CacyBP编码基因对胃癌细胞多药耐药性形成的影响

目的：探讨CacyBP在胃癌多药耐药机制中的作用。方法;构建CacyBP正义核酸真核表达载体pcDNA3.1/hCacyBP ,以脂质体将其导入胃癌药敏细胞SGC7901,以RT－PCR检测hGacyBP mRNA水平的变化。以MTT检测SGC7901及转染细胞对ADR的药物敏感性;以流式细胞仪（FCM）检测SGC7901及转染细胞内ADR的蓄积浓度及细胞周期。结果：转染pcDNA3.1CacyBP 的SGC7901,其hCacyBP mRNA表达水平显著高于空载体转染及未转染之SGC7901,且细胞生存率亦有所增高,未转染之SGC7901及分别转染空载体或pcDNA3.1/hCacyBP 之SGC7901细胞,平均ADR蓄积浓度分别为5．64,5．49和5．17。与未转染之SGC7901相比,转染pcDNA3.1/hCacyBP 和空载体的SGC7901细胞G1期减少G2期及S期增高,结论：CacyBP在胃癌MDR机制中可能有一定作用。     

RPL6/Taxreb107在胃癌耐药细胞系SGC7901/ADR中的差异表达及其与MDR相关性的初步研究

目的 探讨BPL6／Taxreb107在胃癌耐药细胞系SGC7901／ADR与胃癌细胞系SGC7901的差异表达,以及与胃癌多药耐药(MDR)的相关性。方法 提取SGC7901和SGC7901/ADR细胞系总RNA;采用内对照RT-PCR检测RPL6基因和Northem杂交、基因克隆与表达、真核表达载体的构建;以电穿孔法基因转染;以流式细胞仪检测瞬时转染细胞的阿霉素蓄积和潴留。结果 内对照RT-PCR和Northern杂交证实RPL6/Taxreb 107在SGC7901/ADR中高表达。将PCR产物克隆人pUCm-T载体并经测序证实。构建正义和反义真核表达载体(pcDNA3．1)并经酶切鉴定证实后,以电穿孔法将正义真核表达载体转入SGC7901,反义真核表达载体转入SGC7901／ADR。转染48h后检测转染细胞的阿霉素蓄积和潴留,结果提示RPI6／Taxreb107转入细胞后对细胞的耐药性有影响。结论 BPI6／Taxreb107在耐药胃癌细胞中高表达,对胃癌的MDR有影响。     

蛇毒cystatin基因转染抗人胃腺癌细胞体外侵袭作用的研究

目的:探讨蛇毒半胱氨酸蛋白酶抑制剂(sv-cystatin)对人胃腺癌细胞SGC7901侵袭转移的抑制作用.方法:采用人工拼接方法合成蛇毒cystatin cDNA,构建pcDNA3.1/sv-cystatin真核表达质粒,经脂质体转染将pcDNA3.1/sv-cystatin质粒和pcDNA3.1质粒分别导入胃腺癌细胞系SGC7901:利用RT-PCR和Western blot检测SGC7901细胞中sv-cystatin基因的表达;应用细胞-基质粘附实验、细胞运动实验及重建基底膜侵袭实验分析svcystatin表达对SGC7901细胞粘附、运动和侵袭能力的影响.结果:转染sv-cystatin基因后,SGC/sv-cystatin细胞克隆中可检测到sv-cystatin的明显表达,SGC/sv-cystatin细胞的运动能力和体外穿越重建基底膜的能力明显低于转染空载体细胞和未转染的SGC7901细胞,但其体外粘附能力未见明显变化.结论:sv-cystatin基因的表达可使胃癌SGC7901细胞体外运动能力及侵袭能力明显减弱,提示sv-cystatin具有抑制胃癌细胞侵袭转移的作用.     

