大鼠脑胶质瘤干细胞分离及作为树突状细胞治疗抗原来源的研究

脑胶质瘤是原发性恶性脑肿瘤中最常见的一类肿瘤，其具有较差的预后性和高致死率。尽管包括手术、化疗及放疗在内的综合治疗水平在不断提高，但由于胶质瘤细胞的高度侵袭性导致治疗结果仍未得到明显改善，病人常常在治疗后很短时间内发生复发。近年来在肿瘤发生的领域提出了一个新的理论，即肿瘤干细胞理论。这个理论认为，肿瘤干细胞是肿瘤中具有自我更新、无限增殖及分化潜能的细胞，这部分细胞虽然只占少部分，但却是肿瘤发生、发展的关键。本研究主要探讨了利用化疗药物秋水仙素分离大鼠胶质瘤细胞中的干细胞样肿瘤细胞（Stem-like Cancer Cell, SLCC）的可行性，负载胶质瘤SLCC的树突状细胞疫苗在体外对肿瘤细胞的杀伤能力，以及对秋水仙素处理胶质瘤细胞的基因组不稳定性的初步探索。 C6大鼠胶质瘤细胞经不同剂量水平（0、0.5、1.0、2.0μg/ml）的秋水仙素处理后，大部分细胞发生凋亡，剩下存活的细胞经过免疫荧光染色发现其表达干细胞标志分子Nestin，提示可能是SLCC。流式和RT-PCR实验表明，经过秋水仙素处理后的存活的C6细胞表达干细胞标志分子nestin，sox2和bmi1，并且其数量随着秋水仙素浓度的增加而增加。 树突状疫苗是一种脑肿瘤的生物治疗方法。本实验通过分离大鼠的树突状细胞和淋巴细胞，体外培养并用经不同剂量组处理分离的SLCC全蛋白刺激一周后，使用MTT法检测激活的细胞毒T淋巴细胞（cytotoxic T lymphocyte，CTL）体外对肿瘤细胞的杀伤效果。结果表明，经药物分离的SLCC组诱导的CTL效果明显大于对照组，提示脑胶质瘤SLCC可以作为树突状疫苗免疫治疗的新靶点。 此外RT-PCR证明经秋水仙素处理后的胶质瘤细胞中mad2水平明显升高，表明化疗药物可能增加了肿瘤细胞中基因组不稳定性，从而导致SLCC的聚集。 综上，本实验证明了利用化疗药物秋水仙素可以分离大鼠胶质瘤细胞C6中的SLCC细胞，且该分离可能与肿瘤细胞基因组不稳定性提高有关。同时，利用负载SLCC全蛋白的树突状细胞激活的T淋巴细胞在体外具备比对照组更强的细胞杀伤能力。这些结果为以脑胶质瘤SLCC最为靶点的免疫治疗方法开发提供了基础依据。

Indole and carbazole alkaloids from Glycosmis montana with weak anti-HIV and cytotoxic activities

A diprenylated indole, (E)-3-(3-hydroxymethyl-2-butenyl)-7-(3-methyl-2-butenyl)-1H-indole (1), and six known carbazole alkaloids were isolated from the twigs and leaves of Glycosmis montana Pierre (Rutaceae). Their structures were determined on the basis of analysis of spectral evidence including 1D and 2D NMR and MS. The alkaloids (1-3) exhibited weak to moderate take in vitro inhibitory activity against HIV replication in C8166 cells, and they (as well as carbalexine A and B) had cytotoxic activity against the human leukaemia cell line CCRF-CEM. (c) 2005 Elsevier Ltd. All rights reserved.

Effects of Molecular Length, Ortientation and Amino Acid Mutation on Kinetics of Selectin/Ligand interactions

Receptor/ligand interactions are basic issues to cell adhesion, which are important to many physiological and pathological processes such as lymphocyte-mediated cytotoxicity, tumor metastasis and inflammatory reactionl. Selectin/carbohydrate ligand bindings have been found to mediate the fast rolling of leukocytes on activated endothelial monolayer. Kinetic rate and binding affinity constants are essential determinants of cell adhesion...

