RGD肽对肺癌A549 细胞增殖侵袭能力的影响及其机制研究

目的:研究RGD肽对肺癌A549细胞增殖凋亡及侵袭迁移的影响,并探讨其作用机制。方法:不同浓度RGD肽处理肺癌A549细胞后,MTT检测肺癌细胞的增殖能力,流式细胞仪检测肺癌细胞凋亡及周期分布,Transwell检测其迁移及侵袭能力的变化,Western blot检测RGD肽对肺癌A549细胞MMP2、MMP9的表达水平影响。结果:当RGD肽浓度增加至50 mg/L时,肺癌A549细胞增殖明显受到抑制,且这种抑制作用呈剂量依赖关系;RGD肽组A549细胞G0/G1期细胞比例增高,细胞凋亡率由(6.1±0.1)%增至(15.2±0.5)%;在迁移和侵袭试验中,RGD肽组A549细胞的穿膜细胞数分别由123±10和43±10降至45±5和18±5;RGD肽组A549细胞MMP2、MMP9表达水平显著降低。结论:RGD肽对肺癌A549细胞的增殖有明显抑制作用,并促进其凋亡,可能与RGD肽改变其周期分布有关,RGD肽可明显抑制A549细胞的迁移及侵袭,可能与其下调MMP2、MMP9的表达相关。     

连翘叶黄酮的体外抗氧化作用

为研究连翘叶黄酮(Forsythia suspenseleaves flavonoids,FLF)的体外抗氧化作用,用水杨酸法研究FLF清除.OH的效果,并测定了FLF对连苯三酚自氧化体系的抑制作用。用硫代巴比妥酸(TBA)法测定了小鼠4种器官及肝线粒体、微粒体中的丙二醛(MDA)含量,用分光光度法测定了小鼠红细胞溶血和肝线粒体膨胀程度。结果表明,FLF可以清除.OH,抑制连苯三酚自氧化,并抑制.OH所致丙二醛的产生,减少红细胞溶血,减轻肝线粒体膨胀程度。说明FLF具有明显的抗氧化活性。     

大豆肽体外抗氧化活性研究

研究大豆肽的体外抗氧化的作用。采用邻二氮菲-Fe^2＋检测大豆肽对羟自由基（·OH）的清除作用,邻苯三酚检测大豆肽对超氧阴离子（·O2^-）的清除作用,用硫代巴比妥酸法测定小鼠肝匀浆丙二醛（MDA）的含量,用比色法测定小鼠红细胞溶血度,来研究大豆肽的抗氧化效果。结果表明：大豆肽可以清除·OH和·O2^-,抑制·OH所致的丙二醛的产生,减少H2O2所致的红细胞溶血,在2～15g／L内均具有明显的量效关系。表明大豆肽在体外具有明显的抗氧化效果。     

一种特异性识别非小细胞肺癌A549细胞的小分子肽

应用"一个珠子一个化合物"的组合化学肽库,以期筛选得到特异性识别非小细胞肺癌细胞(A549)的小分子肽.初次筛选共得到29个与A549阳性结合的珠子,经氨基酸序列分析后发现含有-NGXG-肽链结构的序列共有10个.选择cNGQGEQc作进一步的细胞特异性研究,发现cNGQGEQc与非小细胞肺癌A549、Calu-1及H178的粘附特异性明显高于其他细胞系,对cNGQGEQc的结构分析显示,-NGXG-及六肽长度对小分子肽与A549细胞的粘附非常重要.标记FITC的小分子肽cNGQGEQc能与A549细胞发生特异性结合.用抗整合素的抗体(!1～6,"v和#1～5)阻断小分子肽与A549细胞表面的相应受体结合,结果显示,α3与$亚单位的任何组合均对cNGQGEQc与A549细胞的粘附有明显的阻断作用.结果表明,小分子肽cNGQGEQc是通过细胞表面整合素α3与非小细胞肺癌A549发生特异性结合.     

