darbufelone对胃癌SGC-7901细胞裸鼠移植瘤血管生成的影响

目的观察环氧合酶-2／5-脂氧合酶双重抑制剂darbufelone对人胃癌皮下移植瘤血管生成的影响,并初步探讨其机制。方法建立裸鼠实体瘤模型,随机分为darbufelone组和对照组,darbufelone及生理盐水分别连续灌服4周。测量肿瘤质量、体积,计算抑瘤率;免疫组化检测CD34并计算微血管密度;RT—PCR法及Western blot法分析移植瘤组织中MMP-9、VEGF的表达。结果darbufelone可明显抑制裸鼠移植瘤的生长,质量抑瘤率为58．42％,体积抑瘤率为67．13％。darbufelone组的微血管密度（MVD）（15．36±0．30）明显低于对照组（29．47±0．63）（P〈0．05）;darbufelone组肿瘤组织中VEGF及MMP-9在基因水平及蛋白水平的表达（P〈0．05）。结论darbufelone能有效抑制裸鼠移植瘤的生长,减少移植瘤组织中VEGF及MMP-9的表达,抑制肿瘤的微血管生成,具有抗血管生成的作用。     

全新结构抗肿瘤药物TNBG对裸鼠移植瘤生长抑制作用及药物毒副作用初步评价

目的观察TNBG对人肝癌细胞株QGY-7701裸鼠移植瘤生长抑制情况,初步评估药物的毒副作用。方法采用裸鼠复制人肝癌移植瘤模型。实验分对照组和给药组,腹腔注射给药,持续5周。治疗期间定期测量肿瘤大小,观察裸鼠生存状况。实验结束时处死裸鼠,测量肿瘤体积计算抑瘤率;眼眶取血,分离血清检测血脂、肝、肾功能;摘取裸鼠肿瘤及主要脏器组织作苏丹Ⅲ染色观察脂质沉积情况。结果TNBG对移植瘤的抑瘤率为60．1％;各实验组裸鼠血脂、肝、肾功检测与对照组比无显著性差异（P〉0．05）;苏丹Ⅲ染色显示给药组裸鼠主要脏器组织内无脂质沉积,而肿瘤组织中可见大量脂质沉积。结论TNBG对人肝癌细胞裸鼠移植瘤有较明显的抑制作用,毒副作用小,抗肿瘤作用具有选择性。     

microRNA-451对人结肠癌细胞SW620裸鼠皮下种植瘤生长的抑制作用及机制探讨

目的 探讨microRNA-451(miR-451)对人结肠癌细胞系SW620裸鼠皮下种植瘤生长的影响,并探讨其可能机制.方法 18只裸鼠随机分为3组,实验组(SW-620-451组)、阴性对照组(SW-620-NC)、空白对照组(SW-620组),每组6只,分别皮下接种转染miRNA-451 agomir的SW620细胞、转染了阴性片段agomir Negative Control(NC)的SW620细胞和不经处理的结肠癌SW620细胞,并每周两次于瘤体内分别注射miRNA-451 agomir、miRNA-451 agomir NC片段及生理盐水.比较各组裸鼠皮下形成瘤体的大小并计算抑瘤率,接种四周后处死裸鼠取组织,用免疫组化法和Western-blot法定性定量分析和比较肿瘤相关基因c-myc的表达.结果 各组裸鼠皮下接种肿瘤细胞6天后均有瘤体形成,成瘤率100％,实验组(SW-620-451组)瘤体生长速度明显低于其他两组(p＜0.05);时间终点为第30天时,实验组(SW-620-451组)裸鼠皮下种植瘤的体积为(0.95±0.13)cm3,阴性对照组(SW-620-NC组)为(2.25±0.50) cm3,空白对照组(SW-620组)为(2.46±0.59)cm3;实验组(SW-620-451组)瘤体体积显著小于其他两组体积(p＜0.05);实验组(SW-620-451组)瘤体质量为(1.15±0.13)g,明显低于SW-620-NC组(2.59±0.46)g及SW-620组(2.76±0.44)g(p ＜0.05).实验组种植瘤中c-myc的表达量较另外两组下降(p＜0.05).结论 转染miR-451后可以抑制结肠癌细胞SW620裸鼠皮下移植瘤的生长,其机制可能与下调c-myc表达有关.     