人核糖体蛋白S13(RPS13)编码基因的克隆及其正反义核酸转染胃癌细胞的初步研究

目的：扩增人核糖体蛋白S13（RPS13）编码基因序列，构建其正，反义真核表达载体，分别转染胃癌细胞SGC7901及胃癌耐药细胞SGC7901/VCR，以进一步研究RPS13基因与胃癌多药耐药的关系。方法：采用RT-PCR法扩增RPS13cDNA片段编码区全长序列，利用DNA重组技术的建正，反义真核表达载体，经脂质体介导转染胃癌细胞SGC7901及胃癌耐药细胞SGC7901/VCR，筛选正，反义基因稳定转染细胞，RNA斑点杂交法检测正，反义稳定转染细胞mRNA水平的变化。结果：从高表达RPS13的SGC7901/VCR耐药细胞中提取细胞总RNA，RT-PCR法成功扩增了RPS13cDNA片段编码区全长序列，并经测序证实；目的基因因按正，反两个方向亚克隆至真核表达载体pcDNA3.1( ),并经酶切鉴定证实；经脂质体介导将正义真核表达载体转染SGC7901细胞，反义真核表达载体转染SGC7901/VCR细胞，G418筛选获得稳定转染细胞；RNA斑点杂交试验证实；正义稳定转染细胞RPS13mRNA水平上调，反义稳定转染细胞RPS13mRNA水平下调。结论：成功克隆了人RPS13编码基因序列，并构建了其正，反义真核表达载体，在胃癌细胞系SGC7901及长春新碱耐药细胞SGC7901/VCR中得到稳定转染细胞，正义转染细胞中RPS13mRNA表达明显增加，反义转染细胞中表达明显受抑。     

转染Bcl-2基因的膀胱癌细胞多药耐药性研究

目的:探讨转染Bcl-2基因后膀胱癌细胞的多药耐药变化。方法:采用基因重组技术构建Bcl-2基因的真核表达载体pcD-NA3.1( )/Bcl-2,经酶切和基因测序分析对构建结果进行鉴定;通过脂质体将含Bcl-2基因的表达载体转染膀胱癌细胞BIU-87,RT-PCR检测基因转录水平;应用四甲基偶氮唑蓝(MTT)比色法检测细胞在不同化疗药物作用下的细胞耐药性。结果:经酶切鉴定和基因测序分析证明真核表达载体pcDNA3·1( )/Bcl-2构建成功。将该表达载体转染BIU-87细胞后,Bcl-2基因的表达水平与野生型BIU-87细胞和转染空载体的BIU-87/neo细胞相比显著提高(P<0·01)。与BIU-87细胞和BIU-87/neo细胞相比,BIU-87/Bcl-2细胞对MMC、PHA和DDP等化疗药物的耐药性也明显增强(P<0·01)。结论:Bcl-2基因高表达是膀胱癌多药耐药的原因之一。     

HSP90β在细胞中的表达水平与化疗敏感性的相关研究

目的:了解胃癌细胞SGC7901及其耐药细胞SGC7901/VCR中HSP90β蛋白的表达,与化疗药物敏感性的关系及降低细胞HSP90β蛋白的表达是否影响化疗药物的敏感性.方法:用免疫组化法检测胃癌细胞SGC7901及其耐药细胞SGC7901/VCR中HSP90β蛋白的表达.MTT法测定SGC7901、SGC7901/VCR细胞及其相应的HSP90β蛋白低表达细胞AH-SGC7901和AH-SGC7901/VCR对化疗药物的敏感性,流式细胞仪测定细胞内药物含量.结果:SGC7901/VCR细胞HSP90β蛋白的表达高于SGC7901细胞.SGC7901/VCR细胞对ADM、VCR、MMC和CTX的IC50分别为0.398、1.25、0.199和3 981.07μg/ml.SGC7901对ADM、VCR、MMC及CTX的IC50分别为1.58×10-6、1.99×10-3、3.1×10-2及630μg/ml.SGC7901/VCR相对于SGC7901细胞对ADM、VCR、MMC和CTX的耐药指数分别为2.52×105、6.28×102、6.42和6.32 AH-SGC7901和AH-SGC7901/VCR对化疗药物的敏感性显著提高.与相应的亲本细胞相比,AH-SGC7901对ADM、VCR和MMC的敏感性分别提高了10、10 000和24.8倍,对CTX无显著变化AH-SGC7901/VCR对ADM、VCR、MMC和CTX的敏感性分别提高了3.98、6.28、3.15和5.01倍.AH-SGC7901和AH-SGC7901/VCR的阿霉素荧光强度较其相应的亲本细胞显著增强(P＜0.05).结论:降低胃癌细胞HSP90β蛋白的表达可提高它们对化疗药物的敏感性.     

