人β防御素-2通过TGF-β/Smad信号通路对胃癌SGC7901细胞增殖、迁移和侵袭的影响

该文探讨了人β防御素-2(hBD2)对胃癌SGC7901细胞的增殖、迁移和侵袭的影响。将真核表达载体pCMV-hBD2转染于人胃癌SGC7901细胞。采用qPCR和免疫印迹法(Western blot)检测转染效率。Western blot检测TGF-β1、p-Smad2/3、Smad2/3、MMP9的蛋白表达水平。Transwell法检测SGC7901细胞迁移和侵袭能力。EdU法和流式细胞术分别检测增殖能力与细胞周期。结果显示,转染真核表达载体pCMV-hBD2的SGC7901细胞, hBD2的表达水平明显高于SGC7901和转染pCMV-Blank的SGC7901细胞。SGC7901-hBD2细胞中TGF-β1、p-Smad2/3和MMP9的蛋白表达水平均低于SGC7901和SGC7901-Blank细胞,而Smad2/3表达水平不变。同时,其迁移侵袭和增殖能力均受到抑制,细胞周期G0/G1期阻滞。该实验结果表明, hBD2可能通过下调TGF-β/Smad信号通路调控SGC7901细胞的迁移侵袭以及增殖能力。     

MDR1基因沉默增强紫杉醇诱导SGC7901/ADM细胞凋亡的敏感性

为了探讨MDR1基因沉默对紫杉醇诱导人胃癌SGC7901/ADM细胞凋亡的影响,本研究构建靶向MDR1基因重组干扰载体,稳定转染SGC7901/ADM细胞,流式细胞术检测P-gp外排泵功能。激光共聚焦显微镜下观察细胞形态变化,琼脂糖凝胶电泳检测DNA断裂情况。结果发现,构建的shRNA表达载体pSilencer 2.1-U6 neo-MDR1稳定转染SGC7901/ADM细胞后,细胞内Rho-123相对荧光强度升高;激光共聚焦显微镜下观察到,细胞形态发生改变,出现凋亡特征,与SGC7901/ADM细胞相比,经紫杉醇处理后SGC7901/ADM-2.1-3细胞的DNA片段化更为明显。结果表明,MDR1基因沉默增强了紫杉醇诱导SGC7901/ADM细胞凋亡的敏感性。     

沉默MDR1基因逆转SGC7901/ADM细胞对紫杉醇的耐药性

本研究利用短发夹sh RNA(short hairpin RNA)沉默SGC7901/ADM细胞MDR1基因,从而逆转SGC7901/ADM细胞对紫杉醇的耐药性。根据MDR1基因序列设计并合成编码sh RNA的DNA模板,构建3种靶向MDR1基因重组干扰载体,稳定转染SGC7901/ADM细胞,q RT-PCR检测MDR1 m RNA表达水平,Western blotting检测MDR1蛋白表达水平,MTT法检测细胞对紫杉醇的敏感性。结果表明,成功构建了靶向MDR1基因的3种重组表达载体,分别转染SGC7901/ADM细胞后均能不同程度沉默MDR1基因的表达,其中p2.1-3对MDR1沉默效果最好,m RNA和蛋白的沉默效率分别为78.5%和45%。紫杉醇对细胞的IC50值由(3.147±0.494)μmol/L降至(0.714±0.059)μmol/L,逆转率达到(78.22±1.906)%。结果表明,靶向MDR1的干扰表达载体能够抑制MDR1基因的表达,从而逆转SGC7901/ADM细胞对紫杉醇的耐药性。     

姜黄素诱导沉默MDR1基因的SGC7901/ADM细胞凋亡

为探讨MDR1基因沉默对姜黄素诱导人胃癌SGC7901/ADM细胞凋亡的影响,将已构建的靶向MDR1基因的RNAi表达载体转染SGC7901/ADM细胞,建立转染细胞单克隆,流式细胞术检测细胞外排功能。姜黄素处理SGC7901/ADM细胞48 h后,通过激光共聚焦显微镜下观察细胞形态结构,琼脂糖凝胶电泳检测DNA片段化。结果显示,各干扰载体转染组细胞内Rho-123荧光强度不同程度增强,细胞经姜黄素处理48 h后,激光共聚焦显微镜下呈明显的凋亡形态结构,DNA琼脂糖凝胶电泳呈梯状条带,说明MDR1基因沉默能促进姜黄素诱导SGC7901/ADM细胞凋亡。     

