生长抑素抑制人低分化胃癌细胞株BGC-823的生长及其机制初探

生长抑素（SS）广泛存在于神经内分泌系统和消化道，研究发现，SS能影响细胞的增殖能力,促使我们探讨外源性SS可否作为肿瘤治疗的一种选择方法。胃癌是消化道常见肿瘤之一，本文旨在观察对人低分化胃癌细胞株BGC-823生长的影响，并进一步探讨SS对细胞内信息传递途径的影响，为临床治疗胃癌提供理论依据。     

采用HepG2细胞株建立裸鼠肝癌模型的方法

目的 :探讨采用HepG2 细胞株建立裸鼠肝癌模型的方法。方法 :采用皮下注射HepG2 细胞 (1 2× 1 0 6)建立裸鼠肝癌模型 ,观察种植细胞后 1 4天内裸鼠的死亡率 ,并在 1 4天时活杀动物测定瘤体重量、直径及血清AFP浓度。结果 :该法造模成功率达 1 0 0 %。结论 :该方法操作简便 ,周期短 ,成功率高等可作为肝癌模型应用     

IFN-α对人胃癌细胞株BGC-823生长的抑制作用

目的：观察干扰素-α(IFN-α)对人胃癌细胞株BGC-823生长和浸润转移的抑制作用及其作用机理。方法:在体外培养体系中检测IFN-α对BGC-823细胞株生长增殖能力的影响，MTT法检测细胞活力，流式细胞仪检测细胞周期。同时采用免疫组化法观察了IFN-α对肿瘤细胞E-cadherin、MMP-2表达的调节作用，电镜下观察肿瘤细胞间连接的超微结构的变化。结果:IFN-α对人胃癌细胞株BGC-823的生长有明显抑制作用，并表现出剂量依赖性，IFN-α浓度≥106 U/L时可有效抑制细胞增殖，抑制率≥12.2%，细胞周期在G1-S期之间发生阻滞；在IFN-α诱导下细胞E-cadherin表达水平升高而MMP-2表达下降，超微结构出现了细胞粘附连接数量增多、结构相对紧密等变化 。结论：IFN-α可通过影响细胞周期来抑制人胃癌细胞株BGC-823的生长；IFN-α能调节E-cadherin、MMP-2的表达，密切细胞间的连接，具有限制胃癌细胞侵袭转移的潜能。     

shRNAs对胃癌细胞系BGC-823胃泌素表达的抑制效应

目的 研究shRNAs对胃癌细胞系BGC-823胃泌素表达的抑制效应.方法 设计4条针对胃泌素基因不同位点的寡核苷酸序列,通过体外转录法合成相应的shRNAs.以10nmol/L、20nmol/L、40nmol/L和80nmol/L的终浓度,将4条shRNAs分别转染胃癌细胞BGC-823.应用原位杂交及免疫细胞化学方法检测胃泌素表达的抑制效果,筛选最有效的shRNA.应用RT-PCR进一步验证其对胃泌素mRNA的抑制效应.应用MTT法检测4条shRNAs在不同浓度下对BGC-823细胞的增殖抑制效应.结果 转染后24h、48h及72h,胃泌素表达均被明显地抑制,并呈现浓度及时间依赖的趋势.shRNA3转染后72h,mRNA及蛋白水平表现出最佳的抑制效率,分别为(54.27±0.042)%和(41.69±0.038)%.RT-PCR结果显示,shRNA3对BGC-823细胞胃泌素mRNA的抑制率为48.1%.MTT结果显示,除shRNA4处理组外,其余3组处理细胞均表现出浓度依赖性的增殖抑制趋势.结论 4条shRNAs在mRNA及蛋白水平均明显地抑制了胃癌细胞BGC-823胃泌素的表达,shRNA3可能为最有效的胃泌素-shRNA.shRNA对胃泌素表达的抑制,显著降低了BGC-823细胞增殖能力.     

