经典Wnt信号通路与弥漫大B细胞淋巴瘤

弥漫性大B细胞淋巴瘤(DLBCL)的发病机制虽未完全阐明,但研究显示与信号通路表达异常有关,如经典Wnt通路。在DLBCL发病机制的研究中观察到经典Wnt通路的重要下游因子β-catenin的表达和核内定位。同时证据显示经典Wnt通路不仅与DLBCL发病机制有关,还和DLBCL临床分期密切相关,经典Wnt通路通路有可能成为治疗DLBCL潜在的有用靶点。     

Role of FGF-2／FGFR signaling pathway in cancer and its signification in breast cancer

Fibroblast growth factor-2 (FGF-2) is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. It has been proved that FGF-2 stimulates the growth and development of new blood vessels (angiogenesis) that contribute to the pathogenesis of several diseases (i.e. cancer, atherosclerosis). However, many of the biological activities of FGF-2 have been found to depend on its receptor抯 intrinsic tyrosine kinase activity and second messengers such as the mitogen activated protein kinases. This review will focus on the mechanism of FGF-2/FGFR induced signaling pathway in tumor and human breast cancer.     

PI3K-Akt和JNK信号通路抑制剂对杆状病毒诱导昆虫细胞凋亡影响的初步研究

为了探讨杆状病毒诱导昆虫细胞凋亡通路与细胞内PI3K-Akt和JNK信号通路的关系,应用PI3K的特异性抑制剂Wortmannin和JNK的特异性抑制剂SP600125处理芹菜夜蛾核型多角体病毒(AfMNPV)感染的斜纹夜蛾SL-1细胞,研究了这些抑制剂对杆状病毒诱导昆虫细胞凋亡的影响.分别使用浓度梯度2.5,25,50μmol的SP600125和0.3,3,30μmol的Wortmannin处理感染了SfaMNPV的SL-1细胞,24h后进光镜观察、DAPI荧光染色,流式细胞术分析显示,抑制PI3K-Akt和JNK信号通路后杆状病毒诱导的细胞凋亡受到明显影响,细胞凋亡水平明显降低.研究结果提示AfMNPV诱导斜纹夜蛾SL-1细胞凋亡过程可能涉及细胞PI3K-Akt和JNK信号通路.     

基于NFAT信号通路的抗LAG3抗体8F-6生物活性检测

淋巴细胞活化基因3（LAG3）在T细胞活化和增殖过程中起负调控作用,通过单克隆抗体封闭LAG3分子可以增强T细胞对肿瘤的杀伤力,因此开发抗LAG3抗体的生物活性检测平台对免疫治疗具有重要意义。前期利用杂交瘤融合及抗体人源化改造技术,得到了一株全人源化的抗LAG3单克隆抗体8F-6,并基于活化T细胞核因子（NFAT）信号通路构建了Jurkat-NFAT-Luc2-LAG3和Jurkat-NFAT-Luc2-LAG3-CD3zeta细胞。本文利用所构建的细胞,通过酶联免疫吸附测定法（ELISA）和流式细胞技术对抗体8F-6进行亲和力及阻断效果检测。结果表明,抗体8F-6与LAG3分子具有良好的亲和力,可以阻断主要组织相容性复合体II （MHC II）分子与LAG3分子的结合;在报告细胞检测平台中,抗体8F-6在CHO-K1-FCGRIA细胞的协助下可以激活Jurkat-NFAT-Luc2-LAG3-CD3zeta细胞;在Daudi/Raji细胞、人金黄色葡萄球菌肠毒素E （SEE）和Jurkat-NFAT-Luc2-LAG3细胞的反应体系中,相比于对照抗体IgG1,抗体8F-6可以明显提高Jurkat-NFAT-Luc2-LAG3细胞的激活效果。     