慢病毒siRNA靶向干扰YAP基因胃癌细胞株的建立

目的：构建并鉴定YAP基因短发夹干扰RNA（shRNA）慢病毒载体,建立稳定干扰YAP基因表达的胃癌细胞株SGC7901。方法：荧光定量PCR检测YAP基因在多种胃癌细胞株中的表达情况。构建重组靶向YAP基因的shRNA慢病毒表达质粒PGC-shRNA-YAP,用脂质体转染的方法将载体导入胃癌细胞。经杀稻瘟菌素筛选后,建立稳定表达siRNA的细胞株。荧光定量PCR检测干扰效率。结果：在胃癌细胞株SGC7901中,YAP基因显示高表达。测序验证PGC-shRNA-YAP重组质粒构建成功。将重组质粒稳定转染入胃癌细胞株SGC7901后能明显抑制YAPmRNA表达水平。结论：成功构建了PGC-shRNA-YAP慢病毒重组质粒,建立了靶向稳定干扰YAP基因表达的siRNA胃癌细胞株SGC7901。     

CD147-shRNA对胃癌细胞CD147表达的抑制作用

目的:构建CD147-shRNA重组质粒并检测其对胃癌细胞SGC7901内源性CD147表达的抑制作用。方法:构建靶向CD147的siRNA表达载体pSilencer-CD147siRNA,稳定转染至SGC7901细胞中,通过Real-time PCR和Western blot法检测转染细胞中CD147表达水平的变化。结果:酶切鉴定和测序证实成功构建pSilencer-siRNA1,pSilencer-siRNA2和pSilencer-negative,转染后SGC7901中CD147表达水平显著降,而以pSilencer-siRNA2抑制作用最为显著。结论:构建pSilencer-CD147siRNA成功,并筛选出基因抑制效果抑制最佳的pSilencer-siRNA2,为进一步实验打下了基础。     

长链非编码RNA UCA1在胃癌多药耐药中的作用研究

目的:研究胃癌耐药细胞及其亲本细胞中长链非编码RNA UCA1的表达差异,探讨UCA1在胃癌多药耐药中的作用。方法:通过实时荧光定量PCR(q RT-PCR)检测胃癌耐药细胞SGC7901/ADR、SGC7901/VCR及其亲本细胞SGC7901中UCA1的表达差异;通过si RNA转染降低SGC7901/ADR中UCA1表达,MTT法检测细胞半数抑制浓度(IC50)的变化,流式细胞仪检测细胞凋亡变化。结果:QRT-PCR结果显示,UCA1在SGC7901/ADR和SGC7901/VCR胃癌耐药细胞表达显著高于SGC7901胃癌亲本细胞;MTT实验表明,干扰UCA1的SGC7901/ADR相对于阴性对照(NC)组的IC50显著降低;凋亡检测结果显示,在相同剂量化疗药物作用下,干扰UCA1后SGC7901/ADR凋亡率显著高于NC组;Western blot证实,干扰UCA1表达可显著降低BCL-2蛋白表达。结论:长链非编码RNA UCA1在胃癌耐药细胞表达显著升高,干扰UCA1表达可明显逆转胃癌耐药,UCA1可作为治疗胃癌耐药的重要分子靶标。     

比较NRP-1反义寡核苷酸与VEGFR-2反义寡核苷酸对人胃癌SGC7901细胞增殖和凋亡的影响

目的:研究比较神经纤毛蛋白1(NRP-1)反义寡核苷酸(ASODN)与血管内皮生长因子受体2(VEGFR-2)反义寡核苷酸(ASODN)对人胃癌SGC7901细胞增殖活性及凋亡水平的影响。 方法:分别及同时将不同浓度经硫代磷酸化修饰的NRP-1 ASODN 和 VEGFR-2 ASODN 转染入人胃癌SGC7901细胞,逆转录-聚合酶链反应(RT-PCR)检测NRP-1基因和VEGFR-2 基因mRNA的转录水平;MTT比色法测量细胞的增殖活性;流式细胞仪测量细胞的凋亡水平。 结果:转染NRP-1 ASODN和VEGFR-2 ASODN后,人胃癌SGC7901细胞NRP-1基因和VEGFR-2 基因mRNA的转录水平均出现降低;NRP-1 ASODN和VEGFR-2 ASODN对SGC7901细胞有明显抑制增殖和促进凋亡的作用,且随着ASODN浓度升高而增强;分别转染时其作用无显著差别,联合转染时其作用明显增强。结论:NRP-1 ASODN和VEGFR-2 ASODN可抑制人胃癌SGC7901细胞 NRP-1基因和VEGFR-2 基因mRNA的转录水平及细胞增殖活性,促进细胞凋亡;与分别转染相比,两者联合转染作用明显增强。     