人前列腺癌裸鼠皮下移植瘤模型的建立

目的 建立人前列腺癌裸鼠皮下移植瘤模型.方法 采用人前列腺癌细胞株PC-3细胞接种于裸鼠的颈背部皮下,计算成瘤潜伏期、成瘤百分率,观察移植瘤大体生长情况,绘制生长曲线,并对移植瘤进行病理鉴定.结果 采用人前列腺癌细胞株PC-3细胞皮下接种方式建立移植瘤的平均成瘤潜伏期为24天,成瘤百分率为100％,瘤体积倍增时间为10天左右,移植瘤的形态和功能特性与原发肿瘤基本相似.结论 本动物模型的建立可为前列腺癌的放射免疫显像以及放射免疫治疗提供一个有价值的实验平台.     

SY86B人胃癌裸鼠移植瘤CEA分布和含量的研究

对我所已建立的SY86B人胃癌组织裸鼠模型进行了CEA定位和定量研究。通过对连续四代带瘤裸鼠皮下移植瘤健康组织和坏死部分以及血清内CEA水平的观察,并与对照鼠比较,发现带瘤鼠血清CEA值明显升高,其程度与皮下移植瘤重量成正比。对皮下移植瘤的抗CEA单克隆抗体ABC免疫组化染色结果表明,CEA广泛存在于瘤组织内,且符合肠型胃癌的某些特点。     

胃肠道间质瘤细胞组织在不同周龄裸小鼠不同部位的成瘤情况

背景：建立合适的人胃肠道间质瘤裸鼠动物模型,可为以后在活体状态下研究胃肠道间质瘤细胞提供一个必不可少的工具。 目的：观察胃肠道间质瘤细胞组织块接种于不同周龄裸小鼠腋下和臀部的成瘤情况。 方法：将胃肠道间质瘤细胞组织块分别接种于4,8周龄BALB/c(nu/nu)裸鼠的腋下和臀部,观察在一定时间内,不同周龄裸鼠不同部位的瘤体成瘤率,生长速度,破溃率和转移率。 结果与结论：4周龄裸鼠的成瘤率及生长速度明显高于8周龄裸鼠(P < 0.05),且接种于腋下的成瘤率较臀部高;4周龄与8周龄裸鼠的肿瘤破溃率差异无显著性意义(P > 0.05),但接种于臀部的肿瘤破溃率大于腋下(P < 0.05);在肿瘤软化消失率方面,8周龄裸鼠明显高于4周龄裸鼠(P < 0.05),同龄裸鼠中,臀部高于腋下。说明不论接种部位如何,周龄越小的裸鼠,移植瘤成瘤率越高、生长速度越快;但对于相同周龄的裸鼠,不论接种部位如何,肿瘤生长速度无明显差异。     

胃癌细胞株中NPRA基因启动子区CpG岛的甲基化分析

目的NPRA-cGMP信号通路系统可激活cGMP依赖的蛋白激酶,活化的蛋白激酶调控离子转运蛋白和转录因子的表达,从而最终影响细胞生长、凋亡、增殖和炎症反应。本研究旨在研究NPRA基因在胃癌细胞中的甲基化状态及其对下游mRNA表达的影响。方法应用亚硫酸氢钠处理基因组DNA后PCR并测序,我们分析了6株胃癌细胞株NPRA基因的甲基化状态及去甲基化试剂对下游mRNA表达的影响。结果在胃癌细胞中发现NPRA基因启动子区存在甲基化畸变并导致下游mRNA表达减少或沉默。去甲基化试剂可逆转mRNA表达沉默。结论胃癌细胞中存在NPRA基因甲基化畸变。     

