抗DR5单抗CTB006联合5-FU对结直肠癌细胞系作用研究

目的探讨肿瘤坏死因子相关诱导配体受体（DR5）激动型单抗CTB006对人结直肠痛细胞系SW1116、SW480、SW620、Colo205的影响。方法采用ATPlite法检测CTB006组、5-FU组及两药物合用组作用后的细胞存活率,流式细胞仪技术检测细胞系表面DR5的表达水平,研究两者之间的关系。结果CTB006和5-FU对四种结直肠癌细胞增殖均表现出抑制作用。CTB006对早期细胞SW1116增殖抑制效果欠佳,对中晚期细胞SW480、SW620、Colo205有着良好的增殖抑制作用（P〈0．05）。当联合使用5-FU后,对后三者有明显的协同作用。流式细胞仪检测DR5表达水平,SW1116细胞表达（47．01±30．4）％,SW480细胞表达（76．11±15．1）％,SW620细胞表达（86．77±9.3）％,Col0205细胞表达（93．55±7．9）％。结论CTB006对中晚期结直肠癌杀伤作用较强,联用5-FU具有显著的协同作用。     

不同人结直肠肿瘤细胞系对常用抗肿瘤药物体外化疗敏感性的比较

 目的：观察5种常用抗肿瘤药物对这些人结直肠肿瘤细胞系的生长抑制作用,探讨5种常用抗肿瘤药物对11株人结直肠肿瘤细胞系的作用强度以及比较其体外敏感性,研究不同抗肿瘤药物对人结直肠癌细胞系HCT116和SW480热休克蛋白27（HSP27）和HSP70表达水平的影响。方法：采用CCK-8（Cell Counting Kit-8）法检测5种常用抗肿瘤药物分别对11株人结直肠肿瘤细胞系的生长抑制效应,计算50%抑制浓度（50% inhibitory concentration, IC50）及敏感指数,并比较不同人结直肠肿瘤细胞系对5种抗肿瘤药物的敏感性,Western blotting检测HSP27和HSP70蛋白表达水平。结果：11株人结直肠肿瘤细胞系对5-氟尿嘧啶(5-FU)和奥沙利铂(OHP)均比较敏感,没有明显耐药性;5株人结直肠肿瘤细胞系对丝裂霉素(MMC)敏感,6株中度敏感;除SW1116 外的10株人结直肠肿瘤细胞系都对多西紫杉醇(DXL)敏感,而SW1116细胞对DXL表现出明显耐药性;除LS174T和SW1116外的9株人结直肠肿瘤细胞系都对伊立替康(IFL)表现出中度敏感,LS174T细胞对IFL表现敏感,而SW1116细胞对IFL表现出明显耐药性。抗肿瘤药物作用于人结直肠癌细胞系HCT116和SW480使HSP27的表达上调,但HSP70的表达水平变化不明显。结论：LS174T是多药敏感细胞株,SW1116是多药耐药细胞株,5-FU和OHP为广谱抗结直肠肿瘤药物;化疗药物的敏感性及HSP27表达量检测对临床选择化疗药物具有一定的提示意义。     

蚯蚓体腔液体外抗呼吸道合胞病毒的实验研究

目的研究蚯蚓体腔液（Earthworm coelomic fluid,ECF）体外抗呼吸道合胞病毒（RSV）的作用。方法以Hep-2细胞为宿主细胞,利巴韦林作为阳性对照药物,通过观察细胞病变效应（CPE）和MTT染色法来研究蚯蚓体腔液体外抗RSV的作用。结果ECF和利巴韦林对Hep-2细胞的CC50分别为3．11mg／ml和1．35mg／ml。在直接杀灭作用实验中,IC50为184．1μg／ml,SI值为16．87;在抑制病毒复制实验中,IC50为1555．8μg／ml,SI值为1．99;在阻断病毒侵入实验中,没有观察到抗病毒的作用。结论蚯蚓体腔液具有直接杀灭和抑制RSV复制的作用,其中直接杀灭作用的抗病毒效果较明显,但不具有阻断RSV入侵的作用。     

