乙型肝炎病毒P22e蛋白经核因子-κB抑制HepG2细胞凋亡

目的 探讨核因子-κB(NF-κB)在乙型肝炎病毒P22e蛋白抑制HepG2细胞凋亡中的作用.方法 用含HBV P22e基因的重组pEGFP-C2HBVP22e质粒的肝癌细胞HepG2,以放线菌素-D(Act-D)、肿瘤坏死因子(TNF)α诱导该细胞凋亡,采用激光共聚焦显微镜、核蛋白电泳迁移率等技术,观察在HBV P22e抑制TNFα诱导HepG2EGFP-C2HBVP22e细胞的凋亡过程中NF-κB的核转移、活化等情况.用NF-κB抑制剂ALLN抑制其信号通路,检测以Act-D、TNFα诱导的HepG2、HepG2EGFP-C2HBVP22e细胞凋亡率的变化.对实验结果的数据分析用秩和检验和t检验. 结果激光共聚焦显微镜及电泳迁移率实验观察到HepG2EGFP-C2HBVP22e细胞在发生凋亡前后,有明显的NF-κB向核内迁移活化现象.NF-κB抑制剂ALLN可使以Act-D、TNF α诱导HepG2EGFP-C2HBVP22e细胞的凋亡率明显升高(6.19%±1.58%与39.99%±7.620/0,t=7.515,P<0.01).结论 在HBV P22e蛋白抑制肝癌细胞凋亡过程中,NF-κB信号途径起着重要作用.     

抗神经节苷脂抗体与神经精神性狼疮相关性研究

目的 探讨抗神经节苷脂抗体在神经精神性狼疮(NPSLE)中的意义.方法 采用酶联免疫吸附试验(ELISA)法,检测68份血清(包括NPSLE患者33例.无神经精神症状的狼疮患者35例)和18例脑脊液(包括NPSLE患者10例,非NPSLE的狼疮患者8例)中的抗神经节苷脂抗体IsG和IgM.结果 血清中抗神经节苷脂抗体IgG、IgM在系统性红斑狼疮患者中的阳件率分别为21%、24%,在NPSLE患者中的阳性率均为18%,在无神经精神症状的狼疮患者中的阳性率分别为22%、29%,二者在抗体水平和阳性率上差异无统计学意义(P＞0.05).脑脊液中抗神经节苷脂抗体IgG、IgM在系统性红斑狼疮患者中的阳性率分别为10/18、9/18,在神经精神性狼疮患者中的阳性率均为7/10、8/10,在非NPSLE的狼疮患者中的阳性率分别为3/8、1/8,二者在抗体水平和阳性率上差异有统计学意义(P＜0.05).结论 脑脊液中AGA与NPSLE密切相关.     

神经节苷脂治疗血管性帕金森综合征的临床疗效观察

目的 观察神经节苷脂治疗血管性帕金森综合征（VP）的临床疗效.方法 将66例VP患者按照入院顺序分为治疗组和对照组,各33例,两组均继续进行基础疾病的治疗,治疗组加用单唾液酸四己糖神经节苷脂钠静脉滴注,对照组加用美多巴口服,3周为1个疗程.观察两组治疗前后帕金森统一量表（UPDRS）评分、Webster评分及疗效.结果 治疗组总有效率为57.6%,高于对照组的21.2%,差异有统计学意义（P＜0.05）;治疗后治疗组Webster积分低于对照组,UPDRS评分中精神状态、日常生活能力及运动指数方面积分低于对照组,差异均有统计学意义（P＜0.05）.结论 神经节苷脂对VP有较好的临床疗效,能有效缓解症状,提高患者生活质量.     