As_2O_3对胃癌细胞SGC7901/ADR阿霉素耐药性逆转作用

目的探讨三氧化二砷(As2O3)对胃癌细胞SGC7901/ADR阿霉素(ADM)耐药性的逆转作用和对Pgp、GSTπ表达的影响。方法以胃癌多药耐药细胞株SGC7901/ADR为靶细胞,用MTT法检测SGC7901/ADR细胞对ADM的敏感性,用流式细胞仪检测细胞内药物浓度及免疫组织化学法检测细胞Pgp、GSTπ表达。结果0.4～0.8μmol/LAs2O3对耐药细胞SGC7901/ADR无明显毒性(P>0.05)。As2O3可下调Pgp、GSTπ表达,提高SGC7901/ADR细胞内ADM浓度,增加SGC7901/ADR细胞对ADM的敏感性。结论As2O3能够抑制SGC7901/ADR细胞Pgp、GSTπ表达,增加细胞内ADM药物浓度而部分逆转其对ADM的耐药性。     

15-羟基前列腺素脱氢酶基因转染对人胃癌细胞生长和迁移的影响

目的:探讨含有NAD+依赖性l5-羟基前列腺素脱氢酶(NAD+-dependent 15-hydroxyprostaglandin dehydrogenase, 15-PGDH)的真核表达质粒(pcDNA3/15-PGDH)转染对人胃癌细胞生长和迁移的影响. 方法:Real time PCR检测多种胃癌细胞株中15-PGDH的表达情况.双酶切方法鉴定15-PGDH真核表达质粒pcDNA3/15-PGDH后,应用Lipofectamine 2000将其转染入胃癌细胞SGC7901中.MTT法检测质粒转染对胃癌细胞增殖的影响,划痕实验观察质粒转染对胃癌细胞迁移的影响,软琼脂克隆形成实验观察质粒转染对胃癌细胞克隆形成的影响.结果:SGC7901细胞中15-PGDH表达显著偏低.pcDNA3/15-PGDH质粒转染入SGC7901细胞的效率较高,转染后24 h及48 h时pcDNA3/15-PGDH组的15-PGDH mRNA表达量分别是pcDNA3空质粒组的188倍和271倍.pcDNA3/15-PGDH质粒转染后SGC7901细胞增殖、迁移和克隆形成能力均显著低于空质粒组和未转染组(P<0.05). 结论:15-PGDH基因转染可显著抑制人胃癌SGC7901细胞的增殖、迁移和克隆形成能力,推测15-PGDH可能是抑制胃癌发生、发展的重要因素之一.     

Af116609基因对胃癌细胞系SGC7901/VCR耐药性的影响

目的 研究Af116609基因转染胃癌耐药细胞系SGC7901／VCR后,对胃癌细胞耐药性的影响。方法 采用RT-PCR法克隆Af116609基因,用Northern blot检测其差异表达,以基因转染技术调节其表达,通过体外药敏实验观察其对胃癌耐药表型的影响。结果 从胃癌耐药细胞SGC7901／VCR中克隆到Af116609基因的CDS序列327bp,该序列与GenBank登录的一致。Northern blot结果显示,该基因在耐药细胞高表达。体外药敏实验发现,转染正义真核表达载体的药敏细胞中该基因上调,对长春新碱、5-氟脲嘧啶和阿糖胞苷的耐药性增加,而对阿霉素的抗性有损害,对氨甲喋呤的抗性无明显影响;转染反义真核表达载体的耐药细胞中该基因下调,对上述5种药物的抗性均减弱。结论 Af116609基因与胃癌MDR表型相关,可作为逆转胃癌MDR的候选靶分子。     

下调CACNA2D1对胃癌耐药细胞系SGC7901/ADR药物敏感性的影响

目的:探讨下调CACNA2D1对胃癌耐药性细胞系SGC7901/ADR耐药的影响。方法:采用实时荧光定量聚合酶链反应(qRT-PCR)的方法检测16对胃癌及癌旁组织以及胃癌细胞系SGC7901和SGC7901/ADR中的CACNA2D1 的mRNA表达水平;蛋白免疫印迹技术（Western blot）检测CACNA2D1的蛋白表达情况;MTT法检测SGC7901、SGC7901/ADR及转染CACNA2D1 siRNA后SGC7901/ADR的IC50值;流式细胞仪检测细胞凋亡。结果:qRT-PCR结果显示:CACNA2D1在胃癌组织中的表达明显高于癌旁组织(P＜0.01);CACNA2D1在耐药细胞系SGC7901/ADR中的表达明显高于其亲本细胞系SGC7901(P＜0.01)。蛋白免疫印迹技术结果显示相对于转染CACNA2D1 negative control（NC）组,转染CACNA2D1 siRNA组的胃癌细胞CACNA2D1表达降低;MTT结果显示细胞系SGC7901与SGC7901/ADR的阿霉素半数抑制浓度（IC50）分别为（1.3±0.6）μg/ml与（5.5±0.45）μg/ml;SGC7901/ADR转染CACNA2D1 siRNA后对阿霉素的IC50明显下降。流式细胞术检测凋亡结果显示,CACNA2D1的表达下调后,SGC7901/ADR细胞的凋亡比明显增加。 结论:CACNA2D1能够促进胃癌SGC7901/ADR细胞系产生多药耐药。     