离子霉素对SW480和SWO-38细胞中E-cadherin裂解的影响

 目的：观察可促进钙离子内流的工具药离子霉素(ionomycin),对不同肿瘤细胞株SW480及SWO-38其C末端片段2（E-cad/CTF2）的表达裂解的影响。方法：应用MTT法确定ionomycin作用于 SW480及SWO-38细胞的最佳浓度;Western blotting检测ionomycin 作用不同时间后E-cadherin全长及其C末端片段2(E-cad/CTF2)的表达水平;共聚焦显微镜动态检测SWO-38细胞胞内Ca2+浓度变化。结果：Ionomycin对SW480及SWO-38细胞均有细胞毒性作用,半数抑制浓度均为12 μmol/L,ionomycin可促进 SW480细胞中Ca2+浓度增加,E-cadherin裂解,E-Cad/CTF2片段水平升高,ionomycin没有引起SWO-38细胞中的大量钙内流,对E-cadherin裂解没有明显作用。结论：Ionomycin可促进钙离子内流,引起SW480肿瘤细胞E-cadherin裂解,但对SWO-38细胞E-cadherin裂解无明显影响。     

奥沙利铂对结肠癌SW480细胞迁移侵袭及细胞凋亡的影响

目的探讨奥沙利铂对结肠癌细胞SW480细胞的增殖、凋亡及迁移作用的影响。。方法体外培养结肠癌SW480细胞,加入不同浓度的奥沙利铂(5, 10, 20, 40μg/mL),分别培养0,24,48,72h后,平板克隆形成实验检测奥沙利铂对结肠癌SW480细胞生长能力的影响;细胞划痕实验观察实验处理前后SW480细胞的迁移能力的变化;Matrigel细胞侵袭实验检测奥沙利铂对恶性肿瘤细胞侵袭和转移的影响;Real-time PCR和Western blot验证Bcl2、Bax、Bcl-Xl、E-Cadherin等基因的表达水平。结果奥沙利铂降低结肠癌SW480细胞的克隆形成率,显著抑制细胞的迁移能力,但奥沙利铂对结肠癌SW480细胞的侵袭能力无抑制作用;下调原癌基因Bcl2和Bcl-Xl的表达水平、上调抑癌基因Bax的表达水平。结论奥沙利铂抑制SW480细胞增殖、促进凋亡与下调Bcl2/Bax比值相关。     

WTP53联合双自杀基因CD和TK抑制结肠癌细胞SW480生长

目的探讨野生型P53(WTP53)基因联合双自杀基因CD、TK对P53突变的SW 480结肠癌细胞的体外生长抑制作用、旁观者效应以及联合基因应用的效果。方法将P53的表达载体pCMV-P53转染SW 480细胞;用含有CD、TK双自杀基因的反转录病毒感染SW 480,得到稳定转染的阳性克隆。RT-PCR鉴定目的基因的表达。绘制细胞生长曲线等方法观察不同混合条件下WTP53、TK、CD系统的联合应用效应和药物相互作用指数。结果SW 480/P53细胞倍增时间明显延长。SW 480/TK-CD细胞对前体药物丙氧鸟苷(GCV)、5-氟胞嘧啶(5-FC)呈剂量依赖性的细胞生长抑制作用。SW 480/P53和SW 480/TK-CD混合细胞的生长抑制作用、旁观者效应和药物相互作用最显著。结论WTP53基因和TK/GCV、CD/5-FC系统对人结肠癌细胞SW 480均具有生长抑制和旁观者效应,联合应用细胞毒性更明显。     

骨桥蛋白在不同大肠癌细胞株中的转移相关功能研究

目的：探索骨桥蛋白（OPN）在大肠癌转移中的相关功能。 方法:分别构建OPN反义和正义真核表达质粒后，转染入Colo205和SW480大肠癌细胞株。经鉴定后，进一步运用流式细胞仪、免疫组化、同质和异质黏附等技术检测转染细胞的转移相关功能变化。结果:OPN高表达的大肠癌细胞，CD44表达量增强，E-cadherin的表达减弱；细胞之间同质黏附减弱，细胞侵袭运动能力增强，细胞与ECV304细胞之间的异质黏附作用增强。均证明了OPN与侵袭转移的密切相关性。结论:提示OPN影响大肠癌细胞同质黏附和异质黏附能力的作用可能是大肠癌肝转移发生发展的相关机制之一。     