COPD大鼠肺组织中 LTB4水平、5 LOmRNA表达及齐留通的干预作用

目的探讨慢性阻塞性肺疾病(chronicobstructivepulmonarydisease,COPD)大鼠模型肺组织中白三烯 B4(LeukotrieneB4, LTB4)含量与5 脂氧合酶(5 LO,5 lipoxygenase)mRNA水平的变化,以及给予齐留通干预的影响.方法36只健康雄性 Wistar大鼠随机分为对照组、模型组、药物组,采用被动吸烟法制作 COPD大鼠模型,给予药物组鼻饲齐留通,测定肺功能,各组肺组织匀浆检测其 LTB4含量及髓过氧化物酶(Myeloperoxidase,MPO)活性,RT PCR法检测5 LOmRAN的表达.结果模型组、药物组的0.3秒用力呼吸容积(Forcedexpiratoryvolumeinthe0.3second,FEV0.3)/用力肺活量(Forcedvitalcapacity,FVC)%较对照组显著下降(P<0.001),吸气相阻力(Inspiratoryphaseresistance,Ri)和呼气相阻力(expiratoryphaseresistance,Re)较对照组显著增加(P<0.001,P<0.05);模型组较药物组 FEV0.3/FVC%下降程度更低,Ri、Re更高,两组间差异有显著性(P<0.05).比较各组大鼠肺组织 LTB4水平,MPO活性及5 LOmRNA表达:模型组、药物组较对照组显著升高(P<0.001);与模型组比较,齐留通干预使得三项指标均有显著降低(P<0.001).结论 LTB4及5 LO参与 COPD的气道炎症过程.5 LOX抑制剂齐留通可部分抑制减少 COPD大鼠肺组织中 LTB4产生,其机制可能为抑制5 LO表达     

胃转流术对2型糖尿病大鼠肾脏氧化应激及蛋白激酶C活性的影响

目的观察胃转流术对2型糖尿病大鼠肾脏氧化应激及蛋白激酶C(PKC)活性的影响。方法40只SD大鼠随机分为正常对照组(8只),糖尿病模型组(32只),模型组注射STZ(35 mg/kg),血糖稳定后确定成模30只,再将其分为糖尿病对照组(8只),假手术组(8只),手术组(14只)。术后8周检测各组大鼠空腹血糖、肾重指数(肾重/体重)、尿素氮(BuN)、血肌酐(Cr),测定各组大鼠肾组织总超氧化物歧化酶(TSOD)、谷胱甘肽过氧化酶(GSHPX)活性,脂质过氧化代谢产物丙二醛(MDA)含量及PKC的活性,HE染色观察肾组织病理变化。结果与糖尿病对照组和假手术组相比,手术组能明显降低空腹血糖、改善肾重指数、BUN、Cr等指标(P0.05),同时肾组织抗氧化酶TSOD、GSH-PX活性增强,MDA含量减少,肾脏PKC活性降低(PO.05)。HE染色显示手术组病变也明显轻于糖尿病对照组和假手术组。结论胃转流术可能通过抑制2型糖尿病大鼠肾脏氧化应激及PKC活性增强发挥肾脏保护作用。     