神经节苷脂的临床应用研究

神经节苷脂（Ganglioside，GMI）是一类含有唾液酸的糖神经鞘脂，存在于哺乳动物细胞，特别是富存在神经元细胞的胞膜中，是神经细胞膜的天然组成部分。外源性GMI能以稳定的方式与神经细胞膜结合，引起膜的功能变化。给药后2h在脑和脊髓测得放射活性高峰。应用剂量一般20-40mg，遵医嘱一次或分次肌注或缓慢静脉注射。在病变急性期（尤急性创伤）：100mg／d，静脉滴注；2—3周后改为维持量,     

核转录因子κB与肝癌的关系

核转录因子（NF）-κB是一种存在于多种细胞内,具有多向性调节作用的核转录因子,参与炎症、免疫、细胞增殖和细胞凋亡等多种生理、病理过程的基因调控。NF-κB还参与肿瘤的生成及其生物学行为,与肝癌的发生发展过程密切相关,现就近年来NF-κB在肝癌相关研究中的进展作一综述。[第一段]     

小干扰RNA抑制核因子-κB活化对HepG2细胞凋亡的影响

目的 探讨小干扰RNA(SiRNA)抑制核转录因子(NF)-κB活化对肝癌细胞凋亡的影响.方法 化学合成NF-κB siRNA和阴性对照siRNA,脂质体法转染HepG2细胞,用巢式RT-PCR和荧光定量PCR检测NF-κB mRNA表达情况;免疫组织化学法、酶联免疫吸附法、Western b10t检测NF-κB蛋白表达情况;用磷脂结合蛋白V-异硫氰酸荧光素法检测细胞凋亡,分析NF-κB表达抑制和细胞凋亡间关系.多个样本均数间的比较先行方差齐性检验,方差齐时行单因素方差分析;计数资料比较采用确切概率法分析.结果 NF-κBp65 mRNA在HepG2细胞相对表达量为1.13±0.03,在正常肝细胞L02为0.29±0.07,两者比较,t=27.02,P<0.05,差异有统计学意义.利用NF-κB siRNA干扰可下调NF-κB表达,且呈剂量、时间依赖;NF-κB siRNA转染HepG2细胞72h后,NF-κBmRNA和蛋白表达分别下降了93%和62%,抑制NF-κB表达使HepG2细胞凋亡增加85%. 结论 NF-κB在肝癌细胞中高表达,NF-κB SiRNA能特异性抑制其在肝癌细胞中活化并促进癌细胞凋亡发生.     

神经节苷脂对脑梗死患者神经功能缺损的影响

目的探讨神经节苷脂对脑梗死患者神经功能缺损的影响。方法选择96例伴有神经功能缺损的脑梗死患者,随机分为治疗组、对照组各48例。对照组给予脱水、抗凝及营养神经等药物治疗,治疗组在此基础上给予神经节苷脂60 mg/d静滴。治疗4周后比较两组患者的临床疗效及神经功能缺损改善情况。结果治疗组临床有效率为89.6%,对照组为77.1%,两组比较P〈0.05;治疗组NIHSS评分为（12.6±3.82）分,对照组为（18.7±5.10）分,两组比较P〈0.05。结论神经节苷脂能够改善脑梗死患者神经功能缺损及临床疗效,具有较好的治疗效果。     

神经节苷脂GM1与神经系统疾病

神经节苷脂是一类含唾液酸的鞘糖脂,在脑内的含量非常丰富.它不但能够促进神经细胞分化、神经突生长以及突触形成,而且参与了神经可塑性的凋节和脑损伤后的功能恢复.GM1是迄今研究最为深入的神经节苷脂,对细胞内C4~(2+)稳态的调节被认为是GM1神经营养/神经保护作用的基础,而它与神经营养因子的相互作用则是其神经保护作用的关键.此外,它还具有抗兴奋性毒性、抗氧化、扩血管等作用.各种神经变性疾病以及缺血缺氧性脑病均与神经元脱失和凋亡有关,而GM1的神经营养/神经保护作用能在这些疾病的治疗中发挥重要作用.目前,GM1已被广泛用于帕金森病、卒中、新生儿缺血缺氧性脑病、脑外伤、脊髓损伤以及周围神经病的治疗,对其作用机制的进一步研究有望为上述疾病的治疗提供新的思路.     

