralized B95a cell line. This further confirms that SLAM is one of the (co) receptors for PPRV. However, the presence of other putative virus receptor(s) is/are not ruled out.     

Selectins and selectin ligands in extravasation of cancer cells and organ selectivity of metastasis

Metastatic spreading is a dreadful complication of neoplastic diseases that is responsible for most deaths due to cancer. It consists in the formation of secondary neoplasms from cancer cells that have detached from the primary site. The formation of these secondary sites is not random and several clinical observations indicate that the metastatic colonization exhibits organ selectivity. This organ tropism relies mostly on the complementary adhesive interactions between the cancer cells and their microenvironment. In particular, several lines of evidence suggest that the organ selectivity of colon cancer cells for the liver involves the binding of the circulating cancer cells to endothelial E-selectin. The aim of this review is to make an integrative up-date of the mechanisms that govern the organ selectivity of the metastatic process focusing more especially on the role of selectins and selectin ligands.     

Cell-cell interaction modulates neuroectodermal specification of embryonic stem cells

The controlled differentiation of embryonic stem (ES) cells is of utmost interest to their clinical, biotechnological, and basic science use. Many investigators have combinatorially assessed the role of specific soluble factors and extracellular matrices in guiding ES cell fate, yet the interaction between neighboring cells in these heterogeneous cultures has been poorly defined due to a lack of conventional tools to specifically uncouple these variables. Herein, we explored the role of cell-cell interactions during neuroectodermal specification of ES cells using a microfabricated cell pair array. We tracked differentiation events in situ, using an ES cell line expressing green fluorescent protein (GFP) under the regulation of the Sox1 gene promoter, an early marker of neuroectodermal germ cell commitment in the adult forebrain. We observed that a previously specified Sox1-GFP+ cell could induce the specification of an undifferentiated ES cell. This induction was modulated by the two cells being in contact and was dependent on the age of previously specified cell prior to coculture. A screen of candidate cell adhesion molecules revealed that the expression of connexin (Cx)-43 correlated with the age-dependent effect of cell contact in cell pair experiments. ES cells deficient in Cx-43 showed aberrant neuroectodermal specification and lineage commitment, highlighting the importance of gap junctional signaling in the development of this germ layer. Moreover, this study demonstrates the integration of microscale culture techniques to explore the biology of ES cells and gain insight into relevant developmental processes otherwise undefined due to bulk culture methods.     

Interferon-gamma limits Th1 lymphocyte adhesion to inflamed endothelium: a nitric oxide regulatory feedback mechanism

CD4(+) T helper (Th1 and Th2) cell localization to a site of inflammation is important for the development, maintenance and regulation of an immune response. The factors that regulate Th1 and Th2 cell recruitment into tissue are not fully understood. The aim of the present study was to examine the effect of different cytokine microenvironments on the recruitment of Th1 and Th2 lymphocytes into tissue. Fluorescently labelled Th1 or Th2 lymphocyte-endothelial interactions were observed via intravital microscopy of the cytokine-treated cremaster muscle. Our results show that TNF-alpha alone is sufficient to maximally recruit Th1 cells. Surprisingly, treatment with TNF-alpha + IFN-gamma significantly decreased Th1 adhesion and emigration in comparison to TNF-alpha treatment alone. The decreased adhesion of Th1 cells in response to TNF-alpha + IFN-gamma reflected a decreased ability to bind to ICAM-1 and was iNOS-dependent. This phenomenon was not observed with Th2 cells. These results suggest that IFN-gamma may play a key immunomodulatory role in the recruitment of different T lymphocyte subsets. Indeed, blockade of IFN-gamma or iNOS function during the Th1-mediated contact hypersensitivity response resulted in an acceleration and exacerbation of the late-phase inflammatory response.     

Neutralizing antibodies decrease the envelope fluidity of HIV-1

For successful penetration of HIV-1, the formation of a fusion pore may be required in order to accumulate critical numbers of fusion-activated gp41 with the help of fluidization of the plasma membrane and viral envelope. An increase in temperature to 40 degrees C after viral adsorption at 25 degrees C enhanced the infectivity by 1.4-fold. The enhanced infectivity was inhibited by an anti-CXCR4 peptide, T140, and anti-V3 monoclonal antibodies (0.5beta and 694/98-D) by post-attachment neutralization, but not by non-neutralizing antibodies (670-30D and 246-D) specific for the C5 of gp120 and cluster I of gp41, respectively. Anti-HLA-II and an anti-HTLV-I gp46 antibody, LAT27, neutralized the molecule-carrying HIV-1(C-2(MT-2)). The anti-V3 antibodies suppressed the fluidity of the HIV-1(C-2) envelope, whereas the non-neutralizing antibodies did not. The anti-HLA-II antibody decreased the envelope fluidity of HIV-1(C-2(MT-2)), but not that of HIV-1(C-2). Therefore, fluidity suppression by these antibodies represents an important neutralization mechanism, in addition to inhibition of viral attachment.     

Trophoblasts and shear stress induce an asymmetric distribution of icam-1 in uterine endothelial cells

PROBLEM: We have used an in vitro co-culture system consisting of early gestation macaque trophoblasts cultured on top of human uterine microvascular endothelial cells (UtMVECs) to investigate the inflammatory response of endothelial cells to trophoblasts under shear stress conditions. METHOD: of study Uterine microvascular endothelial cells and trophoblasts were co-cultured in a parallel plate chamber under shear stress (15 dyn/cm(2)) conditions. The distribution and expression of endothelial intercellular adhesion molecule-1 (ICAM-1) was quantified by immunofluorescence image analysis and flow cytometry. Endothelial regulated upon activation normal T-cell expressed and secreted (RANTES) secretion was measured by enzyme-linked immunosorbent assay and permeability was assessed using fluorescein isothiocyanate-dextran. RESULTS: Intercellular adhesion molecule-1, but not vascular cell adhesion molecule-1 or platelet endothelial cell adhesion molecule-1, was re-distributed towards the downstream edge of endothelial cells when the cells were co-cultured with trophoblasts under shear stress conditions. Changes in ICAM-1 distribution were also observed when UtMVECs were co-cultured with trophoblast-conditioned medium under shear stress conditions. Incubation of UtMVECs with trophoblast-conditioned medium increased endothelial permeability, RANTES secretion, and trophoblast adhesion. CONCLUSION: These data support the idea that trophoblasts induce an inflammatory response in uterine endothelial cells that could enhance trophoblast invasion and transmigration.     

CD56+ cells are recruited to the uterus in two waves: at ovulation and during the first 2 weeks after missed menses

PROBLEM: Uterine natural killer (uNK) cells are enriched in the post-ovulatory uterus and during pregnancy. Whether these cells arise from blood pre-cursors or from stem cells in the uterus is undefined. To support a hypothesis that precursors of uNK cells are recruited from blood, adhesive function of blood CD56+ subsets were assessed during one cycle and during pregnancy. METHOD OF STUDY: Fifteen women of proven fertility provided serial blood samples during one menstrual cycle and thirty women with a history of implantation failure or recurrent spontaneous abortion provided serial samples during infertility treatment. RESULTS: CD56(bright) cells, but not CD56(dim) cells or NKT cells, increased in ligand-binding capacity during ovulation in fertile cycles only and during the first 2 weeks from date of missed menses. CONCLUSION: Enhanced adhesive function at ovulation in CD56(bright) cells in fertile cycles and during early gestation supports a hypothesis of recruitment of pre-uNK cells from the blood CD56(bright) subset.