外源性干细胞因子对糖尿病胃轻瘫大鼠胃窦Cajal细胞的影响

目的探讨外源性干细胞因子（SCF）能否改善糖尿病胃轻瘫（DGP）大鼠胃窦Cajal细胞（ICC）的异常病变。方法sD大鼠随机分为实验组和对照组，实验组腹腔注射链脲佐菌素（STZ）50mg／kg并高糖高脂饲料不规律喂养构建DGP模型，对照组腹腔注射等量生理盐水并普通饲料规律喂养。成模后随机分为糖尿病组（DM组）、糖尿病＋外源性干细胞因子治疗组（DM＋SCF组）；DM＋SCF纽腹腔注射SCF0．4ug／（kg·d），共15天，对照组和DM纽每天腹腔注射等量的磷酸盐缓冲液（pH=7．4）。RT-PCR、Wester Dblot检测胃窦组织中SCF及c—kit的mRNA及蛋白表达；免疫组化、透射电镜观察胃窦组织中Cajal细胞的变化。结果DM组大鼠胃窦组织SCF及c—kit的mRNA及蛋白表达下降，Cajal细胞数量减少，细胞内线粒体肿胀、内质网扩张；予外源性SCF干预后，胃窦组织中SCF及C—kjf的mRNA及蛋白表达上调，caial细胞数量增多、超微结构明显改善。结论外源性SCF能在一定程度上改善或逆转糖尿病胃轻瘫大鼠胃窦Cajal细胞的异常病变：     

甘氨双唑钠对鼻咽癌移植瘤的时辰放射增敏作用

目的 观察甘氨双唑钠（CMNa）联合时辰放疗对鼻咽癌祼鼠移植瘤的时辰放射增敏作用,并探讨其作用机制.方法 将荷瘤裸鼠随机分为3组：放疗组、放疗＋CMNa组、空白对照组,每组分3HALO（光照后小时,hours after light onset）、9HALO、15HALO、21HALO四个时辰进行相应处理.测定肿瘤再生长延长时间（regrowth delay time,TGD）,绘制生长曲线.用免疫组化法检测各组肿瘤标本中HIF-1α、γ-H2AX和凋亡蛋白的表达.结果 通过对各组TGD的比较.以放疗＋CMNa组对肿瘤的抑制效果最好.在该组中,TGD：15HALO＞21HALO＞9HALO＞3HALO,15HALO与3HALO的TGD比较有统计学意义 15HALO与3HALO的HIF-1α、γ-H2AX及凋亡蛋白的表达水平比较有统计学意义.结论 甘氨双唑钠联合时辰放疗对鼻咽癌裸鼠移植瘤有明显的时辰放射增敏作用,以15HALO放疗＋CMNa组对肿瘤的抑制效果最佳.其机制可能与HIF-1α的表达.DNA双链损伤,凋亡有关.     

儿茶素对晚期糖基化终末产物诱导的内皮细胞凋亡的干预作用

目的探讨儿茶素对晚期糖基化终末产物（AGEs）诱导内皮细胞凋亡的干预作用。方法糖孵育法制备晚期糖基化终末产物,分离肾血管内皮细胞,将内皮细胞分成空白对照组、AGE组、浓度分别为10、15、20μg／ml的儿茶素组共五组。实验末,采用AlamarBlue还原法测定同内皮细胞增生活力,TUNEL法测定细胞凋亡率,生化法测定培养上清中·OH、MDA浓度,RT-PCR测定Bcl-2mRNA表达,Western-Blotting测定Bcl-2蛋白活性。结果与对照组相比,AGE组内皮细胞增生活性、Bcl-2基因与蛋白活性显著降低（P均〈0．01）;凋亡率、·OH与MDA浓度显著增加（P均〈0．01）;与AGE组相比,儿茶素各浓度组内皮细胞增生活力与Bcl-2基因与蛋白活性表达增高,凋亡率、·OH与MDA浓度降低;儿茶素各剂量组之间呈浓度梯度效应。结论儿茶素可降低AGEs引起的血管内皮细胞凋亡,其机制可能是通过有效清除活性氧自由基、上调Bcl-2mRNA与活性蛋白表达,阻断内皮细胞内过氧化物的堆积二种途径实现的。     