抗小鼠凝血因子IX的大鼠—小鼠杂交瘤细胞株的建立

1997年 ,Ｓｔａｆｆｏｒｄ博士等建立了凝血因子ＩＸ基因剔除小鼠动物模型 ,薛京伦等 (1997)证实了该模型能极佳地复现人血友病Ｂ临床表型 ,从而为血友病Ｂ基因治疗研究提供了一个良好的模型。为了对该动物模型的遗传稳定性和相应临床表征作深入研究 ,以及提供小鼠凝血因子ＩＸ(ｍＦＩＸ)表达的检测手段 ,我们制备了抗小鼠ｍＦＩＸ单克隆抗体。本实验选择Ｌｏｕ/Ｍ大鼠 ,以复旦大学遗传所制备的基因工程产品ｍＦＩＸ为抗原 ,以 0 2ｍｌｍＦＩＸ(含蛋白 0 6ｍｇ)加0 2ｍｌ福氏完全佐剂腹背部皮下多点免疫 ,2周后以同等剂量ｍＦＩＸ加不完…     

siRNA抑制胃癌细胞BGC-823及裸鼠皮下移植瘤中生存素的表达

目的： 探讨RNA干扰用于胃癌治疗的可行性与特异性。方法: 设计靶向生存素基因的小干扰RNA,并导入胃癌BGC-823细胞株和裸鼠皮下移植瘤,在体内、外诱导RNA干扰,采用MTT、流式细胞检测技术、RT-PCR法、Western bloting、免疫组织化学和TUNEL检测survivin基因表达、细胞周期和细胞凋亡。结果: 重组质粒pTZU6+1-siRNA- survivin导入BGC-823后,survivin mRNA和蛋白表达均明显下调;细胞生长被抑制,细胞周期中S期细胞减少, G1/G0期细胞增加,出现明显细胞凋亡。裸鼠皮下移植瘤体积明显缩小,Survivin表达均下调。体内、外对照组各指标均无明显变化。 结论: RNA干扰明显抑制靶基因survivin在胃癌细胞BGC-823及裸鼠皮下移植瘤中的表达及肿瘤细胞增殖,并诱导细胞凋亡,且特异性好,可能成为肿瘤治疗的新方法。     

Peritoneal metastatic model for human scirrhous gastric carcinoma in nude mice

We established a peritoneal-metastatic model for scirrhous gastric carcinoma. Peritoneal metastasis had developed after intraperitoneal inoculation of OCUM-2MD3 cells in nude mice. This cell line was derived from a peritoneal-metastatic nodule at the mesenterium after orthotopic implantation of OCUM-2M cells which developed no peritoneal metastasis after intraperitoneal inoculation. The histologic findings of orthotopic-implanted tumor in the stomach show scirrhous type while those of subcutaneous-implanted tumor show medullary type. There might be factors, in OCUM-2MD3 cells, which are responsible for peritoneal metastasis. We next investigated the differences in the biological behavior of the original OCUM-2M and the derived variant OCUM-2MD3. Morphology and growth activity of the two cell lines were similar to each other. The specific chromosomes, add(6)(g13), del(7)(q21.2) and inv(11)(pl3q21), were found in OCUM-2MD3 cells but not in OCUM-2M cells. While the oncogenes amplification by OCUM-2M cells was found in K-sam and c-myc, that by OCUM-2MD3 cells was found only in c-myc. The expression of E-cadherin by OCUM-2MD3 cells was decreased compared with that of OCUM-2M cells. Expression level of β1-integrin of OCUM-2MD3 cells was higher than that of OCUM-2M cells. The binding and invasion activity of OCUM-2MD3 cells were higher than those of OCUM-2M cells, and were decreased by anti-β1-integrin antibody. The invasion activity of OCUM-2MD3 cells was increased in the presence of peritoneal fibroblast. In this study, it was suggested that orthotopic implantation of cancer cells might have an effect on the acquisition of metastatic ability. β1-integrin and peritoneal fibroblasts might be correlated with peritoneal metastasis. This peritoneal-metastatic model should be useful for analysing the mechanism of peritoneal metastasis of human scirrhous gastric cancer.     