人tBID蛋白的表达纯化及其偶联的新型分子探针对前列腺癌细胞的促凋亡作用研究

目的:以PQE-30为原核表达载体,构建PQE30-tBID重组表达载体,表达和纯化目的蛋白tBID,并利用金磁纳米微粒将tBID蛋白与人HER2抗体偶联成分子探针Anti HER2-Gold Mag-tBID,以探究其对前列腺癌细胞的促凋亡作用。方法:根据tBID的基因序列设计特异性的上下游引物,利用普通PCR扩增目的基因tBID,构建重组表达载体PQE30-tBID,将其转化到BL21(DE3)中,IPTG诱导表达,经SDS-PAGE凝胶电泳和Western Blot鉴定分析,验证目的蛋白tBID的表达,并对其进行纯化。利用金磁纳米微粒与蛋白质之间的静电相互作用以及疏水相互作用,将人HER2抗体与tBID蛋白偶联在其表面,构建分子探针Anti HER2-Gold Mag-tBID。流式细胞术检测该分子探针与前列腺癌PC-3细胞的特异性亲附结合能力,通过Annexin V-FITC细胞凋亡检测试剂盒分析分子探针对PC-3细胞的促凋亡作用。结果:普通PCR扩增后得到了411 bp的DNA片段,经双酶切鉴定以及菌液测序,表明重组表达载体PQE30-tBID构建成功。促凋亡蛋白tBID成功地在大肠杆菌中表达,蛋白相对分子量约15 KD,经过纯化,得到了纯度较高的tBID蛋白。经过与金磁纳米微粒的偶联,成功构建出一种新型的分子探针Anti HER2-Gold Mag-tBID。该分子探针可与PC-3细胞特异性结合,且经Annexin V-FITC染色分析可见PC-3细胞发生明显凋亡,凋亡率达62.9%,与未处理组(3.79%)和对照组(4.33%)相比,具有显著的统计学差异。结论:PQE30-tBID重组表达载体能在大肠杆菌中高效表达,且成功得到了纯度较高的人促凋亡蛋白tBID。经金磁纳米微粒偶联,该蛋白能够与人HER2抗体重组成新型的分子探针,且能特异性地靶向前列腺癌PC-3细胞并显著促进其凋亡。     

表达血管内皮生长因子-siRNA及双自杀基因yCDglyTK的联合基因载体构建及其应用研究

构建了新型联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK,研究其在人胃癌细胞系SGC7901细胞中的表达和杀伤作用.构建靶向血管内皮生长因子(VEGF)的干扰质粒pGenesil-VEGF-siRNA,采用PCR法从中扩增siRNA表达框(含U6启动子),亚克隆至双自杀基因载体pcDNA3.1(-)CV-yCDglyTK,构建联合基因质粒pcDNA3.1(-)VEGF-siRNA/yCDglyTK;通过酶切、测序等鉴定重组质粒;以磷酸钙纳米颗粒为载体,将干扰质粒、双自杀基因质粒及联合基因质粒转染SGC7901细胞,RT-PCR、Western-blot验证目的基因表达;MTT法检测转染细胞对5-氟胞嘧啶(5-FC)的敏感性.结果表明:酶切及测序证实联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK构建成功;SGC7901细胞转染联合基因质粒后,RT-PCR、Western-blot证实融合自杀基因表达,而VEGF基因表达下调;在前体药物5-FC作用下,转染联合基因组细胞存活率最低,与其他组比较有统计学差异.成功构建联合基因载体pcDNA3.1(-)VEGF-si...     

Mad2beta, an alternative variant of Mad2 reducing mitotic arrest and apoptosis induced by adriamycin in gastric cancer cells

Mad2beta is an alternative splicing variant of spindle checkpoint gene mad2, which was previously found by us and was related to the drug resistance in gastric cancer cells. In this paper, we explored the molecular mechanisms that Mad2beta variant promoted the formation of multidrug resistance in gastric cancer cells. We found that Mad2beta variant was detected only in the two human drug resistant gastric cancer cell sublines SGC7901/VCR and SGC7901/ADR, and it did not appear in its parental cell line SGC7901 and other detected gastric cancer cell lines. Expressions of Mad2 mRNA and protein in SGC7901 cells transfected with Mad2beta, SGC7901/VCR and SGC7901/ADR were significantly lower than that in SGC7901 cells. Moreover, SGC7901 cells overexpressing Mad2beta variant became more resistant to adriamycin, vincristine and mitomycin by abrogating mitotic arrest and apoptosis. This suggests that expression of Mad2beta variant decreases the relative expression of efficient MAD2, which may help gastric cancer cells to develop the phenotype of multidrug resistance.     

