特异性微小RNA抑制剂对胃癌细胞增殖的影响

目的： 探讨特异性微小RNA抑制剂对胃癌细胞增殖的影响。方法: 设计并合成4种微小RNA（miR-17、miR-21、miR-106a和miR-421）的2’-甲氧修饰的RNA寡核苷酸（微小RNA抑制剂）,用脂质体分别转染到SGC-7901和MGC-803胃癌细胞,然后用实时定量逆转录-聚合酶链反应技术检测微小RNA的表达情况,最后用MTT方法检测胃癌细胞的增殖情况。结果: 4种微小RNA抑制剂均有效抑制了SGC-7901和MGC-803细胞中相应微小RNA的表达;除miR-21抑制剂未见抑制胃癌细胞的增殖外,其它3种微小RNA抑制剂均以剂量依赖方式抑制胃癌细胞的增殖且对SGC-7901的效果优于MGC-803。结论: 微小RNA的特异性抑制剂能有效抑制胃癌细胞的增殖,采用这种技术为研究胃癌的发病机制提供了新方法。     

氧化苦参碱对淋巴细胞增殖和调节性T细胞（Tr）数量的影响

目的： 分析氧化苦参碱（OMT）对小鼠外周血调节性T细胞（Tr细胞）数量及其对刀豆蛋白A(Con A) 刺激的小鼠淋巴结T细胞增殖的影响, 探讨OMT治疗ACD的免疫学机制。方法： 建立DNFB诱发的小鼠ACD模型，腹腔注射（ip）不同剂量的OMT、PBS、氢化可的松(HCT)，在实验的第1 d、7 d、14 d、21 d、28 d小鼠尾静脉采血, 应用抗-CD3、抗-CD4、抗-CD25单抗进行免疫荧光标记, 流式细胞术检测各组CD4+CD25+T细胞数量。利用羧基荧光素乙酰乙酸(CFDA-SE)染色，流式细胞术检测OMT对小鼠淋巴结T细胞增殖的影响。结果： 500、125和31 mg/L OMT对小鼠淋巴结T细胞增殖呈剂量依赖性抑制，而16、8、4、2 mg/L OMT可促进小鼠淋巴结T细胞增殖，但剂量依赖关系不明显。腹腔注射OMT能明显提高小鼠外周血中CD4+CD25+T细胞数量, 与HCT组、PBS组比较（P<0.01）。结论: OMT对小鼠淋巴结T细胞增殖呈双向作用；腹腔注射OMT能明显提高小鼠外周血中CD4+CD25+T细胞数量；提示:OMT是一种双向免疫调节剂。     

Annexin A3高表达对胃癌 MGC803细胞增殖及凋亡的作用

目的：探讨Annexin A3高表达对胃癌MGC803细胞增殖及凋亡的影响。方法构建重组质粒pYr-ads-4-Annexin A3,转染至胃癌MGC803细胞,G418筛选稳定表达株,Western b1ot法检测转染前后Annexin A3蛋白表达变化;以空载体转染组及未转染MGC803细胞为对照,采用CCK8、平板克隆和流式细胞仪技术检测Annexin A3高表达对MGC803细胞增殖、克隆形成及细胞周期的作用。结果成功构建重组质粒pYr-ads-4-Annexin A3;pYr-ads-4-Annexin A3稳定转染细胞株中Annexin A3蛋白表达均明显高于空载体转染及未转染MGC803细胞组（ P<0.05）;CCK8实验显示,pYr-ads-4-Annexin A3组中肿瘤细胞数目显著多于空载体转染及未转染MGC803细胞组（ P<0.05）;平板克隆实验发现,pYr-ads-4-Annexin A3组中肿瘤细胞形成克隆数量多于其余两组（P<0.05）;流式细胞仪检测结果提示,Annexin A3高表达抑制了MGC803细胞凋亡（P<0.05）。结论 An-nexin A3在胃癌的发生过程中起重要作用,其机制可能与促进胃癌细胞增殖和抑制肿瘤细胞凋亡有关,提示Annexin A3可能成为胃癌治疗的新靶点。     