Depression of MAD2 inhibits apoptosis and increases proliferation and multidrug resistance in gastric cancer cells by regulating the activation of phosphorylated survivin

Mitotic arrest-deficient 2 (MAD2) is one of the essential mitotic spindle checkpoint regulators, and it can protect cells from aberrant chromosome segregation. The Mad2 gene is very rarely mutated in many kinds of human cancer, but aberrantly reduced expression of MAD2 has been correlated with defective mitotic checkpoints in several human cancers. We have previously found that the MAD2 expression level is also shown to be associated with the multidrug resistance of tumour cells. In this study, we constructed a small interfering RNA (siRNA) eukaryotic expression vector of MAD2 and downregulated MAD2 expression in the gastric cancer cell line SGC7901 by transfection of MAD2-siRNA. SGC7901 cells stably transfected with the MAD2-siRNA exhibited significantly increased expression of phosphorylated survivin protein and enhanced drug resistance. Furthermore, MAD2-siRNA suppressed the proliferation of SGC7901 cells and inhibited tumour formation in athymic nude mice. This study clearly reveals that downregulation of MAD2 could regulate the cell cycle, increase proliferation, and improve the drug resistance of gastric cancer cells by regulating the activation of phosphorylated survivin. It also suggests both that MAD2 might play an important role in the development of human gastric cancer and that silencing the MAD2 gene may help to deal with the multidrug resistance of gastric cancer cells.     

下调PHLDB3对胃癌耐药细胞系SGC7901/ADR药物敏感性的影响

目的:探讨下调PHLDB3对胃癌耐药细胞系 SGC7901/ADR 药物敏感性的影响。方法:采用实时定量聚合酶链反应（qRT-PCR）和蛋白免疫印迹技术（Western blot）的方法检测胃癌及癌旁正常组织中PHLDB3的表达（分别是20对和10对）;qRT-PCR和Western blot检测PHLDB3在胃癌细胞系 SGC7901以及胃癌耐药细胞系SGC7901/ADR和SGC7901/VCR中的表达;MTT法检测SGC7901、SGC7901/ADR及转染PHLDB3 siRNA后 SGC7901/ADR的半数抑制浓度（IC50）值;流式细胞仪检测细胞凋亡。结果:qRT-PCR和Western blot结果显示:PHLDB3在胃癌组织中的表达明显高于癌旁正常组织（P<0.000 1）;PHLDB3在耐药细胞系SGC7901/ADR和SGC7901/VCR中的表达明显高于其亲本细胞系SGC7901（P<0.000 1和P<0.05）。Western blot结果显示:相对于转染 PHLDB3 negative control（NC）组,转染 PHLDB3 siRNA组的SGC7901/ADR细胞中PHLDB3的表达降低（P<0.005）。MTT结果显示:SGC7901与 SGC7901/ADR的IC50值分别为（1.5±0.1） μg/ml与（5.5±0.2） μg/ml（P<0.000 1）;SGC7901/ADR 转染 PHLDB3 siRNA 后对顺铂的IC50值明显下降（P<0.05）。流式细胞术（flow cytometry,FCM）检测凋亡,结果显示:下调PHLDB3的表达后,SGC7901/ADR细胞的凋亡率明显增加（P<0.05）。结论:PHLDB3能够促进胃癌细胞系 SGC7901/ADR产生多药耐药。     

下调RPL23对胃癌耐药细胞SGC7901/ADR药物敏感性的影响

目的:检测人胃癌细胞系SGC7901及其阿霉素（ADR）耐药细胞系SGC7901/ADR中RPL23的表达差异,探讨胃癌细胞中RPL23表达对ADR耐药的影响。方法:qPCR检测RPL23在SGC7901/ADR与SGC7901两种细胞系中的表达差异;MTT法检测下调RPL23后SGC7901/ADR细胞对ADR的敏感性;流式细胞仪检测下调RPL23后对细胞凋亡的影响;Western Blot检测凋亡相关分子Bcl-2和Caspase-3蛋白的表达。结果:qPCR结果显示,RPL23在 SGC7901/ADR细胞系中的表达高于其在SGC7901细胞系中的表达(P＜0.001)。MTT法结果显示,下调RPL23后,在SGC7901/ADR细胞系中ADR的IC50明显升高(P＜0.001)。凋亡检测结果显示,下调RPL23后,SGC7901/ADR细胞凋亡率明显增加(P＜0.001)。Western Blot结果显示,下调RPL23后,细胞中Caspase-3表达明显升高,Bcl-2表达明显降低。结论:RPL23在SGC7901/ADR中的表达高于SGC7901细胞,下调RPL23表达可提高其对ADR的敏感性并促进细胞凋亡,提示RPL23可能通过作用于Bcl-2调节细胞凋亡。     