人参皂苷Rh2对肺癌A549细胞miR-16和Bcl-2表达与生长的影响研究

目的探讨人参皂苷Rh2(G-Rh2)诱导人肺癌细胞A549凋亡作用及机制研究。方法采用2.5、5.0、10.0、20.0、40.0μmol/L的G-Rh2处理A549细胞,MTT法于不同时间点测定G-Rh2对A549的生长抑制作用;倒置显微镜观察G-Rh2对A549细胞作用后的形态改变;Annexin-FITC/PI双染法检测细胞凋亡情况;Real-time PCR和Western-blot法分别检测G-Rh2对A549细胞中miR-16和Bcl-2蛋白表达的影响。结果与阴性对照组相比,不同浓度G-Rh2能够抑制A549肺癌细胞增殖并呈时间-浓度依赖性;GRh2作用后,A549细胞凋亡率明显增加,miR-16的表达显著增加,而Bcl-2蛋白表达显著降低。结论G-Rh2能够显著抑制A549肺癌细胞增殖并通过上调miRNA-16,下调Bcl-2的表达发挥治疗肺癌的作用。     

hTERT启动子调控rVBMDMP基因表达载体的构建与功能研究

为了探讨端粒酶催化亚单位(hTERT)启动子调控重组血管基膜衍生多功能肽(rVBMDMP)基因表达抑制肺癌细胞生长的作用机制,采用PCR方法,克隆hTERT启动子核心区片段,并检测其功能活性.然后,构建pLNSX/hTERT/rVBMDMP逆病毒载体,获取逆病毒,感染肺癌A549细胞,检测hTERT启动子调控rVBMDMP基因表达对细胞形态、细胞生长、细胞凋亡以及Caspase-3表达的影响.另外,观察hTERT启动子调控rVBMDMP基因表达对裸鼠成瘤的抑制作用、瘤组织细胞的凋亡及Caspase-3表达情况.结果发现:a.hTERT启动子核心区能调控rVBMDMP基因的表达(n=3,P<0.05);b.hTERT启动子调控rVBMDMP基因的表达具有抑制肺癌A549细胞的生长和裸鼠成瘤作用(n=6,P<0.05);c.rVBMDMP基因表达能促进肺癌A549细胞凋亡和Caspase-3蛋白表达.以上结果说明,hTERT启动子调控rVBMDMP基因表达后,通过提高Caspase-3的表达水平,促进细胞凋亡而抑制肺癌A549细胞的生长.     

长链非编码RNA SNHG17通过抑制p57表达促进肺癌进展

目的:分析肺癌中长链非编码RNA(lncRNA)小核仁RNA宿主基因17(SNHG17)与p57的表达相关性及功能联系。方法:通过starBase数据库分析SNHG17和p57在肺癌组织和正常组织中的表达差异;采用人正常肺上皮细胞BEAS-2B和人肺癌细胞系A549、H1975进一步分析SNHG17和p57在肺癌细胞和正常肺细胞中的表达差异;采用qRT-PCR和Western印迹检测si-SNHG17在mRNA和蛋白表达方面对SNHG17和p57的影响;采用CCK-8法检测siSNHG17对A549细胞增殖的影响;采用Transwell法检测si-SNHG17对A549细胞迁移和侵袭能力的影响。结果:通过数据分析发现SNHG17在肺腺癌和肺鳞癌组织中显著高表达,而p57在肺腺癌和肺鳞癌组织中却显著低表达;与正常肺细胞相比,SNHG17和p57在肺癌细胞中的表达趋势与在肺癌组织中一致;SNHG17和p57的表达呈负相关;沉默SNHG17使p57的表达一定程度上调,A549细胞的增殖、迁移和侵袭能力受到抑制。结论:SNHG17在肺癌中高表达,促进肺癌细胞增殖,并通过调节p57的表达来抑制肺癌细胞的增殖和迁移。SNHG17和p57在肺癌中的具体作用机制仍有待进一步研究。     

