锌带基因ZNRD1在胃癌耐药细胞中的表达和功能

目的 探讨锌带基因ZNRD1在多药耐药胃癌细胞中的表达和作用。方法 应用Northern blot和半定量RT—PCR,观察锌带基因ZNRD1在胃癌细胞SGC7901及其长春新碱(VCR)诱导的耐药细胞SGC7901／VCR中的表达;将反义核酸转染SGC7901／VCR细胞,通过免疫组化检测转导细胞与非转导细胞ZNRD1蛋白的表达;以流式细胞仪检测细胞周期的变化,以MTT实验检测细胞生长曲线和对VCR、阿霉素(ADM)的药敏性。结果 胃癌耐药细胞SGC7901／VCR与非耐药细胞相比,ZNBDl基因的表达明显增高。转导株antiZNRDl—SGC7901／VCR细胞的ZNRD1蛋白水平明显低于非转导株,C1期细胞比例增加而S期比例减少,生长受到抑制,且对VCR、ADM的敏感性明显增高。结论 胃癌耐药细胞与非耐药细胞相比,ZNRDl基因处于高表达状态。反义核酸转染可有效阻断ZNRDl蛋白的表达,在一定程度上逆转人胃癌耐药细胞SGC7901／VCR的多药耐药性。     

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。     

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。     

Ro60在耐药胃癌细胞系SGC7901/ADR中的克隆及表达

目的:在耐药胃癌细胞系SGC7901/ADR中对Ro60进行克隆,对Ro60在耐药胃癌细胞系SGC7901/ADR和药敏胃癌细胞系SGC7901中的表达情况进行比较分析。方法:应用RT-PCR法克隆Ro60的编码基因,通过DNA序列测定对所克隆的基因进行验证。应用半定量RT-PCR法检测Ro60在耐药胃癌细胞系SGC7901/ADR和药敏胃癌细胞系SGC7901中的表达情况。结果:在耐药胃癌细胞系SGC7901/ADR中,成功克隆了Ro60的编码基因,其DNA序列与Ro60的cDNA序列完全一致。半定量RT-PCR结果显示,Ro60在耐药胃癌细胞系SGC7901/ADR中的表达强度显著高于药敏胃癌细胞系SGC7901细胞中的表达强度,P<0.01。结论:Ro60在耐药胃癌细胞系中高表达,该分子可能是一种胃癌多药耐药相关分子。     

shRNA沉默多药耐药胃癌细胞7901/VCR MDR1基因表达的实验研究

目的利用RNAi技术干扰胃癌多药耐药细胞（7901/VCR）多药耐药基因（MDR1）的表达,为临床肿瘤的基因治疗奠定基础。方法根据MDR1的碱基序列设计并合成两对短发夹RNA,构建重组载体,用阳离子脂质体法体外转染7901/VCR;采用MTT法检测转染胃癌细胞的生长抑制率,流式细胞术检测细胞周期变化,RT-PCR和Western blot检测转染前后MDR1 mRNA和蛋白表达的变化。结果成功构建RNAi质粒载体,转染后细胞IC50明显降低（P〈0.05）;G1期细胞增加,S期细胞减少（P〈0.05）;MDR1 mRNA转录水平下降（P〈0.05）,P-糖蛋白（P-gp）的表达水平降低（P〈0.05）。结论RNAi能明显抑制胃癌细胞7901/VCR MDR1mRNA和P-gp蛋白的表达,进而对肿瘤细胞多药耐药性有明显的逆转作用,为基因治疗提供了一种新的手段。     

靶向人胃癌SGC7901/VCR细胞多药耐药基因1小分子干扰RNA的有效序列筛选

目的筛选靶向人胃癌SGC7901/VCR细胞多药耐药基因1(MDR1)小分子干扰RNA(siRNA)的有效序列。方法设计并体外转录靶向MDR1的4条siRNA(MDR1si326、MDR1si1513、MDR1si2631和MDR1si3071),转染SGC7901/VCR细胞。用RT PCR检测MDR1mRNA表达,免疫印迹检测P糖蛋白(P gp)表达,流式细胞仪检测细胞内阿霉素(ADR)蓄积,四甲基偶氮唑蓝(MTT)法检测细胞对ADR的敏感性。结果4条siRNA均能不同程度逆转SGC7901/VCR细胞MDR1介导的多药耐药。转染后48h,MDR1si326组和MDR1si2631组的mRNA表达水平分别为0.42±0.07和0.49±0.02,较MDR1si1513或MDR1si3071组明显下降(P<0.05);MDR1si326组细胞内ADR蓄积最显著,中位数为30.03,MDR1si2631组次之,MDR1si3071组较少,MDR1si1513组最少(P<0.05);MDR1si2631组对ADR耐药的相对逆转率最高,为(98.12±0.26)%,MDR1si326组次之(P<0.05),MDR1si1513组和MDR1si3071组的差别不大(P>0.05)。转染后72h,MDR1si326组蛋白质表达水平下降最明显。结论MDR1si326对人胃癌SGC7901/VCR细胞MDR1介导的耐药逆转效果最好,MDR1si2631次之,MDR1si3071较差,MDR1si1513最差。     

