大鼠抗胃癌单克隆抗体的制备及鉴定

本文用BGC823人胃癌细胞株为抗原,免疫LOU/M大鼠,取脾细胞与大鼠骨髓瘤细胞IR983F融合获得杂交瘤株1F_1。其Mc-Ab的Ig亚类为IgM。用PAP组织化学染色法检测,1F_1与3株胃癌细胞(BGC 823,MGC803,SGC7901)呈阳性反应。以免     

小分子化合物H-131抑制胃癌细胞增殖及机制

目的研究小分子化合物H-131对人胃癌细胞SGC7901、BGC823、MKN-45增殖的抑制作用及其相关机制。方法应用不同浓度的化合物H-131处理人胃癌细胞SGC7901、BGC823和MKN-45,采用MTT法检测该化合物对细胞增殖的影响,流式细胞术检测其对细胞周期的影响,Westernblot检测化合物对周期调控蛋白CyclinD1和CDK4表达的影响。结果化合物H-131能够以剂量依赖的方式抑制人胃癌细胞SGC7901、BGC823及MKN-45的增殖,并且能够抑制上述细胞G_1期向S期的转化。Westernblot结果显示H-131能够以剂量依赖的方式下调胃癌细胞SGC7901中CyclinD1和CDK4的蛋白表达水平。结论化合物H-131能够通过下调胃癌细胞中CyclinD1和CDK4的表达水平,抑制胃癌细胞G_1期向S期的转化,进而抑制胃癌细胞增殖。     

ZAC基因在奥曲肽抑制胃癌细胞体外增殖通路的作用

目的 探讨ZAC基因在生长抑素类似物奥曲肽（OCT）抑制胃癌细胞增殖通路的作用。方法 分别以不同浓度OCT处理胃癌细胞BGC823和 SGC7901不同时间,MTT法筛选其增殖抑制的有效条件。OCT处理胃癌细胞（有效浓度/不同时间,有效时间/不同浓度）,Western blotting检测
OCT对胃癌细胞ZAC基因的效应。设计3条ZAC基因RNA干扰片段,分别插入pSUPER-EGFP-I载体,构建3个ZAC-shRNA表达载体（pSUPER-EGFP-ZAC/1 pSUPER-EGFP-ZAC/2和pSUPER-EGFP-ZAC/3）。经酶切和序列分析鉴定后,分别转染胃癌细胞BGC823和SGC7901。经G418筛选,RT-PCR鉴定,建立
ZAC基因敲低（knock-down）的胃癌细胞系。以有效条件OCT孵育胃癌细胞（对照组）和ZAC基因敲低胃癌细胞（实验组）,MTT法检测OCT对胃癌 细胞的生长抑制效应。结果 OCT抑制胃癌细胞增殖的有效条件为10nmol/L 孵育24h;OCT以时间和浓度依赖的方式诱导胃癌细胞ZAC基因表达。酶切及序列分析鉴定表明ZAC-shRNA表达载体构建成功。转染shRNA-ZAC/2的胃癌细胞,其ZAC mRNA水平明显降低（P<0.05）,为ZAC基因敲低胃癌 细胞。ZAC基因敲低胃癌细胞的增殖明显高于对应的BGC823细胞和SGC7901细胞（P<0.05）。OCT孵育后,BGC823细胞和SGC7901细胞的增殖明显降低（P<0.05）。然而,ZAC基因敲低胃癌细胞的增殖无明显改变（P>0.05）。结论 OCT以时间和浓度依赖的方式诱导胃癌细胞ZAC基因表达;ZAC 基因在OCT抑制胃癌细胞增殖通路中具有重要作用。     

小分子化合物P-275抑制胃癌细胞增殖及机制研究

目的研究小分子化合物P-275对人胃癌细胞BGC823、SGC7901、MKN-45增殖的抑制作用及其机制。方法分别用不同浓度的化合物P-275处理人胃癌细胞BGC823、SGC7901和MKN-45,MTT法检测化合物对细胞增殖的影响,流式细胞术检测化合物对细胞周期的影响,Western Blot检测化合物对周期调控蛋白Cyclin D1、CDK4表达的影响。结果化合物P-275能够以剂量依赖的方式抑制人胃癌细胞BGC823、SGC7901及MKN-45的增殖,同时能够抑制上述细胞G1期向S期的转化。Western Blot结果显示P-275能够以剂量依赖的方式下调胃癌细胞SGC7901中周期调控蛋白Cyclin D1、CDK4的表达。结论化合物P-275能够通过下调胃癌细胞中周期调控蛋白Cyclin D1、CDK4的表达,从而抑制胃癌细胞G1期向S期的转化,进而抑制胃癌细胞的增殖。     