RNA干扰CD133基因对结肠癌干细胞生物学行为的影响

目的探讨CD133基因表达、活化被阻断后对结肠癌干细胞生物学行为的影响。方法从EpcAMhighCD44＋结肠癌干细胞中流式分选获得CD133＋细胞,感染LV-CD133shRNA载体慢病毒后观察CD133＋结肠癌干细胞在生长方式、成球能力、克隆形成率、成瘤能力以及ABCC2mRNA的变化;Westernblot分析CD133-细胞中CD133蛋白表达情况。结果EpcAMhighCD44＋结肠癌干细胞中CD133＋细胞比例为89．2％。实验组经过LV-CD133shRNA载体病毒感染后,在干细胞养液中细胞改悬浮生长的方式为贴壁生长,不能形成细胞球。MTT法测定发现细胞增殖减慢,克隆形成率明显下降。将感染细胞移植在Balb／C裸鼠体内,在观察期间,感染LV—CD133shRNA载体病毒的CD133＋细胞无肿瘤形成。ABCG2mRNA表达水平明显降低（P〈0．01）。从EpcAMhighCD44＋结肠癌干细胞中流式分选获得CD133-细胞,其中也有CD133蛋白的表达。结论CD133维持结肠癌干细胞生物学特性。     

Roux-en-Y胃旁路术对2型糖尿病大鼠肾脏非对称性二甲基精氨酸代谢的影响

目的研究Roux-en-Y胃旁路术对2型糖尿病大鼠肾脏非对称性二甲基精氨酸(ADMA)代谢的影响。方法 40只SD大鼠,随机分为正常对照组(NC组,n=8)和糖尿病模型组(32只)。糖尿病模型组成模30只,随机分为糖尿病对照组(DC组,n=8)、糖尿病假手术组(DSO组,n=8)及糖尿病手术组(DO,n=14),DO组行胃旁路术,DSO组行胃窦十二指肠离断原位吻合。检测术前、术后1、2、4、8周血胰高血糖素样肽(GLP-1)水平,测定术前、术后8周空腹血糖(FBG),肾脏ADMA、血尿素氮(BUN)、肌酐(Cr)、一氧化氮(NO)水平,一氧化氮合酶(NOS)活性;检测蛋白质精氨酸甲基转移酶1(PRMT1)mRNA及二甲基精氨酸二甲胺水解酶1(DDAH1)mRNA,PRMT1蛋白表达水平,PAS染色观察肾脏组织学变化。结果随着手术时间延长,D0组GLP-1水平增加,其余各组无明显变化(P0.05),术后8周,DC和DSO组较DO组和NC组FBG、BUN、Cr、ADMA水平,PRMT1mRNA和蛋白表达水平均明显升高(P0.05),NO、NOS、水平、DDAH1的mRNA表达明显降低(P0.05);PAS染色示DC组和DSO组肾小球基质沉积,阳性染色物增多,余无明显变化。结论 2型糖尿病肾脏组织中的ADMA升高;胃旁路术促进ADMA代谢,改善了肾功能。     

Inhibition of SRC family kinases facilitates anti-CTLA4 immunotherapy in head and neck squamous cell carcinoma

The immune system plays a critical role in the establishment, development, and progression of head and neck squamous cell carcinoma (HNSCC). As treatment with single-immune checkpoint agent results in a lower response rate in patients, it is important to investigate new strategies to maintain favorable anti-tumor immune response. Herein, the combination immunotherapeutic value of CTLA4 blockade and SFKs inhibition was assessed in transgenic HNSCC mouse model. Our present work showed that tumor growth was not entirely controlled when HNSCC model mice were administered anti-CTLA4 chemotherapeutic treatment. Moreover, it was observed that Src family kinases (SFKs) were hyper-activated and lack of anti-tumor immune responses following anti-CTLA4 chemotherapeutic treatment. We hypothesized that activation of SFKs is a mechanism of anti-CTLA4 immunotherapy resistance. We, therefore, carried out combined drug therapy using anti-CTLA4 mAbs and an SFKs’ inhibitor, dasatinib. As expected, dasatinib and anti-CTLA4 synergistically inhibited tumor growth in Tgfbr1/Pten 2cKO mice. Furthermore, dasatinib and anti-CTLA4 combined to reduce the number of myeloid-derived suppressor cells and Tregs, increasing the CD8⁺ T cell-to-Tregs ratio. We also found that combining dasatinib with anti-CTLA4 therapy significantly attenuated the expression of p-STAT3^Y705 and Ki67 in tumoral environment. These results suggest that combination therapy with SFKs inhibitors may be a useful therapeutic approach to increase the efficacy of anti-CTLA4 immunotherapy in HNSCC.     