糖尿病神经病变血清抗神经节苷脂抗体的临床观察

目的 探讨2型糖尿病周围神经病变（DPN）与血清抗神经节苷脂抗体（anti-GS-ab)之间的关系。方法 受试者分为三组[糖尿病无神经病变组（DM组）、DPN组、对照组],每组30例。采用固相酶免疫法测定各组anti-GS-IgM-ab、anti-GS-IgG-ab,并行糖化血红蛋白（HbAIc)、神经传导速度测定。结果 DPN患者anti-GS-IgM-ab的阳性率为46．67％,明显高于对照及DM组,并与病程、神经病变的临床分级及HbAIc呈显著正相关。结论 anti-GS-ab在DPN的病理过程中起重要作用,对DPN的诊断及病情判断有参考价值。     

老年糖尿病神经病变患者血清抗神经节苷脂抗体水平的观察

目的 探讨老年2型糖尿病（DM）患者周围神经病变（DPN）与血清抗神经节苷脂（GS）抗体之间的关系。方法 受试者分为3组,DPN组32例,DM组25例,对照组30例。采用美国NicoletVikingⅣ型肌电图仪测定神经传导速率,固相酶免法测定各组抗GSIgM抗体、抗GSIgG抗体,同时测定白细胞介素－1β（IL－1β）、白细胞介素－2（IL－2）、肿瘤坏死因子（TNF－α）、糖化血红蛋白（HbA21C）、一氧化氮（NO）。结果 DPN组患者抗GSIgM抗体、抗GSIgG抗体的阳性率分别为65．6％和34．4％,显著高于DM组及对照组（P＜0．01）。DPN组抗GSIgM抗体与糖尿病神经病变临床分级（DPNC）、HbA1C、IL－1β、TNF-α和NO呈显著正相关,而与病程及IL－2呈显著负相关;抗GSIgG抗体与DPNC呈显著正相关。逐渐回归分析,对抗GSIgM抗体有显著作用的因素依次为DPNC、NO、IL－1β,对抗GSIgG抗体有显著作用的因素为DPNC。结论 抗GS抗体的检测可作为了解DPN自身免疫介导损伤的一项免疫学指标,对DPN的诊断及病情的判断有参考价值。     

Ganglioside GD1a enhances immunoglobulin production by human peripheral blood mononuclear cells

We previously reported that ganglioside GD1a greatly enhanced spontaneous immunoglobulin (Ig) production by human peripheral blood mononuclear cells (PBMC) in vitro. We herein examined the mechanism for the stimulatory effect of GD1a.PBMC from healthy volunteers were cultured with GD1a. The amounts of IgG, IgM, and IgA and cytokine activity in the culture supernatants were measured by enzyme-linked immunosorbent assays. Proliferation was determined by [3H] thymidine uptake.GD1a at 10(-6) M increased IgG, IgM, and IgA production by PBMC 2.10-fold, 2.10-fold, and 2.23-fold above the control values, respectively. GD1a did not affect the proliferation and viability of PBMC. GD1a did not alter Ig production of B cells alone. Anti-interleukin-6 (IL-6) or anti-IL-10 antibody each partially blocked the GD1a-induced enhancement of Ig production by PBMC, and the addition of both antibodies completely blocked the enhancement. GD1a increased IL-6 and IL-10 production of monocytes without altering those of T cells or B cells. The supernatant from GD1a-treated monocytes enhanced B cell Ig production to a greater extent than that from medium-treated monocytes. The supernatant-mediated effect of GD1a was partially blocked by anti-IL-6 or anti-IL-10 antibody, and the addition of both antibodies completely blocked the GD1a effect. GD1a-induced increases of IL-6 and IL-10 production in monocytes were both blocked by Ca(2)+/calmodulin (CaM)-dependent phosphodiesterase (PDE) inhibitors, 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetin, but not by other signal-transducing enzyme inhibitors. The culture with GD1a enhanced Ca(2)+/CaM-dependent PDE activity in monocytes.These results suggest that GD1a may indirectly enhance B cell Ig production in whole PBMC by increasing IL-6 and IL-10 production of monocytes via promoting Ca(2)+/CaM-dependent PDE activity.     