小干扰RNA抑制CD97的表达及其对胃癌细胞迁移和侵袭能力的影响

目的: 设计合成有效的siRNA-CD97,体外转染胃癌细胞株,观察CD97表达改变及其与胃癌细胞株迁移、侵袭能力改变的关系。方法: 采用AGS和MGC803胃癌细胞株。针对 CD97 基因设计siRNA,采用化学法合成,筛选出最有效的siRNA-CD97。siRNA-CD97转染胃癌细胞后分别用real-time RT-PCR、免疫荧光流式细胞术检测CD97 mRNA和蛋白表达的改变,MTT法检测细胞活性的改变,并用Transwell细胞迁移和侵袭实验检测细胞迁移和侵袭能力的变化。结果: 在siRNA-CD97转染后48 h,real- time RT-PCR结果显示AGS和MGC803细胞CD97 mRNA的表达量相对于未转染的细胞分别下降了(89.34±9.95)%和(95.42±1.93)%。转染后72 h,流式细胞术结果显示AGS细胞CD97^EGF和CD97^stalk抗原的表达强度相对于未转染的细胞分别下降了(19.29±3.45)%和(30.11±5.93)%, MGC803细胞CD97^EGF和CD97^stalk抗原的表达强度相对于未转染的细胞分别下降了(26.25±5.73)%和(16.22±3.23)%。MTT法检测结果显示,siRNA-CD97转染前后细胞的吸光度值没有显著差异。迁移和侵袭实验结果显示,AGS细胞siRNA-CD97转染组迁移和侵袭细胞相对于未转染组分别下降了(67.63±12.03)%和(68.02±15.63)%,MGC803细胞转染组迁移和侵袭细胞相对于未转染组分别下降了(14.92±2.03)%和(22.09±5.43)%。结论: 胃癌细胞转染 siRNA-CD97能够抑制CD97 mRNA和蛋白表达,随着CD97表达的降低,细胞迁移和转移的能力也明显减弱。     

RegⅠ在胃泌素促胃癌细胞体外增殖通路中的作用

目的 探讨再生蛋白I (RegⅠ)在胃泌素刺激胃癌细胞增殖通路中的作用.方法 根据RegⅠ cDNA已知序列,在线设计3个小干扰RNA,并经基因库同源性比较,构建其RegⅠ-shRNA表达载体(pSUPER-EGFP-REG/1、pSUPER-EGFP-REG/2和pSUPER-EGFP-REG/3),利用脂质体2000转染试剂分别转染胃癌细胞BGC823细胞株、SGC7901细胞株,同时设转染空质粒的实验对照组和无质粒转染的空白细胞组.后经G418筛选建立稳定转染细胞株.RT-PCR检测RegⅠ基因mRNA的转录水平,四甲基偶氮唑盐(MTT)法检测胃泌素孵育对胃癌细胞增殖的效应.结果 酶切及测序鉴定,插入片段正确,3个RegⅠ-shRNA表达载体构建成功;RT-PCR显示,pSUPER-EGFP-REG/1可有效抑制RegⅠ mRNA在胃癌细胞BGC823、SGC7901的转录.Western boltting结果显示,BGC823和SGC7901细胞的RegⅠ蛋白表达率分别下调到空载体对照组的(45±4)%和(53±4)%;MTT结果显示,RegⅠ基因表达抑制胃癌细胞系的增殖效率均降低(P＜0.05).胃泌素孵育后,胃癌细胞BGC823、SGC7901的增殖效率均增加(P＜0.05),而RegⅠ基因表达抑制的胃癌细胞系增殖效率则无明显改变(P＞0.05).结论 实验成功建立了RegⅠ基因表达抑制的胃癌细胞系; RegⅠ基因可能对胃泌素促胃癌细胞的增殖有促进的作用.     