CD24 controls Src/STAT3 activity in human tumors

CD24 is a glycosyl-phosphatidylinositol-anchored membrane protein that is frequently over-expressed in a variety of human carcinomas and is correlated with poor prognosis. In cancer cell lines, changes of CD24 expression can alter several cellular properties in vitro and tumor growth in vivo. However, little is known about how CD24 mediates these effects. Here we have analyzed the functional consequences of CD24 knock-down or over-expression in human cancer cell lines. Depletion of CD24 reduced cell proliferation and adhesion, enhanced apoptosis, and regulated the expression of various genes some of which were identified as STAT3 target genes. Loss of CD24 reduced STAT3 and FAK phosphorylation. Diminished STAT3 activity was confirmed by specific reporter assays. We found that reduced STAT3 activity after CD24 knock-down was accompanied by altered Src phosphorylation. Silencing of Src, similar to CD24, targeted the expression of prototype STAT3-regulated genes. Likewise, the over-expression of CD24 augmented Src-Y416 phosphorylation, the recruitment of Src into lipid rafts and the expression of STAT3-dependent target genes. An antibody to CD24 was effective in reducing tumor growth of A549 lung cancer and BxPC3 pancreatic cancer xenografts in mice. Antibody treatment affected the level of Src-phosphorylation in the tumor and altered the expression of STAT3 target genes. Our results provide evidence that CD24 regulates STAT3 and FAK activity and suggest an important role of Src in this process. Finally, the targeting of CD24 by antibodies could represent a novel route for tumor therapy.     

Immunocytochemical and enzymatic detection of lysozyme in human colon carcinoma cell lines

Six of a total of 14 human colon carcinoma cell lines produce and secrete lysozyme in vitro. Three also produce the enzyme when propagated in vivo in athymic mice. None of the lysozyme positive cells stained in a manner typical of Paneth cells. Additionally, lysozymes from all six colon lines possess identical molecular weights (approximately 14,000 daltons).     

Cross-reactive monoclonal antibodies to alcohol dehydrogenases

Summary Three anti-horse liver alcohol dehydrogenase (HLADH) monoclonal antibodies are described. Two are specific for ADH and cross-react with class I and II enzymes from mouse, horse and Chinese hamster. They are specific for the native enzyme but do not inhibit enzyme activity except when combined at high concentration. The third antibody was isolated as a response to rabbit metallothionein. It binds metalloproteins and inhibits ADH activity.     

Functional impact of PTP1B-mediated Src regulation on oxidative phosphorylation in rat brain mitochondria

Given the presence of Src and PTP1B within rat brain mitochondria, we have investigated whether PTP1B regulates Src activity in mitochondria as in the cytosol. Results showed that Src was stimulated by in vitro addition of ATP to mitochondria, and this stimulation was reversed by a membrane-permeable allosteric inhibitor of PTP1B and by a potent selective Src inhibitor. They also indicated a direct action of PTP1B on phosphorylated tyrosine 527 residue of Src, thus implicating a role for PTP1B in the modulation of Src activity in mitochondria. Putative Src and PTP1B substrates were identified by liquid chromatography tandem mass spectrometry and two-dimensional blue native/SDS-PAGE. Both inhibitors inhibited ADP-stimulated respirations concurrently with Src activation and complex IV activation by ATP, while having no effect or increasing the activity of the other complexes. Our analysis emphasizes the regulatory function of Src and its modulation by PTP1B on oxidative phosphorylation in mitochondria.     