Two-Dimensional Kinetics Of Beta(2)-Integrin And Icam-1 Bindings Between Neutrophils And Melanoma Cells In A Shear Flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta(2)-integrin ( lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta(2)-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta(2)-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Molecular characterization of Trimeresurus stejnegeri venom L-amino acid oxidase with potential anti-HIV activity

A novel L-amino acid oxidase, named TSV-LAO, has been purified and cloned from the snake Trimeresurus stejnegeri. Fifty percentage cytotoxic concentrations (CC50) of TSV-LAO on C8166 cells were 24 and 390 nM in the absence or presence of catalase (400nM), respectively. However, at concentrations that showed little effect on cell viability, TSV-LAO displayed dose dependent inhibition on HIV-1 infection and replication. The antiviral selectivity indexes (CC50/EC50) were 16 and 6, respectively, corresponding to the measurements of syncytium formation and HIV-1 p24 antigen expression. Interestingly, the presence of catalase resulted in an increase of its antiviral selectivity to 52 and 38. Under the same conditions, no anti-HIV-1 activity was observed by exogenous addition of H2O2. The complete amino acid sequence of TSV-LAO, as deduced from its cDNA, exhibits a high degree of sequence identity with other snake venom LAOs. (C) 2003 Elsevier Inc. All rights reserved.

A novel heme-containing protein with anti-HIV-1 activity from skin secretions of Bufo andrewsi

A novel protein, named BAS-AH, was purified and characterized from the skin of the toad Bufo andrewsi. BAS-AH is a single chain protein and the apparent molecular weight is about 63 kDa as judged by SDS-PAGE. BAS-AH was determined to bind heme (0.89 mol heme/mol protein) as determined by pyridine haemochrome analysis. Fifty percentage cytotoxic concentration (CC50) of BAS-AH on C8166 cells was 9.5 mu M. However, at concentrations that showed little effect oil cell viability, BAS-AH displayed dose dependent inhibition oil HIV-1 infection and replication. The antiviral selectivity indexes corresponding to the measurements of syncytium formation and HIV-1 p24 (CC50/EC50) were 14.4 and 11.4, respectively, corresponding to the . BAS-AH also showed an inhibitory effect on the activity of recombinant HIV-1 reverse transcriptase (IC50 = 1.32 mu M). The N-terminal sequence of BAS-AH was determined to be NAKXKADVIGKISILLGQDNLSNIVAM, which exhibited little identity with other known anti-HIV-1 proteins. BAS-AH is devoid of antibacterial, protcolytic, trypsin inhibitory activity, (L)-amino acid oxidase activity and catalase activity. (c) 2005 Elsevier Ltd. All rights reserved.

昆明山海棠片对小鼠淋巴细胞体外增殖活化影响的研究

目的:研究昆明山海棠片对小鼠脾淋巴细胞增殖、活化标志CD69和CD25及细胞因子IL-4及IFN-γ的影响.方法:无菌分离小鼠脾淋巴细胞,加入不同浓度的昆明山海棠溶液,MTT法检测刀豆蛋白A刺激的小鼠脾淋巴细胞增殖;流式细胞仪检测活化标志CD69及CD25;酶联免疫法检测细胞因子IL-4及IFN-γ.结果:昆明山海棠片对脾淋巴细胞增殖活化及分泌均有抑制作用.

IN-VITRO IMMUNOTOXICITY AND CYTOTOXICITY OF TRICHOSANTHIN AGAINST HUMAN NORMAL IMMUNOCYTES AND LEUKEMIA-LYMPHOMA CELLS

Trichosanthin (TCS) is a ribosome-inactivating protein from root tubers of Trichosanthes kirilowii Maxim. In this paper, the effects of TCS on the viability of human peripheral blood immunocytess, on the proliferation of lymphocytes, and its cytotoxicity to twelve cell lines of lymphoma or leukemia had been observed. TCS at high concentration (>12.5 mu g/ml) affected the viability of human B lymphocytes, but not that of human peripheral blood mononuclear cells (PBMCs), T lymphocytes and granulocytes. Human peripheral blood-derived monocytes/macrophages were highly sensitive to TCS (ID50 at 1.70 mu g/ml). TCS suppressed lymphocyte proliferation stimulated by Concanavalin A (Con A) or lipopolysaccharide (LPS). Human T cell lines and macrophage cell lines were more sensitive (ID50 < 0.9 mu g/ml) to TCS than B cell lines and myeloid lines. These results suggest that selective cytotoxicity of TCS to human macrophages/monocytes may be implicated in anti-HIV activity, and that selectively killing some leukemia-lymphoma cells by TCS merit further evaluation in treatment of some lymphoma and leukemia.