人多肽N-乙酰氨基半乳糖转移酶2基因反义RNA表达质粒的构建及其功能的初步研究

反义封闭人多肽N-乙酰氨基半乳糖转移酶2 (pp-GalNAc-T2)的基因表达, 对胃癌细胞SGC7901中转化生长因子-β1(TGF-β1)与基质金属蛋白酶2 (MMP2)基因表达及细胞增殖有影响.在对几株肿瘤细胞的pp-GalNAc-T2基因表达水平进行分析后, 以高表达pp-GalNAc-T2的人胃癌细胞株SGC7901的总RNA为模板, 利用RT-PCR方法扩增两段不同长度pp-GalNAc-T2基因片段, 构建反义表达载体转染胃癌细胞SGC7901, 通过Ｇ418筛选, 建立一系列旨在封闭胃癌细胞SGC7901 ppGalNAc-T2基因表达的亚细胞克隆.通过流式细胞术、荧光显微镜、RT-PCR及Western印迹检测反义封闭pp-GalNAc-T2基因RNA表达后胃癌细胞SGC7901增殖以及TGF-β1、MMP2表达水平的变化. 反义封闭pp-GalNAc-T2基因表达后, 胃癌细胞SGC7901 pp-GalNAc-T2的表达水平明显降低, 细胞分裂增殖减慢, 表明反义封闭pp-GalNAc-T2基因表达对胃癌细胞SGC7901的生长增殖有影响.结果还显示, 反义封闭pp-GalNAc-T2基因表达可使TGF-β1、MMP2基因在mRNA与蛋白质表达水平均增加, 提示pp-GalNAc-T2基因表达可能对胃癌细胞SGC7901浸润转移产生影响.以上结果表明, pp-GalNAc-T2基因在肿瘤细胞中广泛表达, 并可能与肿瘤的增殖及浸润转移相关.     

幽门螺杆菌ureB基因转染胃上皮细胞及其对细胞的作用

研究幽门螺杆菌 (Helicobacterpylori,Hp)ureB基因重组子转染胃上皮细胞后对胃上皮细胞的作用。用PCR方法从Hp标准株NCTC116 37中获取ureB全长基因 ,将其开放读码框架定向克隆入真核表达载体pcDNA3 1;获得的重组子转染SGC 790 1细胞 ,筛选耐潮霉素的细胞克隆 ,用RT PCR方法检测细胞内ureB基因在转录水平的表达 ;分别用荧光染色技术、MTT、流式细胞术检测UreB对细胞表型、增殖、凋亡及细胞周期的影响。UreB阳性表达的细胞 (SureB)胞膜出芽、细胞皱缩 ;用MTT法检测细胞增殖 ,结果表明 ,SureB细胞与SpcDNA3 1细胞比较 (pcDNA3 1转染的细胞 ) ,生长增殖无显著性差异 (P >0 0 5 ) ,流式细胞术检测细胞凋亡结果显示 ,SureB的凋亡率显著高于SpcDNA3 1(P值为 0 0 0 7) ;细胞周期分析显示 ,SureB细胞有S期比率增高、G2 M、G0 G1 期比率下降的趋势。ureB在培养细胞内的表达可促进细胞凋亡     

Depression of MAD2 inhibits apoptosis of gastric cancer cells by upregulating Bcl-2 and interfering mitochondrion pathway

Mitotic arrest deficient 2 (MAD2) is an essential component of the mitotic spindle checkpoint pathway. It was previously shown to be associated with drug resistance of tumor cells. To further explore the roles of MAD2 in responses of gastric cancer cells to chemotherapy drugs, we constructed the siRNA vectors of MAD2 and transfected them into gastric cancer SGC7901 cells to inhibit expression of MAD2. MTT assay showed that the downregulation of MAD2 increased the resistance of SGC7901 cells to spindle inhibitors and DNA damaging agents. The apoptosis rates of gastric cancer cells transfected with MAD2-siRNA were 10.7% and 10%, respectively, after treated by 1.0microg/ml VCR and cisplatin. In contrast, the apoptosis rates of SGC7901 and SGC7901/psilencer3.1 induced by VCR were 43.2%, 38.7%; and that induced by cispaltin were 34.1%, 31.4%. The ratio of Bcl-2 to Bax was much higher in the MAD2-siRNA transfectants compared with the SGC7901/psilencer. In SGC7901/psilencer, cytochrome c and cleaved caspase 3 protein levels increased along with the exposure time increased. However, these protein levels of SGC7901/MAD2-siRNA had no changes during the drug treatment. These results indicate that down regulation of MAD2 could promote the drug resistance of gastric cancer cells and inhibit anticancer drugs induced-apoptosis by upregulating Bcl-2 and interfering the mitochondrion apoptosis pathway.     