Establishment and characterization of two cell lines derived from human diffuse gastric carcinomas xenografted in nude mice

Two human diffuse gastric carcinoma cell lines were established in vitro from xenografted tumours serially passaged in nude mice. Of 12 primary diffuse gastric carcinomas, 7 were successfully xenografted in nude mice (58.3%). Short-term primary cultures were achieved in all the xenografted lines. However, only 2 of the 7 short-term primary cultures were established as long-term cultures (GP202 and GP220). GP202 cells are larger than GP220 cells, show less abundant intercellular junctions at the ultrastructural level and grow in culture as a compact thin monolayer. The GP220 cells grow preferentially in small clusters attached to the monolayer, with a subpopulation of floating cells. Both lines have cells containing small mucin vacuoles in the cytoplasm and cells displaying a typical signet-ring shape. GP202 cells grow as solid tumours in nude mice but GP220 cells do not give rise to tumours. The flow cytometry and karyotype analysis showed aneuploidy in GP202 cells, with many numerical and structural chromosomal abnormalities, and diploidy in GP220 cells, with several structural chromosomal abnormalities. The CDw75 and Tn antigens are more prominently expressed in GP202 cells than in GP220 cells. T antigen is only expressed in GP202 cells, whereas only GP220 cells express EGFR. Sialosyl-Tn is not expressed in either of the cell lines. The gastric cancer cell lines described in this paper represent a valuable addition to the small number of diffuse gastric cancer cell lines currently available and also provide a good model for further in vitro and in vivo studies of gastric carcinogenesis.     

裸鼠人肝癌模型模拟临床化疗实验研究

目的 探讨人肝癌组织裸鼠原位种植模型体内筛选供瘤病人敏感的化疗药物可行性。方法 荷瘤鼠随机分为对照组和表阿霉素（E—ADM）、5氟脲嘧啶（5-FU）、丝裂霉素（MMC）四组。腹腔化疗每周1次，共4次。B超观测移植瘤大小，化疗结束1wk后处死荷瘤鼠．分别用免疫组化（SP）、RT—PCR、Western blot方法检测移植瘤MDR1和LRP蛋白及其mRNA表达情况。选择敏感和低致多药耐药的化疗药物用于供瘤病人。结果 3个化疗组肿瘤体积均逐渐缩小，与对照组相比差异均有显著性（P〈0．05）。各检测方法均示E—ADM组MDR1和LRP蛋白及其mRNA表达升高，差异有显著性（P〈0．05）。5-Fu组LRP表达升高，差异有显著性（P〈0．05）。MMC和5-Fu对供瘤病人化疗一个疗程，病人骨痛症状消失或明显减轻，SPECT示个别骨转移灶消失，多个骨转移灶缩小。结论 E—ADM化疗可以诱导肿瘤组织MDR1和LRP表达升高；5-Fu化疗则可诱导LRP表达升高。裸鼠原位种植瘤敏感的化疗药物同样供瘤病人也敏感。     

建立卵泡刺激素受体单位点基因突变体的卵巢癌SKOV3细胞和裸鼠模型

目的 建立转染卵泡刺激素受体（FSHR）307和680位点突变的上皮性卵巢癌SKOV3细胞系和裸鼠动物模型。方法 将人卵巢颗粒细胞（来自体外受精取卵时废弃的颗粒细胞）,行原代培养后用于FSHR RNA提取;FSHR和FSHR307、680位点突变重组蛋白构建;将FSHR和FSHR307、680位点突变蛋白转染SKOV3细胞株;将处理后的3组SKOV3细胞接种到裸鼠皮下,8周后处死裸鼠,并取瘤组织验证。     

Synchronous primary gastric mantle cell lymphoma and early gastric carcinoma: a case report

Mantle cell lymphoma (MCL) commonly invades the gastrointestinal (GI) tract. However, primary GI MCL is rare. We experienced a case of synchronous early gastric cancer (EGC) with primary gastric MCL found as a single early lesion rather than as multiple lymphomatous polyposis.