P-糖蛋白、谷胱甘肽-s转移酶的表达与胃癌细胞耐药的关系研究

目的初步探索胃癌多药耐药细胞系SGC7901／VCR的耐药机制。方法间歇诱导法,胃癌细胞系SGC7901经长春新碱（VCR）短时间诱导后,对长春新碱产生耐药。MTT法检测对VCR、5-氟尿嘧啶、表阿霉索的耐药性。Western blot检测P-糖蛋白（P—glucoprotein,P—gp）、谷胱甘肽-s转移酶（ghtathione—s transferring enzyme,GST—s）的表达。结果耐药细胞SGC7901／VCR对长春新碱耐药提高16．56倍,此耐药株同时对5-氟尿嘧啶、表阿霉素呈交叉耐药,耐药指数分别达6．9、13．05。SGC7901／VCR的P—gP、GST—s出现表达增强。结论长春新碱短时间诱导后,SGC7901产生多药耐药性（multi—drug resistance,MDR）,且P—gp、GST—s表达增强。反之,抑制P—gP、GsT—s蛋白的表达,则有可能降低细胞耐药从而逆转胃癌MDR。     

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。     

KLF8在缺氧诱导胃癌细胞多药耐药中的作用

目的:探讨KLF8（Kruppel-like factor 8）在缺氧诱导胃癌细胞多药耐药表型中的作用。方法:利用Western blot和实时定量PCR的方法检测三株胃癌细胞系MKN28、MKN45、SGC7901在常氧及缺氧条件下KLF8的表达水平。Western blot和实时定量PCR检测胃癌耐药细胞系SGC7901/VCR、SGC7901/ADR和亲本细胞系SGC7901中KLF8的表达水平。构建KLF8正义表达载体及KLF8小干扰RNA的慢病毒载体,将其转染入实验细胞中,用Western blot和实时定量PCR的方法检测KLF8表达量的变化。MTT、Annexin V/PI染色法及阿霉素蓄积量与潴留实验检测KLF8在缺氧诱导胃癌细胞多药耐药表型中的作用。结果:缺氧可诱导胃癌细胞系MKN28、MKN45、SGC7901中KLF8的表达升高。与胃癌亲本细胞系相比,KLF8在胃癌耐药细胞系中的表达升高,并且在SGC7901/VCR细胞中表达升高的更为明显。外源性转染KLF8后可显著增加胃癌细胞中KLF8的表达量,KLF8小干扰RNA可有效降低常氧及缺氧条件下胃癌细胞中KLF8的表达。常氧条件下,外源性转染KLF8的正义表达载体可增加胃癌细胞对不同化疗药物的耐受性,降低化疗药物诱导的胃癌细胞凋亡指数,同时可增加细胞内阿霉素的泵出率。缺氧条件下,KLF8小干扰RNA能够逆转胃癌细胞中上述现象的发生。结论:初步实验结果发现了一个新的缺氧反应分子-KLF8。表型试验证实该分子在缺氧诱导胃癌多药耐药中发挥作用。     

Overexpression of sorcin results in multidrug resistance in gastric cancer cells with up-regulation of P-gp

Sorcin, a calcium-binding protein was found up-regulated in the vincristine-induced multi-drug resistance (MDR) gastric cancer cell line SGC7901/VCR, over its parental SGC7901 cells in our previous proteomic studies. The present study explored the role and mechanism of sorcin in the development of MDR in gastric cancer. We constructed the recombinant plasmids FLAG-sorsin-pcDNA3.1 containing the full open reading frame of sorcin and a FLAG affinity tag. Overexpression of sorcin by gene transfection was able to confer drug resistance to vincristine, adriamycin, taxol and 5-fluorouracil in SGC7901 cells. Down-regulation of sorcin expression by sorcin antisense oligonucleutides, (ASO) increased sensitivity to vincristine. The intracellular concentration of vincristine in SGC7901 cells decreased in sorcin transfected cells and increased in sorcin ASO-transfected cells, indicating that sorcin had a direct or indirect function on pumping the drug out of cells. Overexpression of sorcin up-regulated the expression of P-gp and P-gp inhibitor verapamil partially reversed the sorcin-mediated MDR in SGC7901 cell, suggesting that regulation of P-gp might be one of the mechanisms of sorcin-mediated MDR. The further study of the interaction protein of sorcin may be helpful for understanding the mechanisms of MDR in gastric cancer and developing possible strategies to treat gastric cancer.     