胃癌多药耐药细胞株BGC823/5-FU的建立及其耐药机制的研究

目的：建立5-氟尿嘧啶（5-FU）诱导的胃癌多药耐药细胞株BGC823/5-FU，探讨凋亡相关蛋白Survivin、Bcl-2、Bax及caspase-3与其耐药性产生的关系。 方法:采用反复短期暴露并逐渐增加5-FU浓度的方法建立胃癌耐药细胞株BGC823/5-FU，MTT法检测此耐药细胞株对5-FU的耐药倍数及其对临床常用化疗药物阿霉素、丝裂霉素和顺铂的交叉耐药性，流式细胞术检测细胞P-糖蛋白的表达和柔红霉素积累量；Western blotting法检测耐药胃癌细胞株BGC823/5-FU与其亲代药物敏感胃癌细胞株BGC823凋亡相关蛋白Survivin、Bcl-2、Bax及caspase-3的表达。 结果:成功诱导出胃癌多药耐药细胞株BGC823/5-FU，较其亲代细胞BGC823对5-FU、阿霉素、丝裂霉素和顺铂的耐药性分别提高10.82、2.50、22.23和2.00倍。其P-糖蛋白表达较BGC823细胞增高（P<0.01），柔红霉素积累量较BGC823细胞减低（P<0.01）。与亲代药物敏感BGC823细胞相比，耐药细胞株BGC823/5-FU细胞Survivin表达上升（P<0.05），Bcl-2表达升高（P<0.05），Bax表达下降（P<0.05），caspase-3表达减低（P<0.05）。结论:胃癌细胞株BGC823在5-FU的诱导下可形成多药耐药细胞株BGC823/5-FU，P-糖蛋白、凋亡相关蛋白Survivin、Bcl-2、Bax及caspase-3可能参与其耐药性的形成。     

Snai2在人胃癌细胞系中的差异表达及生物学意义

目的 分析Snai2在人胃癌细胞系中的差异表达与胃癌细胞分化的关系，以探讨Snai2在肿瘤发生发展中的作用。方法培养7株胃癌细胞系，TRIzol法抽提总RNA，逆转录后用RT—PCR和实时荧光定量PCR检测Snai2mRNA在胃癌细胞系中的表达，提取蛋白，用Westernblotting测定不同细胞系Snai2蛋白的表达。结果Snai2mRNA在中低分化、致瘤性强的细胞系BGC823、SGC7901、MGC803、PAMC82、MKN45内呈高表达；在高分化，致瘤性弱的细胞系内N87呈低表达，在AGS几乎不表达。结论Snai2mRNA的表达与胃癌细胞的分化与致瘤性强弱有关，可以作为一个判断肿瘤分化程度和预后的分子标志。     

EZH2在胃癌组织中的表达及对胃癌细胞增殖的影响

目的探讨EZH2表达与胃癌临床病理特征、预后的关系,及其对胃癌细胞增殖的影响。方法采用免疫组化SP法检测EZH2在65例胃癌组织及20例正常胃黏膜组织中的表达,分析其表达与胃癌临床病理特征的相关性,应用Kaplan-Meier法及Log-rank检验分析EZH2表达与胃癌患者生存率的关系。采用Western blot法检测胃癌细胞株SGC7901、BGC823中EZH2的表达。EZH2 Sh RNA转染胃癌细胞株后,采用MTT法检测细胞的增殖率。结果胃癌组织中EZH2蛋白阳性率为63.07%,正常胃黏膜组织中EZH2蛋白阳性率为10%;胃癌组织中EZH2蛋白表达明显高于正常胃黏膜组织(P0.000 1)。胃癌组织中EZH2蛋白表达与肿瘤直径(P0.000 1)、肿瘤浸润深度(P0.000 1)、淋巴结转移(P=0.001)及分期相关(P=0.001)。EZH2阳性者5年总生存率明显低于阴性者(P=0.001)。胃癌细胞株SGC7901和BGC823中EZH2蛋白高表达,Sh RNA介导的EZH2表达沉默可明显抑制胃癌细胞的增殖。结论 EZH2高表达是胃癌患者预后不良的重要因子,EZH2可明显促进胃癌细胞的增殖。     

芹菜素诱导人胃癌细胞凋亡作用及机制研究

目的：研究芹菜素（apigenin, API）致人胃癌细胞凋亡作用及其机制。方法：培养人胃癌BGC823细胞株，加入不同浓度的API，孵育48 h。PI染色流式细胞术（FCM）分析测定凋亡率；罗丹明染色FCM分析测定细胞线粒体跨膜电位(Δψm)；Caspase-9分光光度法检测试剂盒测定caspase-9活性；Western印迹检测线粒体凋亡信号转导通路相关蛋白的表达，包括bax，bcl-2，caspase-9和caspase-3。结果： API（20，40和80 μg/mL）作用48 h能呈浓度依赖性地诱导BGC823细胞凋亡。而且，API也能降低BGC823细胞的Δψm，增加caspase-9活性，促进细胞色素c（Cyt c）释放，上调bax，caspase-9和caspase-3蛋白的表达，同时下调bcl-2蛋白表达，且呈剂量依赖性。结论：API通过活化线粒体信号转导途径诱导人胃癌细胞凋亡。     