慢病毒siRNA靶向干扰YAP基因胃癌细胞株的建立

目的:构建并鉴定YAP基因短发夹干扰RNA(shRNA)慢病毒载体,建立稳定干扰YAP基因表达的胃癌细胞株SGC7901。方法:荧光定量PCR检测YAP基因在多种胃癌细胞株中的表达情况。构建重组靶向YAP基因的shRNA慢病毒表达质粒PGC-shRNA-YAP,用脂质体转染的方法将载体导入胃癌细胞。经杀稻瘟菌素筛选后,建立稳定表达siRNA的细胞株。荧光定量PCR检测干扰效率。结果:在胃癌细胞株SGC7901中,YAP基因显示高表达。测序验证PGC-shRNA-YAP重组质粒构建成功。将重组质粒稳定转染入胃癌细胞株SGC7901后能明显抑制YAPmRNA表达水平。结论:成功构建了PGC-shRNA-YAP慢病毒重组质粒,建立了靶向稳定干扰YAP基因表达的siRNA胃癌细胞株SGC7901。     

胃癌SGC7901 表柔比星耐药细胞亚系的建立与耐药机制研究

目的:建立人胃癌SGC7901表柔比星耐药细胞系,探讨其对表柔比星的耐药机制。方法:采用逐步增加表柔比星浓度,间歇作用体外诱导法,建立人胃癌SGC7901表柔比星耐药细胞亚系SGC7901/EPI。用MTT法测定药物敏感性;流式细胞仪检测其药物排除能力和凋亡抵抗能力等生物学指标的改变,western blot检测相关蛋白的表达。结果:经过12个月建成人胃癌SGC7901表柔比星耐药细胞系SGC7901/EPI,其对表柔比星明显耐药,且对其他多种抗癌药具有不同程度的交叉耐药性,阿霉素蓄积潴留实验显示SGC7901/EPI的阿霉素含量明显低于亲本细胞,Western blot显示MRP1的表达上调;SGC7901/EPI凋亡抵抗能力明显上升,Bcl-2表达比亲本细胞增高,而Bax的表达下调。结论:SGC7901/EPI细胞具有多药耐药表型,其可能通过MRP1的上调增加药物排出和上调Bcl-2/Bax的比值促进凋亡抵抗等机制产生耐药。该胃癌多药耐药细胞亚系为进一步研究胃癌耐药机制及逆转方法奠定基础。     

ZNRF3 acts as a tumour suppressor by the Wnt signalling pathway in human gastric adenocarcinoma

E3 ubiquitin ligases regulate a variety of biological processes through the ubiquitin–proteasome system, together with ubiquitin activating enzyme E1 and ubiquitin-conjugating enzyme E2. Previous studies have demonstrated that zinc and ring finger 3 (ZNRF3), which belongs to the E3 ubiquitin ligases family is involved in the Wnt signalling pathway, which plays an important role in causing cancer. However, the expression and function of ZNRF3 in human gastric adenocarcinoma still remains unclear. Immunohistochemical and western blot analysis showed a significant down-regulation of ZNRF3 protein in gastric adenocarcinoma tissues compared with adjacent normal gastric tissues. In addition, there was a correlation between the down-regulation of ZNRF3 and poor tissue differentiation in gastric adenocarcinoma. To investigate the potential function of ZNRF3 in cell proliferation and apoptosis, a gastric cell line SGC7901 was employed. The over-expression of wild-type ZNRF3, which was accomplished by the transient transfection of recombinant pEGFP-ZNRF3 (or empty plasmids as control) into the cell line SGC7901, was confirmed by western blot analysis. Flow-cytometry-based and Cell Counting Kit-8 assays showed that over-expression of wt ZNRF3 induced apoptosis and suppressed proliferation. ZNRF3-overexpressing gastric cells displayed partly attenuated protein levels of beta-catenin and TCF-4 compared with those transfected with the empty plasmid. Our study demonstrates a novel gastric adenocarcinoma suppressor and reveals that ZNRF3 inhibits gastric cancer cell growth and promotes the cell apoptosis by affecting the Wnt/beta-catenin/TCF signalling pathway.