叶酸受体在血液肿瘤细胞株的表达及叶酸-聚乙烯亚胺共聚物的制备

目的:研究叶酸受体(folate receptor,FR)在血液肿瘤细胞表达的特点,合成叶酸-聚乙烯亚胺(FA-PEI)聚合物,探讨其作为靶向性基因输送载体的可行性.方法:用实时荧光定量PCR(real time PCR)法检测α、β、γ3种FR在血液肿瘤细胞K562、K562/A02、U266细胞株上的表达;利用叶酸(FA)活性酯在微碱性条件下与聚乙烯亚胺(polyethylenimine,PEI)的支链氨基反应,合成FA-PEI共聚物;用葡聚糖凝胶柱G-25分离、纯化;用紫外分光光度计波长扫描法及氢核磁共振谱(~1H NMR)法验证交联是否成功.结果:α、β、γ3种FR在K562、K562/A02、U266 3种细胞株上均表达,其中,α-FR的表达占优势,较β-FR和γ-FR表达量高;紫外分光光度计扫描图谱在365 nm处出现叶酸的特征性吸收峰;核磁共振(~1H NMR)示:在2.5～3.2处出现PEI亚甲基质子的特征性化学位移,在6.5～9.0处出现叶酸芳香质子的特征性氢信号.结论:FR在K562、K562/A02、U266 3种细胞株上均有较高表达;FA-PEI偶联成功,为其作为血液肿瘤治疗中1种潜在的靶向性基因输送载体提供了依据.     

Enhancement of T cell immune response to lymphoma cells by CD28/CD80 alternative pathway

The aim of this study is to determine whether myeloma and lymphoma tumor cells can function as efficient antigen presenting cells (APC) to enhance the co-stimulation of T cells. The expression and function of T cell activation-related molecules, especially CD80, CD28, CD40 and CD40 ligand (CD40L), were studied on nine human myeloma cell lines (HMCL) and two B lymphoma cell lines. In the case of myeloma cell lines, the cells generally lacked CD80 antigen and expressed a heterogeneous CD40, and the expressions of CD40 and CD80 molecules could not be induced by either CD28 stimulation or CD40 ligation. Conversely, in the two B lymphoma cell lines, tumor cells expressed both CD80 and CD40 to some extent. CD28 stimulation could obviously increase the expression of CD80, CD40 and some adhesion molecules, and therefore generate a more efficient anti- tumor cell immunity. In conclusion, CD28 stimulation combined with CD40 antibody or soluble CD40 ligand may be a promising immunotherapeutic approach to B lymphoma.     

新型姜黄素结构衍生物抗肝癌细胞增殖作用的研究

采用MTT法对姜黄素结构衍生物（CCM系列化合物）进行抗人肝癌细胞Bel-7402和SMMC-7721活性筛选;利用流式细胞技术和荧光显微镜检测SMMC-7721细胞凋亡及周期分布;采用Western-Blot方法检测SMMC-7721中蛋白caspase-3和剪切后p17的表达.结果表明,化合物CCM-5和CCM-14抗肿瘤活性较好,其对SMMC-7721细胞的凋亡作用呈剂量依赖性,且凋亡率与阴性对照组相比有显著差异（P<0.01）;化合物浓度增高时,G0/G1期细胞减少,S期以及G2/M期细胞增加,凋亡峰SubG1峰增大;两个化合物均可增强caspase-3的表达,随着浓度的提高,caspase-3的表达趋势减弱,而剪切形式p17亚基表达量逐渐提高.因此,姜黄素结构衍生物CCM-5和CCM-14能抑制人肝癌细胞SMMC-7721细胞的增殖,促进凋亡,其作用机制可能与化合物诱导caspase-3活性的增强有关.     

Inhibition of PKB/Akt activity involved in apigenininduced apoptosis in human gastric carcinoma cells

Apigenin is a flavonoid widely distributed in fruits and vegetables. It possesses growth inhibitory properties against numerous cancer cell lines. However,the molecular mechanism(s) by which api-genin elicits its effects have not been fully elucidated. Here we studied whether apigenin inhibits growth and induces apoptosis in human gastric carcinoma cells. We showed that the flavonoid inhibited growth of the cells and caused apoptosis,as evidenced by DNA Ladder,cleavage of pro-caspase-3 in a time-dependent manner. Induction of apoptosis was dependent on inhibition of the PKB/Akt activity. We found that while apigenin had no effect on the expression of Akt and Bad,it inhibited specific phosphorylation of the two proteins that are associated with pro-survival mechanisms. We propose that this important flavonoid induces apoptosis in gastric cancer cells by inhibiting Akt activity. Since Akt is often activated in cancers,our findings may have clinical implications.     