Rapamycin enhances the anti-tumor effect of gemcitabine in pancreatic cancer cells

BACKGROUND/AIMS: Rapamycin is a potent inhibitor of PI3K/Akt pathway activation and its chemotherapeutic effect against pancreatic cancer has been demonstrated. In the present study, the combined effect of rapamycin with gemcitabine was examined and a screening method for detecting sensitivity to combined effects was investigated. METHODOLOGY: Four pancreatic cancer cell lines, MIA, PK-1, PANK-1, and PK-45H, were cultured with or without rapamycin (200, 100, 50, 25nM), or gemcitabine (2, 1, 0.5, 0.25 microM), or with rapamycin and gemcitabine in combination. Growth inhibition of each cell line in response to the agents was examined using the MTT assay. Data were expressed as percentage inhibition at each concentration. Sensitivity to the combined effect of rapamycin and gemcitabine was investigated with real-time PCR (Akt1, 2, and 3, PTEN, mTOR) and western blotting (Akt, Akt/ Ser473, Akt/ Thr308, PTEN). RESULTS: Rapamycin inhibited the growth of MIA (% inhibition: 36.2+/-9.0, 19.5+/-9.0, 10.8+/-6.8, 8.8+/-5.9), PK-1 (41.3+/-0.1, 33.5+/-0.1, 25.2+/-0.1, 22.6+/-0.1), PANK-1 (27.3+/-0.1, 21.7+/-0.1, 15.9+/-0.1, 14.9+/-0.1), and PK-45H (30.0+/-0.1, 24.4+/-0.1, 23.5+/-0.1, 20.5+/-0.1), whereas gemcitabine inhibited the growth in MIA (37.5+/-11.1, 11.5+/-7.8, 3.5+/-1.7, 0.1+/-0.1) and PK-1 (29.2+/-2.8, 26.7+/-0.8, 26.2+/-1.0, 25.1+/-1.0), but only weakly in PANK-1 (7.5+/-5.2, 1.2+/-0.9, 4.2+/-0.8, 2.7+/-0.6) and PK-45H (16.1+/-5.8, 11.8+/-10.0, 7.9+/-1.8, 10.1+/-1.8). However, gemcitabine administered with rapamycin had an enhanced inhibitory effect in PK-45H (61.3+/-12.0%), and no change in PANK-1, when compared to the inhibition by rapamycin or gemcitabine alone. CONCLUSIONS: As a screening method for assessing the combined effects of rapamycin and gemcitabine, the decrease in expression of Akt/Ser473 and Akt/Thr 308 with rapamycin treatment was promising. Rapamycin enhances the anti-tumor effect of gemcitabine. Detecting a decrease in Akt/ Ser473 and Akt/Thr308 after rapamycin treatment, by western blotting, may be an effective way for assessing the combined effect of rapamycin and gemcitabine.     

Ganglioside GD3 promotes cell growth and invasion through p130Cas and paxillin in malignant melanoma cells