rhsBLyS对B淋巴细胞增殖的影响

目的测定可溶性重组人B淋巴细胞刺激因子(rhsBLyS)对B细胞的促增殖活性。方法通过FluroBeadsB荧光磁珠法体外分离并培养人B淋巴细胞,以CD20的特异性抗体标记之,借助流式细胞术检测。结果rhsBLyS非融合蛋白在引发剂AntiIgM的协同作用下表现出显著刺激B淋巴细胞增殖的活性。结论流式细胞术检测简便高效,在验证样品活性的同时为进一步开展工作奠定了基础。     

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。     

Hsa_circ_0020014调控胃癌细胞的增殖、迁移的作用研究

目的 探究hsa_circ_0020014在胃癌中的生物学功能及可能的调控机制.方法 利用实时荧光定量聚合酶链反应检测hsa_circ_0020014在胃生理上皮细胞及多种胃癌细胞系中的表达水平.CCK8方法及transwell实验检测hsa_circ_0020014对胃癌细胞增殖和迁移能力的作用.结果 Hsa_circ_0020014在MGC-803、HGC-27、SGC-7901、AGS等胃癌细胞系中显著高表达,且敲除hsa_circ_0020014能够明显抑制胃癌细胞的增殖、迁移能力.与GES-1相比,hsa-miR-199a/b-3p在多种胃癌细胞系中低表达,且敲除hsa_circ_0020014能上调hsa-miR-199a/b-3p的表达.结论 Hsa_circ_0020014能通过竞争性结合hsa-miR-199a/b-3p调控胃癌的增殖与迁移.     

康莱特注射液对胃癌患者细胞凋亡、增殖及T细胞亚群的影响

目的：探讨康莱特对胃癌细胞凋亡对增殖及胃癌患者T细胞亚群的影响。方法：进展期胃癌患者30例，随机分为3组（每组各10例）：A组（对照组）：手术前后常规给予全胃肠外营养（total parenteral nutrition,TPN)治疗；B组（康莱特治疗组）：术前5d及术后9d，给予康莱特注射液200mL/d静脉滴入加常规TPN治疗；C组（化疗组）：术前给予5d化疗，甲酰四氢叶酸钙（calcium folinatefor,CF)200mg及5氟脲嘧啶（5－FU）750mg/d静脉滴注加常规TPN治疗。分别于治疗前及术后1d,5d及10d，采集外周静脉血，应用免疫荧光法检测CD3^ 、CD4^ 、CD8^ T细胞亚群。手术中取胃癌病理组织，用末端转移酶介导的dUTP切口末端标记法和免疫组织化学染色法检测胃癌细胞的凋亡（AI）与增殖（PI）及二者之比（AI／PI）。结果：B组与C组相比较，胃癌细胞的AI、PI及AI／PI，无显著差异（P＞0．05）；B组与A组相比较，上 3种指标则具有显著差异（P＜0．01）。治疗前3组CD3^ 、CD4^ 、CD8^ T细胞亚群的百分率无显著差异（P＞0．05）；术后1d,5d3组CD3^ 、CD4^ 、CD8^ T细胞亚群的百分率差异显著（P＜0．01）；术后10d,CD3^ 、CD4^ 、CD8^ T细胞亚群的百分率，B组与A组以及B组与C组相比较，差异性分别为显著（P＜0．05）及非常显著（P＜0．01）。结论：康莱特可明显促进胃癌细胞凋亡和抑制其增殖，有助于提高围手术期患者的免疫功能。     