重组蛋白rLj-112对人非小细胞肺癌A549细胞增殖、迁移和凋亡的影响及其机制研究

该文研究了重组蛋白r Lj-112对人非小细胞肺癌A549细胞增殖、迁移和凋亡的影响及其作用机制。采用MTT(四甲基偶氮唑蓝)法检测r Lj-112对人非小细胞肺癌A549细胞增殖的影响,采用Transwell方法检测r Lj-112对A549细胞迁移和侵袭的影响,Hoechst和吉姆萨染色的方法检测r Lj-112对A549细胞凋亡的影响;采用Western blot法检测r Lj-112对A549细胞信号通路相关蛋白质水平的影响。结果表明,r Lj-112能够抑制A549细胞的增殖,半抑制浓度IC50值为1.64μmol/L;r Lj-112能抑制A549细胞的迁移和侵袭并呈剂量依赖性;r Lj-112能诱导A549细胞的凋亡;r Lj-112处理过的细胞cleaved-caspase3蛋白质和cleaved-PARP蛋白质水平升高,证明r Lj-112通过caspase3/PARP途径执行对A549细胞的凋亡的诱导,p-AKT、p-PI3K和p-Erk1/2的水平下降,提示r Lj-112的作用方式与PI3K/AKT相关信号通路有关。     

雷公藤内酯醇对人肺癌细胞增殖抑制及放疗增敏作用

目的:探讨雷公藤内酯醇对人肺癌细胞A549增殖抑制及放疗增敏作用。方法:以人肺癌细胞株A549为研究对象,用CCK-8法和流式细胞技术检测雷公藤内酯醇对肺癌细胞增殖的影响和肺癌细胞放射敏感性的改变。结果:CCK-8法检测雷公藤内酯醇对A549的生长有显著抑制作用且具有剂量依赖性,与阴性对照组对比差异有统计学意义(p0.05)。雷公藤内酯醇能使处于G0/G1、G2/M期的细胞减少,S期细胞增加。同时,雷公藤内酯醇对A549放疗具有增敏作用。结论:雷公藤内酯醇可抑制人肺癌细胞增殖,可能与影响细胞周期有关;雷公藤内酯醇能增强肺癌细胞放射敏感性。     

Periostin在非小细胞肺癌中的高表达可增强肺癌细胞的增殖和侵袭能力

为了研究非小细胞肺癌(non-small cell lung cancer,NSCLC)患者的血浆和肺癌组织中Periostin蛋白表达水平以及对癌细胞增殖和侵袭的影响。本研究选取了40例非小细胞肺癌患者作为研究组,同时选取40例同期体检的健康人群作为对照组,采用Real-time PCR的方法比较肺癌患者和健康人群的血浆中,以及肺癌患者的癌组织和癌旁正常组织中Periostin表达水平;采用小分子干扰RNA(small-interfering RNA,si RNA)抑制非小细胞肺癌细胞系A549中Periostin的表达,使用CCK-8法检测A549增值能力的变化,使用Transwell方法观察A549侵袭能力的变化。研究结果显示,Periostin蛋白在非小细胞肺癌患者的血浆中表达水平显著高于正常人群(p0.05),同时在肺癌组织中的含量显著高于癌旁组织(p0.05);导入Periostin的si RNA后,A549细胞的增殖和侵袭能力显著下降(p0.05)。本研究表明,Periostin在非小细胞肺癌患者的血浆和肺癌组织中表达量提高,可以增强肺癌细胞的增殖和侵袭能力。     