Although ganglioside GD3 levels are highly elevated in malignant melanomas, the role of GD3 in melanomas' malignant properties has not been clearly shown. To investigate this problem, we genetically generated GD3-positive (GD3+) transfectant cells from a GD3-negative (GD3-) mutant line SK-MEL-28-N1 and analyzed the phenotypic changes in the transfected cells. GD3+ cells showed markedly increased cell growth and invasive characteristics. Two bands that underwent stronger tyrosine phosphorylation in GD3+ cell lines than in controls after treatment with FCS were found with molecular masses of 130 and 68 kDa. They were identified as p130Cas and paxillin by sequential immunoprecipitation. Their roles in cell growth and invasion were analyzed with a small interfering RNA (siRNA) approach. Cell growth, as analyzed by BrdUrd uptake, was strongly suppressed in GD3+ cells to near the levels of GD3- cells when treated with siRNA for p130Cas but not when treated with siRNA for paxillin. However, treatment with siRNAs of either p130Cas or paxillin resulted in the marked suppression of the invasive activity of GD3+ cells almost to the levels of control cells. These results suggested that these two molecules function as effectors of GD3-mediated signaling, leading to such malignant properties as rapid cell growth and invasion.     

Ganglioside GD1b supresses immunoglobulin production by human peripheral blood mononuclear cells.

Gangliosides are sialic acid-containing glycolipids, that have various immunomodulatory effects. We previously reported that various gangliosides in vitro either inhibited or enhanced spontaneous immunoglobulin (Ig) production by human peripheral blood mononuclear cells (PBMC). GD1b was one of the inhibitory gangliosides. In this study, we further examined the mechanism for the inhibitory effect of GD1b. The inhibitory effect of GD1b was revealed at 0.1 microM, increased dose dependently, and was maximized at 10 microM, which reduced spontaneous IgG, IgM, and IgA production of human PBMC by 50.5%, 52.0%, and 48.3% compared with controls, respectively. GD1b did not affect the proliferation and viability of PBMC. GD1b did not alter Ig production of B cells alone. Interleukin 6 (IL-6) and IL-10 each partially reversed the GD1b-induced inhibition of Ig production by PBMC, and the addition of both cytokines completely reversed the inhibition. When endogenous IL-6 and IL-10 were neutralized by specific antibodies, GD1b did not reveal inhibitory effects on the Ig production. GD1b inhibited IL-6 and IL-10 production of CD4+ T cells, without affecting those of CD8+ T cells, monocytes, or B cells. When CD4+ T cells were preincubated with GD1b and washed and cultured with B cells and monocytes, Ig production was also suppressed. These results suggest that GD1b may indirectly suppress Ig production of B cells in whole PBMC by reducing IL-6 and IL-10 production of CD4+ T cells. GD1b may act as an important inhibitor of human humoral immune responses.     

Clobenpropit enhances anti-tumor effect of gemcitabine in pancreatic cancer

AIM: To evaluate the anti-tumor effect of clobenpropit, which is a specific H₃ antagonist and H₄ agonist, in combination with gemcitabine in a pancreatic cancer cell line.METHODS: Three kinds of human pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and AsPC-1) were used in this study. Expression of H₃ and H₄ receptors in pancreatic cancer cells was identified with Western blotting. Effects of clobenpropit on cell proliferation, migration and apoptosis were evaluated. Alteration of epithelial and mesenchymal markers after administration of clobenpropit was analyzed. An in vivo study with a Panc-1 xenograft mouse model was also performed.RESULTS: H₄ receptors were present as 2 subunits in human pancreatic cancer cells, while there was no expression of H₃ receptor. Clobenpropit inhibited cell migration and increased apoptosis of pancreatic cancer cells in combination with gemcitabine. Clobenpropit up-regulated E-cadherin, but down-regulated vimentin and matrix metalloproteinase 9 in real-time polymerase chain reaction. Also, clobenpropit inhibited tumor growth (gemcitabine 294 ± 46 mg vs combination 154 ± 54 mg, P = 0.02) and enhanced apoptosis in combination with gemcitabine (control 2.5%, gemcitabine 25.8%, clobenpropit 9.7% and combination 40.9%, P = 0.001) by up-regulation of E-cadherin and down-regulation of Zeb1 in Panc-1 xenograft mouse.CONCLUSION: Clobenpropit enhanced the anti-tumor effect of gemcitabine in pancreatic cancer cells through inhibition of the epithelial-mesenchymal transition process.     