黄芩素抑制胃癌MGC-803细胞增殖和迁移

目的:探讨黄芩素(BAI)对胃癌MGC-803细胞增殖和迁移的作用及机制。方法:MGC-803细胞用不同浓度BAI处理后,采用MTT法检测细胞的存活率;平板集落形成实验检测细胞的集落形成能力;划痕愈合实验和Transwell小室迁移实验检测细胞的迁移能力;ELISA检测12-羟基二十碳四烯酸(12-HETE)的浓度;Western blot实验检测血小板型12-脂氧合酶(p12-LOX)、血管内皮生长因子(VEGF)、p-ezrin和上皮-间充质转化(EMT)标志物蛋白的表达。结果:BAI可显著抑制MGC-803细胞的增殖、平板集落形成及迁移(P0.05或P0.01),显著下调p12-LOX代谢产物12-HETE的浓度(P0.05或P0.01),并显著下调p12-LOX、VEGF、p-ezrin、vimentin和Snail蛋白的表达水平(P0.05或P0.01),上调E-cadherin蛋白的表达水平(P0.01)。结论:BAI可有效抑制胃癌MGC-803细胞的增殖和迁移,其机制与BAI调控p12-LOX、VEGF、p-ezrin及EMT相关蛋白的表达变化有关。     

Effect of bone morphogenetic protein-2 on proliferation and apoptosis of gastric cancer cells

Objective: To investigate the effects of bone morphogenetic protein-2 (BMP-2) on the proliferation, differentiation and apoptosis of normal human gastric mucosal cells and gastric cancer cells.Methods: Poorly differentiated gastric cancer BGC823 cells, moderately differentiated gastric cancer cells and normal human gastric mucosal epithelial GES-1 cells were independently treated with recombinant human BMP-2 or its inhibitor Noggin. MTT assay was performed to detect the proliferation, flow cytometry done to measure the cell cycle and apoptosis and immunohistochemistry carried out to determine the expression of cyclin-dependent kinase 4 (CDK4).Results: BMP-2 exerted inhibitory effect on the growth of all types of cells and the inhibition become more evident with the increase of BMP-2 dose. After treatment with 200 ng/ml BMP-2, cancer cells arrested in G1 phase and those in S phase reduced. Gastric cancer cells had higher CDK4 expression than GES-1 cells. BMP-2 decreased CDK-4 expression in cancer cells but had no influence in GES-1 cells. Noggin conferred promotive effect on the growth of 3 types of cells. In 2 types of cancer cells, treatment with 2000 ng/ml Noggin significantly increased the proportion of cells in S phase but reduced that in G1 phase. However, Noggin did not affect the cell cycle of GES-1 cells. The CDK4 expression was markedly increased in 2 types of cancer cells but that of GES-1 remained unchanged after treatment with 2000 ng/ml Noggin.Conclusions: BMP-2 may inhibit the proliferation of both normal and malignant gastric epithelial cells, down-regulate CDK4 expression in gastric cancer cells and arrest gastric cancer cells in G1-phase in cell cycle. Through antagonizing BMP-2, Noggin, may accelerate the proliferation of gastric cancer cells. Thus, the abnormality of BMP signaling pathway may play an important role in the pathogenesis of gastric cancer.     

Dual effect of ciliary body cells on T lymphocyte proliferation

The interaction between organ-resident cells from the anterior uvea of the eye and T helper (Th) cells was investigated. Cells from Lewis rat ciliary body processes (CB cells), grown in tissue culture using an explant technique, could be induced to express major histocompatibility complex class II (Ia) antigens by incubation with rat interferon-gamma. Ia+ CB cells only poorly functioned as antigen-presenting cells (APC) for a syngeneic, uveitogenic Th cell line specific for the retinal soluble antigen (SAg). Moreover, if added to an Ag-driven lymphocyte proliferation assay in the presence of conventional APC, the rat CB cells had an inhibiting effect on Th proliferation. This inhibitory activity was not species specific, since similar effects were observed with bovine and human ciliary epithelial cells. The suppressive activity of CB cells was composed of a soluble factor, as well as a membrane-associated inhibitor. The soluble activity did not appear to be related to transforming growth factor-beta (TGF-beta), since no reversal of inhibition by a neutralizing antibody to TGF-beta was found. Part of the soluble inhibitory activity could be reversed by indomethacin treatment. The membrane-associated component was trypsin sensitive, suggesting a protein molecule. After abrogation of the inhibitory capacity by trypsin treatment and fixation by glutaraldehyde, CB cells effectively presented SAg to Th cells. These data suggest that CB cells are capable of mediating both Ag presentation and inhibition of Th cell proliferation.     