细胞外信号调节激酶1/2信号通路在小鼠T细胞增殖、周期和活化中的作用

目的:探讨细胞外信号调节激酶1/2(ERK1/2)信号通路在小鼠T淋巴细胞增殖、周期和活化中的作用.方法:分离小鼠淋巴结细胞,藉多克隆刺激剂刀豆蛋白(ConA)或佛波醇酯(PDB)加离子霉素(Ion)刺激,流式细胞术分析ERK1/2信号通路的特异性阻断剂PD98059对小鼠T淋巴细胞增殖、周期和活化的影响.结果:活体染料羧基荧光素乙酰乙酸染色分析显示,不同浓度(5、10、20、30、40 μmol/L)的PD98059对ConA诱导的T淋巴细胞增殖具有明显的抑制作用,呈现剂量依赖关系(r=0.985,P＜0.01).碘化丙锭染色分析表明,PD98059可阻止ConA刺激的T淋巴细胞进入S期和G2/M期,PD98059对PDB Ion刺激的T淋巴细胞细胞周期的影响与ConA刺激相似,不同的是S期和G2/M期的变化较ConA作用更显著.荧光标记单克隆抗体染色显示,不同浓度的PD98059仅能轻微影响T淋巴细胞表面活化标志CD69和CD25的表达.结论:PD98059对小鼠T淋巴细胞的增殖有明显抑制作用,并阻止其进入S期和G2/M期,但不能明显抑制小鼠T淋巴细胞的早期和中期活化.     

S100A8 mediates the activation of P65/HLA-B/S100A8/BCL-2/Caspase-9 (-3) pathway in laryngeal carcinogenesis

S100 calcium binding protein A8 (S100A8),a possible novel member of NF-kappa B signal pathway in laryngeal squamous cell carcinoma (LSCC),interacts with human leukocyte antigen B (HLA-B) which carries an NF-kappa B binding site within the enhancer A. The objective of this study was to explore the molecular mechanism of S100A8 in laryngeal carcinogenesis. RT-PCR,Western blotting and immuno-histochemistry staining were applied to evaluate the expression levels of IKKα,P65,REL-B,S100A8,APAF-1 and BCL-2 genes. The signal transduction passway in which S100A8 might participate was explored by RNA interference. Flow cytometry,TUNEL assay and cell invasion in vitro were used to detect the biological behavior of Hep2 cells induced by S100A8 gene. Our results showed that high expression of S100A8 was related to tumorigenesis in LSCC and negatively correlated with the degree of differentiation,indicating that S100A8 gene could inhibit apoptosis and promote metastasis in LSCC. Additionally,the suppression of S100A8 by RNA interference down-regulated BCL-2 but not APAF-1,P65 and IKKα,while,the suppression of P65 could significantly down-regulate the expression of S100A8 gene. In conclusion,S100A8 plays an important role in P65/HLA-B/S100A8/BCL-2/Caspase-9 (-3) pathway in laryngeal carcinoma.     