Midkine confers Adriamycin resistance in human gastric cancer cells

Midkine (MDK) is a heparin-binding molecule involved in the regulation of growth and differentiation during embryogenesis, which is overexpressed in most of human malignant tumors and may act as an oncoprotein. The aim of the current study was to investigate the mechanism of MDK involved in the Adriamycin (ADR) resistance in human gastric cancer cells in vitro. We found that Adriamycin-resistant SGC7901 (SGC7901/ADR) exhibited 58.6-fold greater resistance to ADR compared with Adriamycin-sensitive SGC7901 cell line. MDK mRNA and protein expression levels were significantly higher in SGC7901/ADR than in SGC7901. To gain a deeper insight into the role of MDK in SGC7901/ADR, we stably transfected Adriamycin-sensitive SGC7901 with viral vector expressing MDK. Our result showed that multidrug resistance type I (MDR1) was found in SGC7901/ADR, not in SGC7901 by RT-PCR regardless of MDK transfection. P-Glycoprotein, which is the MDR1-coded protein, was found in SGC7901/ADR, not in SGC7901 by Western blot regardless of MDK transfection. We investigated whether an activation of the tyrosine kinase pathway would change the drug resistance phenotype with MDK transfection. Western blot results showed the upregulation of phosphorylated protein kinase B (AKT) and phosphorylated extracellular signal-regulated protein kinase (ERK) in Adriamycin-sensitive SGC7901 cell by MDK transfection accompanied with drug resistance to ADR, although the level of AKT and ERK protein expression did not change, so our results suggested that MDK, which can activate AKT and ERK by phosphorylation, induced the Adriamycin resistance in gastric cancer cells. Understanding the molecular mechanisms, driving MDK-induced ADR resistance, will provide benefits in developing new therapies for gastric cancer.     

miR-187通过抑制胃癌细胞凋亡影响胃癌多药耐药

目的:探讨miR-187对胃癌多药耐药的影响。方法:应用qPCR检测miR-187在胃癌SGC7901细胞和SGC7901ADR细胞中的表达。miR-187 mimics转染上述细胞,MTT检测转染后细胞对多种化疗药物的敏感性,流式细胞仪检测细胞凋亡。结果:SGC7901ADR细胞中miR-187的表达明显高于SGC7901细胞。MTT实验显示转染miR-187 mimics后胃癌细胞对化疗药物的敏感性均明显降低(P＜0.05)。流式细胞检测表明miR-187 mimics转染可以减少SGC7901细胞的凋亡(P＜0.05)。结论:miR-187通过抑制胃癌细胞凋亡参与胃癌多药耐药,可能成为逆转胃癌多药耐药的新靶点。     

Regulation of multidrug resistance by ribosomal protein l6 in gastric cancer cells

Ribosomal proteins (RP) L6 was previously identified as an up-regulated gene in multidrug-resistant gastric cancer cells SGC7901/ADR comparing to its parental cells SGC7901 by subtractive hybridization. The aim of this study was to explore the roles of RPL6 in multidrug resistance (MDR) in gastric cancer cells. Northern and Western blot analysis confirmed that RPL6 was overexpressed in SGC7901/ADR cells. By gene transfection, RPL6 was genetically upregulated in SGC7901 or down-regulated in SGC7901/ ADR cells. Upregulation of RPL6 was associated with enhanced resistance to multiple anticancer drugs (adriamycin, vincristine, etoposide, 5-fluorouracil and cisplatin) and to adriamycin-induced apoptosis. Downregulation of RPL6 reversed MDR and sensitized cells to adriamycin-induced apoptosis. Alteration of RPL6 showed no obvious influence on intracellular adriamycin accumulation, glutathione content and expression of glutathione S-transferase. RPL6 could upregulate Bcl-2 and downregulate Bax in cells. Together, this work demonstrates that RPL6 could regulate MDR in gastric cancer cells by suppressing drug-induced apoptosis.