Xanthohumol, a novel anti-HIV-1 agent purified from Hops Humulus lupulus

Xanthohumol, prenylchacone flavonoid, is a natural product with multi-biofunctions purified from Hops Humulus lupulus. Its anti-HIV-1 activity was tested in the present study. Results showed that xanthohumol inhibited HIV-1 induced cytopathic effects, the production of viral p24 antigen and reverse transcriptase in C8166 lymphocytes at non-cytotoxic concentration. The EC50 values were 0.82, 1.28 and 0.50 mug/ml, respectively. The therapeutic index (TI) was about 10.8. Xanthohumol also inhibited HIV-1 replication in PBMC with EC50 value of 20.74 mug/ml. The activity of recombinant HIV-1 reverse transcriptase and the HIV-1 entry were not inhibited by xanthohumol. The results from this study suggested that xanthohumol is effective against HIV-1 and might serve as an interesting lead compound. It may represent a novel chemotherapeutic agent for HIV-1 infection. However, the mechanism of its anti-HIV-1 effect needs to be further clarified. (C) 2004 Elsevier B.V. All rights reserved.

Enhanced apoptotic action of trichosanthin in HIV-1 infected cells

Trichosanthin (TCS) is a type 1 ribosome-inactivating protein (RIP) effective against HIV-1 replication. The mechanism is not clear. Present results suggested that the antiviral action tray be partly mediated through enhanced apoptosis on infected cells. TCS induced apoptosis in normal H9 cells and this action was more potent in those infected with HIV-1. In flow cytometry study, TCS induced larger population of apoptotic H9 cells chronically infected with HIV-1 in a dose-dependent manner. At TCS concentration of 25 mu g/ml. 8.4% of normal H9 cells were found to be apoptotic whereas the same concentration induced 24.5% in HIV-1 chronically infected cells. Such difference was not found in the control experiments without TCS treatment. Two other studies supported this action. Cytotoxic study showed that cell viability was always lower in HIV-1 infected cells after TCS treatment, and DNA fragmentation studs confirmed more laddering in infected cells. The mechanism of TCS induced apoptosis in normal or infected H9 cells is not clear. Results in this study demonstrated that TCS is snore effective in inducing apoptosis in HIV-1 infected cells. This may explain in part the antiviral action of TCS. (c) 2005 Elsevier Inc. All rights reserved.

Anti-HIV Activities of the Compounds Isolated from Polygonum cuspidatum and Polygonum multiflorum

The 70% EtOH extract of Polygonum cuspidatum showed inhibitory action against HIV-1-induced syncytium formation at non-cytotoxic concentrations in vitro with a 50% effective concentration (EC50) of 13.94 +/- 3.41 mu g/mL. Through bioactivity-guided fractionation, 20 phenolic compounds, including eight stilbenoids, were isolated from the roots of Polygonum cuspidatum, and their anti-HIV-1 activities were evaluated. Results showed that compounds 1, 13, 14, and 16 demonstrated fairly strong antiviral activity against HIV-1-induced cytopathic effects in C8166 lymphocytes at non-cytotoxic concentrations, with EC50 values of 4.37 +/- 1.96 mu g/mL, 19.97 +/- 5.09, 14.4 +/- 1.34 mu g/mL, and 11.29 +/- 6.26 mu g/mL and therapeutic index (TI) values of 8.12, > 10.02, > 13.89, and > 17.71, respectively. Other compounds showed either weak or no effects. Compound 6 also showed weak inhibition (153.42 +/- 19.25 mu g/mL); however, it possesses very good water solubility and showed almost no cytotoxicity (> 2000 mu g/mL), therefore achieving a fairly good TI (13.04). The activities of the two compounds (3 and 18) from Polygonum multiflorum were also assayed. The relationship between molecular structures and their bioactivities was also discussed.

Alpha-momorcharin inhibits HIV-1 replication in acutely but not chronically infected T-lymphocytes

AIM: To identify the anti-human immunodeficiency virus type 1 (HIV-1) activities of alpha-momorcharin ( alpha-MMC) from Momordica charantia in acutely and chronically infected lymphocytes. METHODS: The anti-HIV activities of alpha-MMC were examined by 1) the inhibition of syncytia formation induced by HIV-1 III B; 2) reduction of p24 core antigen expression level and decrease in numbers of HIV antigen positive cells in acutely and chronically infected cultures. The cytotoxic effects of alpha-MMC was tested by trypan blue dye exclusion or colorimetric MTT assay. RESULTS: alpha-MMC was found to obviously inhibit HIV-1 III B-inducing C8166 syncytia formation and markedly reduced both expression of p24 core antigen and the numbers of HIV antigen positive cells in acutely but not chronically HTV-1-infected culture. The median effective concentration (EC50) in these assays were 0.016, 0.07, and 0.32 mg.L-1, respectively. CONCLUSION: alpha-MMC is a unique component of momorcharin with anti-HIV activity, and markedly inhibited HIV-1 replication in acutely but not chronically HIV-1-infected T-lymphocytes.