骨桥蛋白基因真核表达载体构建及对结肠癌细胞的作用

目的：构建骨桥蛋白（Osteopontin,OPN）基因真核表达载体,转染人结肠癌细胞SW480,分析其对SW480细胞株增殖及生存能力的作用。方法：构建重组表达载体pEGFP-N1/OPN,经测序鉴定无误后,转染人结肠癌SW480细胞。RT-PCR检测SW480细胞转染后OPN mRNA表达;Western blot检测SW480细胞转染后OPN蛋白表达量;Cell Counting Kit-8（CCK-8）测定细胞增殖;软琼脂克隆形成实验观察细胞的锚定非依赖性生长能力。结果：pEGFP-N1/OPN转染SW480后,OPN基因获得很好转录,OPN蛋白表达增高,转染OPN后SW480细胞增殖率（光吸收值）显著高于转染阴性组（P〈0.05）,软琼脂克隆形成速度增快,数量明显多于对照组和空载体组（P〈0.05）。结论：OPN基因真核表达载体pEGFP-N1/OPN构建成功,OPN具有促进SW480细胞增殖、存活的作用,为进一步研究OPN作用机制奠定了重要基础。     

鼠抗人CXCR3单克隆抗体的研制及生物学功能研究

目的:获得持续分泌鼠抗人CXCR3单克隆抗体(mAb)的杂交瘤细胞株;以鼠抗人CXCR3 mAb作为工具,研究人CXCR3分子的表达特性及CXCR3信号转导对L929 -huCXCR3和结肠癌细胞株的迁移及增殖的影响.方法:以高表达人CXCR3膜型分子的L929-huCXCR3细胞作为免疫原免疫BALB/c小鼠,采用B淋巴细胞杂交技术,将免疫小鼠的脾脏细胞与同种系小鼠的骨髓瘤细胞sp2/0进行融合.以L929-huCXCR3作为筛选细胞,转染空载体的L929 -mock细胞作为阴性对照细胞,采用间接免疫荧光和流式细胞术,筛选能持续分泌抗人CXCR3 mAb的杂交瘤细胞株.采用Ig亚类快速定性试纸法和间接免疫荧光法对所获得的杂交瘤细胞株和mAb进行鉴定;用间接免疫荧光法分析CXCR3分子在肿瘤细胞表面的表达;Transwell隔离小室检测mAb对L929 -huCXCR3和结肠癌细胞株Colo205、HCT116及HT29迁移的影响;MTT法分析mAb对结肠癌细胞株Colo205增殖的影响.结果:获得了1株能持续分泌鼠抗人CXCR3 mAb的杂交瘤细胞株,命名为9B5.经快速定性试纸分析显示,该mAb重链为IgG1亚类,轻链为链;间接免疫荧光和流式细胞术分析显示,mAb 9B5可识别活化T淋巴细胞和结肠癌细胞株Colo205、HCT116及HT29表面的CXCR3分子.通过阻断CXCR3信号转导,mAb 9B5可抑制L929-huCXCR3细胞和结肠癌细胞株Colo205、HCT116和HT29的定向迁移及IP-10对Colo205的促增殖作用.结论:成功获得了1株能持续分泌鼠抗人CXCR3 mAb的杂交瘤细胞株,为研究CXCR3的表达特性及深入探讨CXCR3信号转导在肿瘤生长与转移过程中的作用及机制奠定了物质基础,并且有望为治疗肿瘤转移提供新的思路和新型药物.     

具有相同遗传背景的人结肠癌细胞系生物学特性的鉴定

目的 对三种取自不同转移部位的结肠癌细胞系进行生物学特性的鉴定.方法 从体内成瘤性、原位移植后的自发性转移能力,以及体外生长、克隆形成率、粘附、运动、侵袭能力等方面,探讨具有相同遗传背景的SW480、SW620以及SW480/M5三种细胞系生物学特性的差异.结果 体内实验证明SW480具有多器官转移的潜能,SW620只具有淋巴结转移的能力,SW480/M5只具有肝脏转移的能力,SW480/M5的皮下瘤生长速度最快,其次是SW620细胞;体外实验证明W48/M5和SW620的体外生长、克隆形成能力均强于SW480细胞,而两者对纤维黏连蛋白的粘附能力较SW480细胞弱,SW480/M5的运动及侵袭能力强于SW480及SW620细胞.结论 与SW480细胞相比,SW480/M5和SW620细胞具有一定的器官亲和力,三者的遗传背景一致,是研究结肠癌晚期演进的遗传改变的重要资源.     