Cisplatin pretreatment enhances anti-tumor activity of cytokine-induced killer cells

AIM:To investigate whether cisplatin (DDP) enhances the anti-tumor activity of cytokine-induced killer (CIK) cells in a murine colon adenocarcinoma model.METHODS:Tumor size and weight served as indicators of therapeutic response.Immunohistochemistry was performed to observe intratumoral lymphocyte infiltration and tumor microvessel density.Changes in the percentage of regulatory T (Treg) cells within the spleens of tumor-bearing mice preconditioned with DDP were monitored using flow cytometry.RESULTS:A mark...     

银耳多糖对小鼠移植性肝癌治疗作用及机制的实验研究

目的研究银耳多糖(Trem ella Polysaccharide,TP)对荷HAC小鼠T细胞表面分化抗原、细胞因子、体外肿瘤细胞的杀伤作用及小鼠移植性肝癌的影响。方法用环磷酰胺和TP分别作用于荷HAC肝癌小鼠,采用免疫荧光法、流式细胞仪及酶标仪观察各组免疫功能的变化及细胞因子的表达,并分离其脾细胞,将诱导的细胞毒性T细胞(CTL细胞)分别作用于体外培养的3H-TdR标记的肿瘤细胞P815、HAC和B16,4 h后用γ-闪烁记数仪进行检测,并记算杀伤程度。检测各组肿瘤重量、体积、组织学改变和主要脏器的形态学改变。结果TP实验组总T细胞、T杀伤细胞及肿瘤坏死因子(TNF)分别为(62.3±1.06)、(25.6±0.87)及(87.5±0.5),高于对照组,其中对总T细胞及TNF上调明显。在对HAC、P815、B16肿瘤细胞的杀伤作用中,TP实验组的杀伤活性明显高于对照组,分别为23.85%、20.81%、25.43%(P<0.01),其中对B16杀伤作用最强,HAC次之,P815最弱。环磷酰胺+TP实验组上述指标与TP实验组相比有所下降,但与对照组相比则明显增高(P<0.05)。治疗组肝肿瘤重量、体积均低于模型组。组织学上肿瘤坏死程度重且范围广,淋巴细胞、巨噬细胞浸润明显,纤维组织增生显著。环磷酰胺组较实验组表现的更为明显。TP实验组除脾脏外,其他主要脏器无明显形态学变化。结论TP具有激活和提高特异性和非特异性杀伤细胞的抗肿瘤作用。     

Sodium butyrate enhances Fas-mediated apoptosis of human hepatoma cells

BACKGROUND/AIMS: Human hepatoma cells have been reported to be resistant to Fas-mediated apoptosis. Sodium butyrate (SB) induced apoptosis of several cancer cells. We investigated the effects of SB on Fas-mediated apoptosis of hepatoma cells. METHODS: In hepatoma cells (HuH-6, HuH-7, Hep-G2, and PLC/PRF/5), susceptibility to Fas-mediated apoptosis and Fas expression were assessed. Caspase-3 activation and cell cycle progression were evaluated in HuH-6. A cDNA microarray assay was performed to screen the changes in the expression of mRNAs. RESULTS: Pretreatment with SB caused an enhancement of the sensitivity to anti-Fas-mediated cytotoxicity, though it did not increase the expression of Fas. The cDNA microarray assay revealed up-regulation of pro-apoptotic Bik, Bak, Bid and c-Jun N-terminal protein kinase-1, and down-regulation of anti-apoptotic Bag-1 and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitor protein. In some molecules, expression of the proteins was confirmed by Western blotting. An increase in truncated-Bid accompanying the reduction in Bid was also observed. CONCLUSIONS: SB enhances the susceptibility of hepatoma cells to anti-Fas-mediated cytotoxicity by altering the mRNA and protein expression and/or the activation status of proteins that could be involved in the Fas signaling pathway. SB may have an important role in the elimination of hepatoma cells.