鼠李素（Rhamnetin）对新型分子靶向药物安罗替尼（Anlotinib）杀伤非小细胞肺癌细胞的增敏作用

目前,安罗替尼(Anlotinib)是非小细胞肺癌(NSCLC)新的和有希望的分子靶向药物,有助于改善与提高NSCLC患者的生存期与生存质量,但安罗替尼治疗费用昂贵,也存在潜在的毒副作用。研究与开发安罗替尼增敏的联合与辅助治疗策略具有重要意义。使用从鼠李科植物中分离得到的天然产物鼠李素与安罗替尼联合作用杀伤非小细胞肺癌细胞,确定鼠李素是否具有对安罗替尼的增敏作用。使用非小细胞肺癌细胞系A549(非小细胞肺癌中腺癌亚型)、以及H460(非小细胞肺癌中大细胞肺癌亚型)建立裸鼠皮下肿瘤模型,对动物灌胃给予安罗替尼联合尾静脉注射鼠李素,观察二者单独与联合作用对肺癌细胞皮下肿瘤体积与肿瘤重量的影响;在此基础上,使用非小细胞肺癌细胞系A549以及H460经由肝门静脉接种进入肝脏建立非小细胞肺癌的肝脏转移模型,对动物灌胃给予安罗替尼联合尾静脉注射鼠李素,观察二者单独与联合作用对肺癌细胞在肝脏形成肿瘤的影响,使用图像处理软件确定肝脏肿瘤病灶所占肝脏面积的比例。在肿瘤组织中检测上皮-间质转化相关因子的表达情况,确定药物对非小细胞肺癌细胞上皮间质转化作用的影响。安罗替尼具有对非小细胞肺癌细胞明确的杀伤作用,鼠李素与安罗替尼联合使用,能够显著提高安罗替尼对非小细胞肺癌细胞的杀伤作用;鼠李素能够显著抑制非小细胞肺癌细胞的上皮间质转化作用;鼠李素能够显著抑制非小细胞肺癌细胞的上皮-间质转化作用,发挥对安罗替尼的增敏作用。     

维药异常黑胆质成熟剂对人肝癌HepG2细胞生物行为和Rho/ROCK信号通路的影响

 为探讨维药异常黑胆质成熟剂(ASM)对人肝癌细胞(HepG2)增殖、侵袭转移的影响及Rho/ROCK 信号传导通路相关蛋白表达影响,采用四甲基偶氮唑蓝(MMT)法检测不同浓度ASM(10、20、25、50 mg/mL)和10 μmol/L Y-27632 作用24、48、72 h后,对HepG2 细胞增殖的影响;采用扫描电镜技术和细胞侵袭实验测定ASM 不同剂量组和10 μmol/L Y-27632 作用24 h 癌细胞侵袭运动能力,Western Blot 检测ASM 不同剂量组和10 μmol/L Y-27632 作用24 h 癌细胞RhoA、ROCK1、ROCK2 的表达。结果显示,ASM 对肝癌细胞增殖有明显抑制作用,且表现为有明显的剂量效应关系:在10、20 mg/mL ASM 剂量组,ASM 药物作用24、48、72 h 后,HepG2 细胞增殖抑制作用随时间的延长而抑制增加,而25、50 mg/mL 剂量组,ASM 抑制细胞增殖作用不明显;ASM 抑制瘤细胞侵袭运动能力,扫描电镜结果显示ASM 抑制肿瘤细胞伪足的生长,ASM 中高剂量组ROCK1、ROCK2 的表达明显降低,RhoA 表达无明显变化。由此推论,ASM 对人肝癌细胞生长增殖和侵袭运动能力有抑制作用,其机制可能与ROCK酶表达降低有关。     

齐墩果酸对衰老大鼠睾丸间质细胞胰岛素/IGF1通路关键基因表达的影响

为了探讨齐墩果酸对衰老大鼠睾丸间质细胞的作用,采用MTT法确定齐墩果酸最适药物浓度,氧化应激创建大鼠睾丸间质细胞衰老模型,RT-qPCR、免疫荧光和免疫印迹检测胰岛素/胰岛素样生长因子1(insulin-like growth factor 1, IGF1)信号通路关键基因转录情况和蛋白翻译情况,采用INSR抑制剂处理细胞,流式细胞术检测细胞凋亡情况.结果显示:与正常组相比,INSR、IRS1、IRS2、IGF1的mRNA转录水平和蛋白翻译水平显著下降(P<0.05);IGFBP3的mRNA转录水平显著提高(P<0.05),免疫荧光和免疫印迹均未检测到此基因表达;与衰老组相比,齐墩果酸组明显逆转此现象(P<0.05);齐墩果酸能明显抑制衰老组细胞的凋亡(P<0.05),INSR抑制剂处理后,细胞凋亡率明显高于齐墩果酸组(P<0.05).综上可知,齐墩果酸通过调控胰岛素/IGF1信号通路关键基因的转录和翻译延缓Leydig细胞衰老.     