An EGC was found in the cardia of a 64-year-old male on a routine GI endoscopic examination. A specimen obtained by total gastrectomy revealed another slightly elevated lesion in the pylorus. Microscopically, monotonous small- to medium-sized atypical lymphocytes with angulated nuclei formed a mass beneath the gastric mucosa. On immunohistochemical staining, the tumor cells revealed strong positivity for cyclin D1, positivity for both CD20 and bcl-2, and weak positivity for CD5, suggesting MCL. Clinically, there was no lymphoma in any other part of the body.

This is the first case of an EGC accompanying a primary gastric MCL. Further investigation of a relationship between MCL and EGC and of factors that may affect GI involvement of MCL is necessary.     

Merkel cell carcinoma of the skin: pathological and molecular evidence for a causative role of MCV in oncogenesis

Merkel cell carcinoma (MCC), a skin tumour with neuroendocrine features, was recently found to be associated with a new type of human polyomavirus, called Merkel cell virus (MCV). We investigated the specificity of this association as well as a causal role of MCV in oncogenesis. DNA and RNA from ten cases of MCC were analysed using PCR and RT‐PCR. DNA from 1241 specimens of a wide range of human tumours was also analysed. The DIPS technique was used to identify the integration locus of viral DNA sequences. Array CGH was performed to analyse structural alterations of the cell genome. MCV DNA sequences were found in all ten cases of MCC and in none of the 1241 specimens of other tumour types. Clonal integration of MCV into the host genome was seen in all MCC cases and was checked by FISH in one case. A recurrent pattern of conserved viral sequences which encompassed the replication origin, the small tumour (ST), and the 5′ part of the large tumour (LT) antigen DNA sequences was observed. Both ST and LT viral sequences were found to be significantly expressed in all MCCs. Neither recurrent site of integration nor alteration of cellular genes located near the viral sequences was observed. The tight association of MCV with MCC, the clonal pattern of MCV integration, and the expression of the viral oncoproteins strongly support a causative role for MCV in the tumour process. This information will help the development of novel approaches for the assessment and therapy of MCC and biologically related tumours. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

乙醇诱导人胃上皮细胞癌细胞株（BGC—823）细胞凋亡

为进一步阐明了乙醇引起胃粘膜损伤的机理,本实验在离体培养的人胃上皮癌细胞株（ＢＧＣ－８２３）建立了乙醇诱导细胞凋亡的模型。从细胞形态、ＤＮＡ梯状条带及流式细胞仪检测等方面,观察了不同剂量的乙醇作用不同时间后ＢＧＣ－８２３细胞凋亡的发生情况。结果表明,较低浓度的乙醇（１．５％￣４％）能有效诱导细胞凋亡,且随剂量增大凋亡发生时间缩短。较高浓度（大于４％）则引起细胞坏死,２％乙醇诱导时,６ｈ即有凋亡出现     

两株肺癌细胞系体外培养细胞和裸鼠体内移植瘤细胞增殖状态的比较研究

目的：比较两株肺癌细胞系（经实验证明为高、低侵袭系）体外培养细胞和裸鼠体内移植瘤细胞的增殖状态；方法：采用激光流式细胞仪，测量单个完整细胞群体的ＤＮＡ含量，分析其各细胞周期的比率；结果：体外培养的高增殖系细胞，移植入裸鼠体内后其Ｇ２／Ｍ期细胞比率反而降低，体外培养的低增殖系细胞，移植入裸鼠体内后其Ｇ２／Ｍ细胞比率反而升高，两株肺癌细胞系的增殖指数间无显著差异；结论：两株肺癌细胞系在体内、外增殖状况的差异性，说明肿瘤细胞在体外增殖速度的快慢不能准确反映其在体内的增殖状态，必须结合肿瘤细胞的其它生物学特性，才能比较准确地分析和判断肿瘤细胞的恶性程度。