运用亚细胞蛋白质组学方法发现胃腺癌潜在标志物UCHL1

目的:通过对胃腺癌细胞系SGC7901和永生化胃正常上皮细胞系GES胞浆组分蛋白进行差异蛋白质组学分析,为胃腺癌诊断提供候选生物标志物,进而为阐明胃腺癌发病机理提供新的线索和思路。方法:运用亚细胞蛋白组份分离结合双向凝胶电泳和基质辅助激光解吸电离飞行时间质谱(MALD I-TOF-MS)技术筛选鉴定出SGC7901和GES细胞系胞浆蛋白组分间的差异蛋白质,并且利用免疫印迹和半定量RT-PCR方法对得到的差异蛋白进行验证。结果:筛选得到10个差异蛋白,进一步证实,差异蛋白泛素羧基末端水解酶-L1(UCHL1)在蛋白质和mRNA水平上,在胃癌细胞系SGC7901、AGS、BGC823和MKN45中的表达水平均低于胃正常上皮细胞系GES;其在胃癌组织中的表达水平也远远低于癌旁正常胃组织中的水平。结论:UCHL1蛋白质分子具有作为胃腺癌诊断检测标志物的潜在应用价值,为开发胃腺癌诊断标志物提供了新的候选蛋白。     

Gastric cancer cells in collagen gel matrix: three-dimensional growth and differential expression of adhesion molecules (CD44s, CD54, E-cadherin)

To characterize the growth of human gastric cancer cells in collagen gel matrix and adhesive status of the cells in comparison with conventional monolayer cells. Three kinds of human gastric cancer cell lines (BGC823, SGC7901, and MKN28) were cultured alone or co-cultured with normal human fibroblasts in collagen gel matrix, and their cell cycle, metabolic function, and the expression of adhesive molecules (CD44s, CD54, and E-cadherin) were analyzed by flow cytometry or other methods. Two of three cell lines (BGC823 and SGC7901) and their co-cultures showed multilayer growth in collagen gel matrix, and their growth and metabolism rate became slow and the cell adhesion molecules (CAMs) expression was down regulated. Gastric cancer cell alone or with fibroblasts in collagen gel matrix showed distinct growth feature when compared with monolayer cells, which represent two kinds of different experimental models. BGC823 and SGC7901 cells growing in three-dimension may recur some characteristics of their original solid tumor in vivo with the invasive or metastatic ability. According to different aims, it should pay great care in choice of experimental model to get more reasonable results.     

LncRNA-AP001631.9 promotes cell migration in gastric cancer

Recently, an increasing number of reports have revealed that long non-coding RNAs (LncRNAs) play important roles in a variety of aspects of cell activity, and their aberrant expression is closely associated with multigenetic diseases, including carcinoma. In the present study, through microarray analysis, we screened out a new LncRNA (LncRNA-AP001631.9), which was regulated by FOXM1, a well-known carcinogenetic factor and aimed to reveal the functional roles of this novel LncRNA in gastric cancer development. The data from qRT-PCR confirmed that the expression level of LncRNA-AP001631.9 was positively correlated with that of FOXM1. The transwell and wound healing assays indicated that LncRNA-AP001631.9 was required for the migration of gastric cancer cells. The downregulation of LncRNA-AP001631.9 by small interference RNA suppressed the migratory ability of MGC803 and AGS cells, while the overexpression of LncRNA-AP001631.9 promoted the movement of BGC823 and SGC7901 cells. Furthermore, a tail vein injection was administered, and the obtained results suggested that LncRNA-AP001631.9 contributed to the distant metastasis of SGC7901 cells. Moreover, we also collected 36 paired samples of gastric cancer tissues to explore the expression levels of LncRNA-AP001631.9. The qRT-PCR data indicated that the LncRNA-AP001631.9 expression was frequently increased in gastric cancer tissues. Taken together, our findings established that LncRNA-AP001631.9 plays critical roles in gastric cancer progression and can serve as a potential new target for the treatment of gastric cancer.     