Bacteria Associated with Immunoregulatory Cells in Mice

This study examined bacteria-immune interactions in a mouse model possessing microbiota-dependent immune regulatory features similar to those occurring in human atopy, colitis, and immune regulation. Associations between the abundance of several bacterial phylotypes and immunoregulatory target cell types were identified, suggesting that they may play a role in these phenotypes.Bacteria are involved in critical aspects of immune system development and regulation (5, 23, 26, 29). Mice raised under germfree conditions exhibit a variety of abnormalities, including hypoplastic Peyer''s patches, reduced numbers of IgA-producing cells, relatively unstructured spleen and lymph nodes, and hypogammaglobulinemic serum (23). Remarkably, after several weeks of exposure to standard intestinal microbiota, normal immune structure and function are restored. Mechanistic details underlying microbe-immune interactions have been recently elucidated for two common intestinal bacteria. Bacteroides thetaiotaomicron was shown to induce the angiogenin Ang4, a component of innate immunity possessing microbicidal activity against a wide range of intestinal microbes, including both bacterial and fungal pathogens (16). In addition, studies of the Bacteroides fragilis zwitterionic capsular polysaccharide A have established it as a cognate antigen of certain CD4⁺ T cells, which programs immune effector polarization (24) and protection of mice from infection by Helicobacter hepaticus through several immune-mediated mechanisms (25). Resident microbiota also modify the interaction of dendritic cells with regulatory T-cell populations, with resultant susceptibility to chronic inflammatory disease, like colitis (15, 28).Recent work by Braun and colleagues has characterized a mouse model with unique immunologic features linking resident microbiota with levels of regulatory CD8⁺ T cells (13, 17, 39). This model is comprised of two physically isolated colonies of isogenic mice harboring distinct microbial communities: conventional floras (CF) and restricted floras (RF). CF refers to C57BL/6 mice housed in a standard specific-pathogen-free facility, while RF refers to C57BL/6 mice containing a different complement of intestinal microorganisms (13, 30), originally created by transferring several nonpathogenic anaerobic bacteria into antibiotic-treated mice (13). RF mice differ from CF mice in several immunologic phenotypes, including selective reduction of marginal zone (MZ) B cells (39), plasmacytoid dendritic cells (pDC) (13), and invariant natural killer (iNK) T cells (38a), as well as naïve CD4⁺ and CD8⁺ T cells (17). In addition, RF mice were shown to be resistant to colitis under genetic or adoptive transfer conditions that permit disease activity in CF mice (2). RF mice also cleared experimental infections by Campylobacter jejuni more slowly than did their CF counterparts (6). The resulting concept is that certain resident microbiota, which may be more abundant in RF mice than in CF mice, induce invariant Qa-1 T cells, with resultant changes in host immunoregulation and microbial surveillance (2).An important issue raised by the foregoing observations is the identity of resident microbiota responsible for this host immunoregulatory response. The objective of this study was to develop a methodology, based on bacteria-immune interactions in the RF/CF mouse model, to identify candidate microbiota. In this study, we employed a series of experiments examining associations between the population densities of bacterial rRNA genes and several immunologic features that differ between CF and RF mice.     

人肝癌细胞与肝星形细胞裸鼠皮下共培养体系的建立

目的:建立裸鼠皮下共培养人肝癌细胞与肝星形细胞模型,观察人肝癌细胞与肝星形细胞间相互作用后超微结构的改变.方法:将16只裸鼠分为两组,肝癌细胞单独培养组和癌细胞与肝星形细胞共培养组,40天后将荷瘤组织切片行光镜及透射电镜观察.结果:肝癌细胞单独培养组中可观察到肝癌细胞的胞质液化及早期细胞凋亡现象,而共培养组中可见肝星形细胞时肝癌细胞的趋化现象,可观察到肝癌细胞结构完整且有增殖趋势.结论:裸鼠皮下荷瘤三维立体模型建立成功,该模型能够模拟肝癌微环境中肝癌细胞与肝星形细胞问的作用,为进一步研究肝癌细胞与肝星形细胞间的相互作用奠定了基础.     

Bioelectromagnetic Field Effects on Cancer Cells and Mice Tumors

We present possibilities and trends of ELF bioelectromagnetic effects in the mT amplitude range on cancer cells and on mice bearing tumors. In contrast to invasive electrochemotherapy and electrogenetherapy, using mostly needle electrodes and single high-amplitude electropulses for treatment, extremely low-frequency (ELF) pulsating electromagnetic fields (PEMF) and sinusoidal electromagnetic fields (SEMF) induce tumor cell apoptosis, inhibit angiogenesis, impede proliferation of neoplastic cells, and cause necrosis non invasively, whereas human lymphocytes are negligibly affected. Our successful results in killing cancer cells—analyzed by trypan blue staining or by flow cytometry—and of the inhibition of MX-1 tumors in mice by 15–20?mT, 50?Hz treatment in a solenoid coil also in the presence of bleomycin are presented in comparison to similar experimental results from the literature.