支架蛋白活化蛋白激酶C受体1在胃癌细胞中的表达及其与胃癌细胞增殖的关系

目的 探讨活化蛋白激酶C受体1C(RACK1, GNB2L1)在胃癌细胞HGC27中的表达及过表达RACK1对HGC27生长增殖的影响。方法 体外培养胃癌未分化细胞HGC27和正常胃黏膜上皮细胞系GES-1,收集细胞48 h后,提取mRNA和蛋白,利用RT-PCR检测RACK1 mRNA在HGC27和GES-1细胞中的表达;利用Western blotting法检测RACK1蛋白在两种细胞中的表达;以人胚肾HEK293细胞cDNA为模板,构建pcDNA3.1A-flag-RACK1重组质粒,利用Lipo2000转染入HGC27细胞中,Western blotting法检测质粒的转染效率,MTT法检测过表达RACK1对HGC27胃癌细胞系生长增殖的影响。 结果 HGC27胃癌细胞中RACK1的mRNA和蛋白水平表达低于GES-1细胞（P<0.01）。双酶切鉴定和测序分析表明,pcDNA3.1A-flag-RACK1重组质粒构建成功。将该质粒转入HGC27细胞后,与未转染组相比,空载转染组RACK1蛋白表达无明显区别（P>0.05）,pcDNA3.1-RACK1转染组RACK1蛋白表达明显升高（P<0.01）;转染pcDNA3.1A-flag-RACK1转染组细胞存活率在72 h和96 h明显少于pcDNA3.1空载对照组（P<0.01）。结论 支架蛋白RACK1的mRNA和蛋白水平在HGC27细胞中低表达,上调RACK1的表达可明显抑制HGC27细胞增殖。     

A novel long non-coding RNA NFIA-AS1 is down-regulated in gastric cancer and inhibits proliferation of gastric cancer cells

Gastric cancer is one of the most common malignant gastrointestinal tumors whose morbidity and mortality account for the second and third place respectively in malignant tumors in China. As an important participant in tumor biology, the abnormal expression of long non-coding RNA (lncRNAs) in cancer cells is closely related to the occurrence and development of tumors and plays the role of oncogenes or tumor suppressor genes. In this study, we identified a novel lncRNA NFIA antisense RNA 1 (NFIA-AS1) and explored its role and clinical significance in gastric cancer. Real-time quantitative PCR was performed to detect the expression of NFIA-AS1 in tumor tissues and corresponding normal tissues from 42 pairs of gastric cancer samples. The lower expression of NFIA-AS1 was significantly associated with larger tumor size, lower histological grade, and advanced TNM stage. Kaplan-meier analysis showed that NFIA-AS1 expression could be used as an independent predictor of overall survival. We also demonstrated that overexpression of NFIA-AS1 significantly inhibited the proliferation of gastric cancer cells through affecting p16 levels. In conclusion, our results suggest that the lncRNA NFIA-AS1 may play the role of tumor suppressor gene, and serve as a biomarker for prognosis or progression of gastric cancer.     