Downregulation of AKT and MDM2, Melatonin Induces Apoptosis in AGS and MGC803 Cells

Melatonin, a neurohormone secreted by the pineal gland, has a variety of biological functions, such as circadian rhythms regulation, anti-oxidative activity, immunomodulatory effects, and anittumor, etc. At present, its antitumor effect has attracted people's attention due to its extensive tissue distribution, good tissue compatibility, and low toxic and side effects. In the gastrointestinal tract, there is high level of melatonin and many studies showed melatonin has effects of anti-gastric cancer. In this experiment, human gastric cancer cell lines AGS and MGC803 were used to investigate the intracellular molecular mechanism of melatonin against gastric cancer. After AGS and MGC803 have been treated with melatonin, the changes of cell morphology and cellular structure were observed under electron microscope. Flow cytometer and apoptosis detection kits were used to analyze the effect of apoptosis on AGS and MGC803. The alterations of apoptosis-related proteins Caspase 9, Caspase 3, and upstream regulators AKT, MDM2 including expression, phosphorylation, and activation were detected to analyze the intracellular molecular mechanism of melatonin inhibiting gastric cancer. In AGS and MGC803 cells with melatonin exposure, cleaved Caspase 9 was upregulated and Caspase 3 was activated; moreover, MDM2 and AKT expression and phosphorylation were downregulated. Melatonin promoted apoptosis of AGS and MGC803 cells by the downregulation of AKT and MDM2. Anat Rec, 302:1544–1551, 2019. © 2019 American Association for Anatomy     

miR-199a-3p targets ETNK1 to promote invasion and migration in gastric cancer cells and is associated with poor prognosis

PurposeTo investigate the prognostic significance of miR-199a-3p and its role in invasion and metastasis in gastric cancer.MethodsmiR-199a-3p expression in 436 formalin-fixed and 39 frozen gastric cancer tissues was investigated by in situ hybridization and RT-PCR, respectively. The role of miR-199a-3p in the migration and invasion of gastric cancer cells was determined in overexpression and inhibitor studies using transwell assays and the SGC-7901, BGC-823 and MGC-803 gastric cancer cells lines. The effect of miR-199a-3p expression on ethanolamine kinase 1 (ETNK1) levels was determined by western botting.ResultsmiR-199a-3p was significantly up-regulated in AGS, SGC-7901, BGC-823 and MGC-803 gastric cancer cells, when compared with GES-1 non-malignant gastric epithelial cells. In situ hybridization studies revealed that human non-tumor gastric mucosa samples were negative for miR-199a-3p expression, while 162 of 436 (37.16%) cases of gastric cancer demonstrated positive expression. miR-199a-3p overexpression was associated with tumor size, Lauren classification, depth of invasion, lymph node and distant metastasis, TNM stage and prognosis. In patients with I, II and III stage tumors, high miR-199a-3p expression was associated with a significantly lower 5-year survival rate. miR-199a-3p overexpression was associated with increased cell migration and invasion. ETNK1 expression was inhibited following miR-199a-3p overexpression in BGC-823 and SGC-7901 cells, and elevated following miR-199a-3p suppression in MGC-803 cells.ConclusionmiR-199a-3p is highly expressed in gastric cancer, and correlates with invasion, metastasis and prognosis. miR-199a-3p regulates the invasion and migration of gastric cancer cells by targeting ETNK1. Consequently, miR-199a-3p may serve as a prognostic indicator in gastric cancer.     

Over expression of amphiregulin promoted malignant progression in gastric cancer

Human Amphiregulin (AREG) was found to be over expressed in many types of cancer patients including gastric cancer (GC). However, its role in GC development remained unknown. In this study, over expression of AREG in MGC803 and SGC7901 GC cells were conducted by gene transfection through utilizing gateway recombinant cloning technology. Meanwhile, AREG knocked down in BGC823 and MKN45 GC cells were achieved by lentvirus-mediated shRNA oligos infection. The results showed that over expression of AREG promoted cancer cells proliferation, invasion and migration, prevented apoptotic cell death and facilitated cell cycle progression. However, the above AREG-mediated oncogenic phenotypes were reversed while AREG was knocked down in cancer cells. Furthermore, the expression of AREG in GC cells was associated with higher level of activation of both ERK/JNK/p38 and PI3K/Akt signaling pathways, which were also diminished by AREG knocked down. Finally, the result from study in vivo showed that the silencing AREG expression by AREG knocked down inhibited tumor growth in nude mice. Collectively, the data provided a convincingly laboratory evidence that over expression of AREG promoted malignant procession via activation of ERK/JNK/p38 and PI3K/Akt signaling pathways. Thus AREG possessed an oncogenic activity and could be served as a target for gastric cancer therapy.