Novel regulation and function of Src tyrosine kinase

Src tyrosine kinase is a critical signal transducer that modulates a wide variety of cellular functions. Misregulation of Src leads to cell transformation and cancer. Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are another group of signaling molecules that transduce signals from cell-surface receptors to generate physiological responses. Recently, it was discovered that Gαs and Gαi could directly stimulate Src family tyrosine kinase activity. This novel regulation of Src tyrosine kinase by G proteins provides insights into the adenylyl cyclase-independent signaling mechanisms involved in ligand-induced receptor desensitization, internalization and other physiological processes. Received 17 August 2001; received after revision 22 October 2001; accepted 24 October 2001     

pEGFPN1-dnEGFR对胃癌细胞的化疗增敏作用

目的研究人 EGFR显性负性突变体真核表达载体(pEGFPN1 dnEGFR)对人胃癌细胞株 SGC 7901和 NCI N87化疗敏感性的影响,并探讨其可能机制.方法 MTT法测定奥沙利铂对稳定转染 pEGFPN1 dnEGFR和 pEGFP N1载体的两种胃癌细胞的量效反应.奥沙利铂作用各组细胞24h后,RT PCR检测各组细胞中 Caspase 3和 CyclinD1的 mRNA表达情况;Westernblot检测各组细胞中 Caspase 3和 CyclinD1蛋白表达情况.结果转染 pEGFPN1 dnEGFR后,两种胃癌细胞对奥沙利铂的敏感性增加,奥沙利铂对 pEGFPN1 dnEGFR转染组细胞的增殖抑制率(VI)与对照组相比有显著提高(P<0.05).RT PCR显示 pEGFPN1 dnEGFR转染组细胞 CyclinD1mRNA表达较对照组下降,而 Caspase 3mRNA表达较对照组升高(P<0.05);Westernblot显示 pEGFPN1 dnEG FR转染组细胞 CyclinD1蛋白表达较对照组下降,而 Caspase 3蛋白表达较对照组升高(P<0.05).结论 EGFR显性负性突变体能提高胃癌细胞对化疗药物奥沙利铂的敏感性,其机制可能与 Caspase 3和 CyclinD1有关     

Antizyme inhibitor: mysterious modulator of cell proliferation

In contrast to the considerable interest in the oncogene ornithine decarboxylase (ODC) and in the family of antizymes with regard to cell proliferation and tumorigenesis, the endogenous antizyme inhibitor (AZI) has been less well studied. AZI is highly homologous to the enzyme ODC but does not possess any decarboxylase activity. Elevated ODC activity is associated with most forms of human malignancies. Antizymes bind ODC, inhibit ODC activity and promote the ubiquitin-independent degradation of ODC. Consequently they are proposed as tumor suppressors. In particular, the most studied member of the antizyme family, antizyme 1, has been demonstrated to play a role in tumor suppression. AZI inactivates all members of the antizyme family, reactivates ODC and prevents the proteolytic degradation of ODC, which may suggest a role for AZI in tumor progression. Received 9 December 2005; received after revision 13 April 2006; accepted 1 June 2006     

Factor(s) from nonmacrophage bone marrow stromal cells inhibit Lewis lung carcinoma and B16 melanoma growth in mice

Bone marrow stroma produces positive and negative growth regulators which constitute the hematopoietic microenvironment. As many tumors metastasize to the bones, these regulators may also influence tumor growth. Hematopoietic cytokines may indeed exert both positive and negative effect on tumor growth. We report that, when mixed with tumor cells. adherent bone marrow cells inhibit primary tumor growth and metastases formation in mice transplanted with Lewis lung carcinoma or B16 melanoma. Peritoneal macrophages or lymph node cells did not exert any influence. The tumor inhibition was apparently due to soluble factor(s) released by marrow stromal cells. In cocultures with B16 melanoma cells, adherent bone marrow cells exerted a significant antiproliferative effect which was increased by previous culture of the bone marrow cells with granulocyte-macrophage colony-stimulating factor but not with macrophage colony-stimulating factor. Neither neutralizing antibodies against tumor necrosis factor-alpha, transforming growth factor-beta or interferon alpha/beta nor addition of Escherichia coli lipopolysaccharide to generate inflammatory cytokines could affect the antiproliferative effect of bone marrow stromal cells. The bone marrow stroma factor(s) which inhibit tumor growth might, therefore, be a novel growth regulator.