EZH2 promotes gastric cancer cells proliferation by repressing p21 expression

EZH2 is a core component of the polycomb repressive complex 2 (PRC2), which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) and promotes carcinogenesis by epigenetically silencing many tumor suppressor genes. Increased EZH2 expression is a marker of advanced and metastatic in many cancers, including lung, prostate and breast cancer, and it has been considered as a potential novel therapeutic target. However, the clinical significance and molecular mechanisms of EZH2 controlling gastric cancer cell proliferation and invasion are not well documented. In this study, immunohistochemical analysis was conducted to investigate the EZH2 expression in gastric cancer. We found that EZH2 levels were increased in gastric cancer tissues compared with adjacent normal tissues. Moreover, patients with high levels of EZH2 expression had a relatively poor prognosis. Furthermore, knockdown of EZH2 expression by siRNA could impair cell proliferation and invasion both in vitro and vivo. Finally, we found that EZH2 influences gastric cancer cells proliferation partly through regulating p21 expression. Our findings present that EZH2 over-expression can be identified as a poor prognostic biomarker in gastric cancer.     

大黄素对人胃癌BGC-823细胞体外增殖和迁移的影响

目的 探讨大黄素对人胃癌BGC-823细胞增殖和迁移的影响。 方法 采用四甲基偶氮唑盐（MTT）比色法测定不同浓度大黄素对体外培养人胃癌BGC-823细胞增殖的影响;采用流式细胞术测定大黄素对人胃癌BGC-823细胞的细胞周期影响;采用划痕损伤实验测定大黄素对人胃癌BGC-823细胞迁移的影响。 结果 MTT法显示,大黄素能抑制人胃癌BGC-823细胞増殖,呈现浓度依赖性;流式细胞术显示,人胃癌BGC-823细胞的细胞周期主要停滞在G₀/G₁期;划痕损伤实验表明,人胃癌BGC-823细胞迁移受到明显的抑制。 结论 大黄素对人胃癌BGC-823细胞的增殖和迁移具有明显的抑制作用,且呈浓度依赖性,并可使人胃癌BGC-823细胞增殖周期停滞于G₀/G₁期。     

逆转录病毒载体介导的IL-4基因修饰对HL-60细胞增殖及T细胞功能的影响

目的观察ＩＬ－４基因转染对ＨＬ－６０增殖及Ｔ细胞功能的影响及其可能机制。方法采用逆转录病毒载体介导基因转染法将人ＩＬ－４基因导入髓系白血病细胞ＨＬ－６０,观察高表达外源性ＩＬ－４基因ＨＬ－６０株的增殖反应及其诱导的正常人ＰＢＭＣ增殖,ＩＬ－２、ＩＬ－６、ＩＦＮ－γ基因表达及肿瘤特异性ＣＴＬ杀伤活性。结果ｒＩＬ－４或瘤细胞产生的ＩＬ－４早期促进ＨＬ－６０细胞增殖,培养５天后则明显抑制其增殖;与ｒＩＬ－４处理的ＨＬ－６０细胞比较,ＩＬ－４基因修饰的ＨＬ－６０细胞明显促进正常人ＰＢＭＣ增殖并合成ＩＬ－２、ＩＬ－６及ＩＦＮ－γｍＲＮＡ,其诱导的特异性ＣＴＬ杀伤活性明显高于野生型ＨＬ－６０、仅导入载体的ＨＬ－６０和加入ｒＩＬ－４共育的ＨＬ－６０细胞诱导者。结论ＩＬ－４基因转染在影响ＨＬ－６０细胞增殖和分化过程的同时,可能促进某些抗原（如ＭＨＣⅠ类抗原）的抗原性,从而有利于机体建立有效的抗肿瘤免疫反应。     

共培养条件下乳腺癌细胞对淋巴管内皮细胞增殖的影响

目的建立乳腺癌细胞和淋巴管内皮细胞共培养模型,探讨乳腺癌细胞在共培养条件下对淋巴管内皮细胞增殖的影响。方法培养并鉴定淋巴管内皮细胞;利用transwell小室建立乳腺癌细胞与淋巴管内皮细胞体外共培养模型;采用MTT法检测乳腺癌细胞对淋巴管内皮细胞增殖情况。结果VEGFR-3抗体对培养的细胞进行鉴定,为典型淋巴管内皮细胞;MTT法结果显示：在共培养24~72 h时,乳腺癌细胞能够促进共培养条件下淋巴管内皮细胞的增殖（P〈0.05）。结论成功建立乳腺癌细胞和淋巴管内皮细胞共培养模型,乳腺癌细胞能够促进共培养条件下淋巴管内皮细胞的增殖。     