非甾体类抗炎药对结肠癌细胞NAG-1 基因表达的诱导

研究非甾体类抗炎药(Non-steroidal anti-inflammatory drug,NSAID)对结肠癌细胞生长的影响及NSAID活化基因-1(NAG-1)的诱导作用。体外培养HT-29、SW480及LS174-T三种结肠癌细胞,分别加入不同浓度的aspirin、celecoxib及meloxicam作用于HT-29及SW480细胞,采用MTT法检测结肠癌细胞增殖;蛋白质印迹技术检测三种结肠癌细胞COX-2的表达;采用半定量RT-PCR技术分析NSAID对三种结肠癌细胞NAG-1基因表达的影响。aspirin、celecoxib及meloxicam均能有效抑制体外培养的HT-29、SW480结肠癌细胞生长,并具有良好的量-效关系。Western blot表明,HT-29细胞表达COX-2,而SW480细胞不表达COX-2。三种结肠癌细胞均表达NAG-1基因mRNA,其中LS174-T细胞NAG-1基础水平较低;NSAID能不同程度上调结肠癌细胞NAG-1基因表达。NSAID能有效抑制结肠癌细胞生长,这种作用可能部分通过诱导结肠癌细胞NAG-1基因表达实现,NAG-1基因表达不受肿瘤细胞是否表达COX-2的影响。     

Low density lipoproteins and Lovastatin modulate the organ-specific transendothelial migration of primary and metastatic human colon adenocarcinoma cell lines in vitro

Tumor cell arrest and tumor migration are two of the critical steps in the metastatic cascade. We hypothesized that these steps may be facilitated by the low density lipoprotein (LDL)-induced activation of microvessel endothelial cells (MVEC). The purpose of our study was to investigate the biological effects of an LDL-enriched milieu and the effects of the anticholesterol drug Lovastatin on metastatic behavior. The SW480 and SW620 are primary and metastatic human colonic adenocarcinoma cell lines derived from the same patient. We investigated the effect of LDL on adhesion and migration of the two tumor cell lines across human brain, lung, liver and dermal endothelial monolayers. Adhesion and migration assays were done before and after pretreat-ment of the MVEC or tumor cells with LDL (100 mg/ml) for 24 h. Although metastatic SW620 cells were more adherent to MVEC compared with primary SW480 cells, LDL pretreatment of SW480 and SW620 cells did not affect tumor cell adhesion to MVEC. In contrast, tumor cell migration was significantly increased across endothelial monolayers when MVEC were pretreated with LDL. Transendothelial cell migration was not sig-nificantly affected by pretreatment of the tumor cells with LDL. Lovastatin is an inhibitor of HMG-CoA reduc-tase, the rate-limiting enzyme in cholesterol biosynthesis. It has been shown to have anti-tumor activity in vitro. We investigated the effect of Lovastatin on tumor cell kinetics and tumor cell migration across MVEC. Growth curves and migration assays were done before and after pretreatment of the tumor cells with Lovastatin (30 mg/ml). Migration assays were also done after treatment of unstimulated or LDL-stimulated MVEC (100 mg/ml) for 24 h with Lovastatin. Lovastatin inhibited the in vitro growth of the metastatic SW620 cell line to a greater extent than the invasive SW480E cell line. On the other hand, pretreatment of tumor cells with Lovastatin (30 mg/ml) did not suppress transendothelial tumor cell migration of tumor cells. Finally, Lovastatin given to mice effectively suppressed the number of MCA-26 tumor colonies in the liver of Balb/c mice com-pared with untreated mice. ©Lippincott Williams & Wilkins     