利用经修饰的survivin短肽体外诱导对胃癌细胞系具有限制性杀伤作用的特异性细胞毒性T细胞

采用一种简单有效的方法体外诱导经修饰的survivin短肽特异性细胞毒性T细胞,并观察其对胃癌细胞的杀伤作用。选取对HLA-A2分子亲和力更高的survivin短肽(96—104,LLLGEFLKL,HLA-A2限制性)以更有效地诱导特异性T细胞。选取正常志愿者外周血单核细胞,筛选其中HLA-A2+者,利用HLA-A2/survivin短肽有效诱导特异性细胞毒性T细胞。Pentamer实验检测特异性细胞毒性T细胞比率。Elispot实验检测细胞毒性T细胞IFN-γ分泌能力。选取胃癌细胞系MKN28、SGC7901及KATO Ⅲ,检测其HLA-A2和survivin表达情况。检测特异性细胞毒性T细胞对三种细胞系的杀伤效果。Pentamer实验结果显示,HLA-A2/survivin短肽刺激之前短肽/HLA Pentamer和CD8双阳性细胞仅为0.06%,接受刺激后双阳性细胞增加到27.5%。Elispot实验结果显示HLA-A2/survivin短肽刺激后IFN-γ分泌细胞比率明显强于阴性对照组。PCR结果显示,KATO Ⅲ为HLA-A2+,MKN28和SGC7901为HLA-A2-。Western Blotting结果显示KATO III和SGC7901为survivin阳性,MKN28为survivin阴性。特异性细胞毒性T细胞杀伤结果显示,细胞能有效杀伤KATO III细胞,但对其他两种细胞杀伤效果不明显。p<0.01。经修饰的survivin短肽能够有效诱导特异性细胞毒性T细胞,并对胃癌细胞系有HLA-A2/survivin限制性杀伤。     

Effects of sense and antisense centromere/kinetochore complex protein-B （CENP-B） in cell cycle regulation

This paper investigates the effects of sense and antisense centromere/kinetochore complex protein-B （CENP-B） in cell cycle regulation. Full-length cenpb cDNA was subcloned into pBI-EGFP eukaryotic expression vector in both sense and antisense orientation. HeLa-Tet-Off cells were transfected with sense or antisense cenpb vectors. Sense transfection of HeLa-Tet-Off cells resulted in the formation of a large centromere/kinetochore complex, and apoptosis of cells following several times of cell division. A stable antisense cenpb transfected cell line, named HACPB, was ob- tained. The centromere/kinetochore complex of HACPB cells became smaller than control HeLa-Tet-Off cells and scattered, and the expression of CENP-B was down-regulated. In addition, delayed cell cycle progression, inhibited malignant phenotype, restrained ability of tumor formation in nude mice, and delayed entry from G2fM phase into next G1 phase were observed in HACPB cells. Furthermore, the expression of cyclin-dependent kinases （CDKs）, cyclins, and CDK inhibitors （CKIs） were modulated during different phases of the cell cycle. CENP-B is an essential protein for the maintenance of the structure and function of centromere/kinetochore complex, and plays important roles in cell cycle regulation.     

六亚甲基二乙酰胺对粘液表皮样癌细胞P21和P53基因的转录激活作用

为了研究分化诱导剂六亚甲基二乙酰胺（hexamethylene bisacetamide,HMBA)在体外诱导人粘液表皮样癌细胞（MEC－1）时P^21（cip1/waf1),P^53基因调控的机理，从而为临床治疗提供理论依据。选用0．002mol/L HMBA和0．001g/L5-FU对培养的MEC－1细胞外诱导72h，分别采用光镜，TUNEL染色，免疫组化，图像分析等方法进行凋亡细胞的检测及P^21(cip1/waf1),P^53表达的定量分析，结果表明HMBA诱导的MEC－1细胞P^21(cip1/waf1)表达强度显著高于对照组（P＜0．01），P^53的表达强度显著低于对照组（P＜0．01），提示HMBA通过激活转录因子P^21(cip1/waf1)，P^53的表达而发挥对粘液表皮样品部细胞诱导分化，促进凋亡的作用。