RARG对胃癌细胞活力和迁移能力的影响

目的:探讨视黄酸受体γ(retinoic acid receptor gamma,RARG)对胃癌细胞活力和迁移能力的影响及其机制。方法:采用生物信息学技术分析在线数据库中胃癌和正常胃组织的RARG表达水平及其与胃癌患者总生存率的相关性;利用过表达质粒转染和RNA干扰技术,在体外促进和抑制胃癌细胞中RARG的表达,并采用MTT和Transwell实验检测RARG对胃癌细胞活力和迁移能力的影响;通过Western blot和TOP/FOP双萤光素酶报告基因检测RARG对Wnt/β-catenin信号通路的调控;利用免疫共沉淀和免疫荧光共定位实验检测RARG与β-catenin蛋白的相互作用情况。结果 :过表达RARG能够增强胃癌细胞SGC7901的活力和迁移能力(P0.05),敲减RARG能够减弱胃癌细胞MGC-803的活力和迁移能力(P0.05)。同时,过表达RARG能够增加β-catenin、c-Myc、cyclin D1、Twist和Snail的蛋白表达水平(P0.05),以及TOP/FOP双萤光素酶报告基因活性(P0.05)。另外,RARG与β-catenin蛋白存在相互作用。结论 :RARG通过激活Wnt/β-catenin信号通路促进胃癌细胞的活力和迁移能力,其基因在胃癌的发生发展中扮演着癌基因的角色。     

大鼠骨髓间充质干细胞对淋巴细胞增殖影响的体外实验

目的：探讨大鼠骨髓间充质干细胞对淋巴细胞增殖的影响。方法：常规分离培养大鼠的骨髓间充质干细胞（MSCs）及淋巴细胞,灭活后的骨髓间充质干细胞和淋巴细胞混合培养48h后,MTT法检测淋巴细胞的相对细胞数,Hoechst 33258染色观察细胞核变化。结果：MTT结果显示,加入MSCs组OD值明显低于未加MSCs的对照组（p〈0．05）。Hoechst 33258染色显示MSCs组淋巴细胞有核回缩及凋亡小体出现。结论：MSCs对淋巴细胞的增殖有抑制作用,其作用机制可能与细胞凋亡有关。     

Flagellin promotes the proliferation of gastric cancer cells via the Toll-like receptor 5

Signaling of the Toll-like receptor (TLR) is closely associated with tumor development and progression processes including cell proliferation, angiogenesis, metastasis, and immunosuppression. In this study, we examined the expression of TLR5 in gastric cancer cells and its function in cell proliferation. RT-PCR revealed that the TLR5 gene was expressed in all gastric cancer cell lines examined, SNU638, SNU601, SNU216, and AGS. The TLR5 agonist, flagellin, induced IL-8 production and NF-κB activation in the gastric cancer cell lines. In addition, flagellin enhanced the proliferation of all gastric cancer cells examined, whereas LPS did not affect that of SNU638 cells. Blockade of TLR5 using an antibody, restored the proliferation of SNU638 cells enhanced by flagellin, indicating that TLR5 is essential for cell proliferation by flagellin. Flagellin also led to phosphorylation of ERK in SNU638 cells. The ERK inhibitor, PD98059, restored the proliferation ability of SNU638 cells enhanced by flagellin, suggesting that ERK may play an important role in the proliferation of gastric cancer cells. These findings suggest that TLR5 may play an important role in tumor progression of gastric cancer via the regulation of cell proliferation.