肿瘤细胞中两种TNFR的表达及新型rhTNF对肿瘤细胞生长的影响

目的 探讨ＴＮＦＲｐ５５和ＴＮＦＲｐ７５在各种肿瘤组织中的表达与新型ｒｈＴＮＦ对肿瘤细胞生长的抑制作用的关系。方法应用免疫组织化学染色法，观察１２０例肿瘤组织和１０株体外培养的肿瘤细胞中，ＴＮＦＲｐ５５和ＴＮＦＲｐ７５的表达及分布。同时应用结晶紫摄入法，观察ｎｒｈＴＮＦ对肿瘤细胞生长的抑制。结果 在肿瘤组织和肿瘤细胞中，ＴＮＦＲｐ５５的阳性表达率分别为８２．５％和９９％，ＴＭＦＲｐ７５分别为 ８４．２％和９８．８％，不同类型肿瘤间的表达无明显差异（Ｐ＞０．０５）．ｎｒｈＴＮＦ可抑制ＳＷ４８０、ＳＧＣ７９０１、８９１０ＳＭＭＣ７７２１和ＢＥＬ７４０２这５株肿瘤细胞生长，但是不能抑制另外５株肿瘤细胞Ａ５４９、ＰＬＡ８０１、Ｈｅｌａ、Ｅｃａ１０９和ＧＲＣ－ｌ的生长。结论ＴＮＦＲ广泛存在于各种肿瘤细胞上，ｎｒｈＴＮＦ抑制肿瘤细胞生长的程度与ＴＮＦＲｐ５５的表达率无相关性。     

Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells.

In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Colo205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited. The antibodies also mediated complement-dependent cytotoxicity. All antibodies tested recognized a cell surface molecule of 55 kDa. Southern blot hybridization analysis of hybridoma DNA with a human JH probe showed that the hybridomas were derived from clonally unrelated B cells. These results demonstrate that human foetal and adult B cells from non-tumour-bearing individuals are able to produce IgM antibodies recognizing defined cell surface molecules expressed on some tumour cells.     

Hypomethylation of the metastasis-associated S100A4 gene correlates with gene activation in human colon adenocarcinoma cell lines

The DNA methylation status of the metastasis-associated S100A4 gene in S100A4-positive and -negative human colon adenocarcinoma cell lines was examined. Northern and Western blot analyses revealed that HT-29, SW480, SW620, WiDr and Colo201 cells expressed S100A4, whereas SW837, LoVo and DLD-1 cells expressed little S100A4. Using CpG methylation-sensitive and -insensitive restriction enzymes and PCR-based methylation assay, it was found that the S100A4 gene in HT-29, SW480, SW620, WiDr and Colo201 cells, but not in SW837, LoVo and DLD-1 cells, was hypomethylated and that the hypomethylation of the second intron was correlated well with the expression of S100A4. 5-Aza-2'-deoxycytidine, an inhibitor of the eukaryotic DNA methyltransferase, induced the expression of the S100A4 gene in SW837, LoVo and DLD-1 cells, while it showed no effect on the expression of the gene in WiDr cells. These results indicate that hypomethylation of the S100A4 gene results in the expression of the gene in colon adenocarcinoma cells.     

Gamma interferon augments the intracellular pathway for lipopolysaccharide (LPS) recognition in human intestinal epithelial cells through coordinated up-regulation of LPS uptake and expression of the intracellular Toll-like receptor 4-MD-2 complex

Although some intestinal epithelial cell lines are known to respond to lipopolysaccharide (LPS), understanding of the relationship between LPS responsiveness and the expression of LPS receptors or factors regulating LPS responsiveness of intestinal epithelial cell lines is incomplete. In this study, we demonstrate that commonly studied human intestinal epithelial cell lines can be classified into at least three different types on the basis of LPS responsiveness, Toll-like receptor-4 (TLR4) expression, and the effects of gamma interferon (IFN-gamma) on LPS responsiveness. The first phenotype, which includes the HCT-116 and Caco-2 cell lines, is characterized by relative hyporesponsiveness to LPS and diminished expression of TLR4 protein. In these cells, IFN-gamma does not induce LPS responsiveness. The second phenotype, which includes cell line SW480, exhibits a highly LPS-responsive phenotype and surface expression of TLR4 protein even in unprimed conditions. These lines are functionally similar to cells of monocytic lineage. In the third phenotype, which includes the HT-29 and Colo205 cell lines, TLR4 protein is largely present in the cytoplasmic fraction and the cells are hyporesponsive to LPS in an unprimed condition. However, priming of these cells with IFN-gamma can induce LPS responsiveness through augmentation of LPS uptake and expression of MD-2 mRNA and intracellular TLR4 proteins. Finally, these findings suggest that the Th1 cytokine IFN-gamma modulates LPS responsiveness through several mechanisms in intestinal epithelial cells and that these cells may comprise different subpopulations with distinct roles in innate immune responses.