环氧合酶-2选择性抑制剂塞莱昔布调节胃癌细胞多药耐药性的实验研究

 目的 观测环氧合酶-2（COX-2）抑制剂塞莱昔布（Celecoxib）对多药耐药细胞株SGC7901／VCR多药耐药（MDR）的逆转及P-糖蛋白（P-gp）的调变作用，探讨COX-2对胃癌细胞MDR调节作用。方法 以长春新碱（VCR）诱导的人胃癌多药耐药细胞SGC7901／VCR为研究对象，应用流式细胞术、MTT法及免疫细胞化学方法研究COX-2抑制剂塞莱昔布对耐药性的逆转作用及对P-gp的调变作用。结果 MTT显示塞莱昔布明显抑制SGC7901、SGC7901／VCR细胞的生长，并呈时间、剂量依赖性。经非细胞毒剂量（2.5 μmol／L）的塞莱昔布作用后，SGC7901／VCR细胞的多药耐药性得到部分逆转，表现为靶细胞对多种化疗药物的敏感性显著增加，逆转倍数为1.09 ~ 6.28；流式细胞仪分析发现，塞莱昔布介导的SGC7901／VCR细胞耐药性的逆转伴有胞内多柔比星浓度的升高；免疫细胞化学研究显示，经塞莱昔布作用后耐药株SGC7901／VCR表达P-gp强度下降。结论 塞莱昔布能有效地抑制肿瘤细胞的生长，且部分逆转SGC7901／VCR细胞的多药耐药性，其可能主要通过影响P-gp的药物泵功能实现。     

Reversal of multidrug resistance of gastric cancer cells by downregulation of TSG101 with TSG101siRNA

We have previously identified tumor susceptibility gene101 (TSG101), overexpressed in vincristine-resistant human gastric adenocarcinoma cell line SGC7901/VCR using a modified subtractive hybridization method and Western blot. In the present study, we constructed two siRNA eukaryotic expression vectors of TSG101 and transfected them into SGC7901/VCR cells to examine whether the down-regulation of TSG101 increased cell sensitivity towards chemotherapeutic drugs. After transfection, the expression of TSG101 was dramatically decreased in TSG101 siRNA transfectants compared with that in parental cells and empty vector control cells. The down-regulation of TSG101 could significantly enhance the sensitivity of SGC7901/VCR cells to vincristine and adriamycin. The capacity of cells to efflux adriamycin markedly decreased in TSG101 siRNA transfectants. These observations suggested that the siRNA constructs of TSG101 we made could effectively down-regulate the expression of TSG101 and reverse the resistant phenotype of gastric cancer cells. The preliminary study suggested that TSG101 might play an important role in multidrug resistance of gastric carcinoma.     

MGr1-Ag is associated with multidrug-resistant phenotype of gastric cancer cells

BACKGROUND: MGr1-antigen (Ag) was previously reported as an upregulated protein in multidrug-resistant (MDR) gastric cancer cells. The aim of this study was to characterize the role of MGr1-Ag in the multidrug resistance of gastric cancer cells. METHODS: Laser scanning confocal microscopy (LSCM), two-dimensional electrophoresis, and Western blot were used to detect MGr1-Ag in gastric cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine the sensitivity of the MDR gastric cancer cells, SGC7901/VCR, to chemotherapeutic drugs. Adriamycin accumulation and retention in SGC7901/VCR cells were analyzed using flow cytometry. RESULTS: LSCM showed that MGr1-Ag localized mainly on the membrane and partly in the cytoplasm of SGC7901/VCR cells. Western blot showed that the expression level of MGr1-Ag in SGC7901/VCR cells was higher than that in its parental cells, SGC7901, and that the apparent molecular weight and isoelectric point of MGr1-Ag were 42 kDa and pH 4.8, respectively. After incubation with MGr1 antibody, SGC7901/VCR cells showed significantly decreased IC(50) values for adriamycin (from 0.887 +/- 0.081 mg/l to 0.607 +/- 0.084 mg/l; P, 0.05), vincristine (from 0.707 +/- 0.055 mg/l to 0.557 +/- 0.042 mg/l; P, 0.05), and 5-fluorouracil (from 4.367 +/- 0.407 mg/l to 2.630 +/- 0.644 mg/l; P, 0.05), as well as slightly increased IC(50) values for mitomycin (from 0.183 +/- 0.045 mg/l to 0.198 +/- 0.048 mg/l; P. 0.05). In addition, incubation with MGr1 significantly enhanced adriamycin accumulation and retention in SGC7901/VCR cells. CONCLUSION: Overexpression of MGr1-Ag is associated with the MDR phenotype of gastric cancer cells.