In conclusion, the synergistic combinations of PEMF or SEMF with hyperthermia (41.5°C) and/or cancerostatic agents presented in the tables for cells and mice offer a basis for further development of an adjuvant treatment for patients suffering from malignant tumors and metastases pending the near-term development of suitable solenoids of 45–60?cm in diameter, producing >20?mT in their cores.     

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma^1-4. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker⁵, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered. Therefore, it is essential to determine whether there is an alternative medulloblastoma tumor cell-of-origin based on which cell-type specific therapeutic modality can be developed. To this end, intracranial orthotopic allografting of genetically marked tumor cell types followed by subsequent analyses of secondary tumor development in recipients will allow determination of the cellular origin of tumor-initiating cells. Here we describe the experimental protocol for intracranial orthotopic allografting of medulloblastoma cells derived from primary tumor tissue, and this procedure can also be used for transplanting cells from established cell lines.Download video file.^{(31M, mov)}     

小鼠睾丸支持细胞的生物学特性研究

目的:获得高纯度的小鼠支持细胞,用以研究睾丸支持细胞在诱导胚胎干细胞向雄性生殖细胞分化过程中的作用,同时借助睾丸支持细胞减少进行体内诱导试验时可能产生的免疫排斥反应。方法:用胶原酶和胰蛋白酶组合消化结合选择性贴壁法从1周龄昆明白小鼠睾丸分离获得睾丸支持细胞,纯化后进行体外培养,观察其体外培养的生物学特性。结果:睾丸支持细胞体外培养3～4h即贴壁,贴壁后伸出3～4个突起,为成纤维型细胞,在体外培养2～3d即长满全瓶。油红Ο染色显示,其胞质含有大量脂滴。透射电镜观察结果表明,支持细胞核仁周围有卫星核小体。RT-PCR结果显示获得的细胞表达缪勒管抑制物,不表达促黄体素受体和小鼠VASA同源物。结论:获得了较高纯度的小鼠睾丸支持细胞,可以用于诱导小鼠胚胎干细胞向雄性生殖细胞分化的体内和体外试验。     

肿瘤坏死因子相关的凋亡诱导配体(TRAIL)基因抑制小鼠子宫基质细胞的蜕膜化

为了观察肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因对体外培养的小鼠蜕膜基质细胞增殖及凋亡的作用,探讨TRAIL对小鼠子宫蜕膜化进程的影响,构建TRAIL过表达及干扰质粒,转染小鼠基质细胞后诱导蜕膜化发生.转染72h后,应用半定量RT-PCR和Western blotting检测蜕膜基质细胞中TRAILmRNA和蛋白质的表达情况、MTT法观察蜕膜基质细胞的生长和增殖能力、流式细胞术检测蜕膜基质细胞的细胞周期分布情况和凋亡率.经酶切和核苷酸测序证实,TRAIL基因正确克隆入真核表达载体且能够上调TRAIL的表达,干扰质粒能有效地抑制TRAIL基因的表达.TRAIL过表达和RNA干扰的结果表明:TRAIL具有将蜕膜基质细胞阻滞在G0/G1期、抑制蜕膜基质细胞增殖并促使其凋亡的功效,提示TRAIL可能参与调节胚胎植入后基质细胞的有序蜕膜化进程.     

Engineering Antigen-Specific T Cells from Genetically Modified Human Hematopoietic Stem Cells in Immunodeficient Mice

There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, “transgenic” human anti-HIV T cell receptor (TCR). Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.     

镉对雄性小鼠生精细胞的影响

为了研究镉对小鼠生殖力和生精细胞的作用,本文对氯化镉处理后的雄性小鼠进行了交配实验,以观察其对怀孕率、每窝产仔数及子代性比的影响。测定比较了注射镉后,小鼠成熟精子的总LDH酶和LDH-X酶的活性;还用双向电泳方法分析了成熟精子的蛋白质变化。结果表明,处理组在怀孕率、每窝产仔数及子代性比方面无统计学意义的差异。成熟精子的总LDH活性经镉处理后未发现明显变化,但镉能显著地抑制与精子运动的能量有关的LDH-X酶的活性。双向电泳图谱表示,镉处理后,精子中含量较少的三组蛋白质或消失不见,或发生明显变化。