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。     

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。     

二藤散结方对SGC7901/VCR的多药耐药影响及与P-gp表达的关系

目的 观察二藤散结方对人胃癌多药耐药细胞SGC7901/VCR的影响及与P-糖蛋白(P-gp)表达的关系.方法 体外培养SGC7901/VCR,采用MTT法、流式细胞仪、免疫组化法检测二藤散结方对SGC7901/VCR的影响及与细胞膜P-gp表达的关系.结果 二藤散结方能逆转SGC7901/VCR的多药耐药性,增加SGC7901/VCR细胞内ADM浓度,下调SGC7901/VCR细胞膜P-gp的表达.结论 二藤散结方能逆转SGC7901/VCR的多药耐药性,逆转机制与细胞膜P-gp表达减弱有关.     

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。     

罗格列酮逆转丝裂霉素对人胃癌SGC7901/VCR细胞株耐药的作用

目的探讨过氧化物酶体增殖子活化受体γ(PPARγ)配体罗格列酮(ROS),对耐药胃癌SGC7901/VCR细胞药物敏感性的影响。方法以长春新碱(VCR)诱导的人胃癌多药耐药细胞SGC7901/VCR及其亲代SGC7901为研究对象,应用MTT比色法及集落形成实验,研究罗格列酮对丝裂霉素(MMC)抑制人胃癌SGC7901细胞及其耐药亚系SGC7901/VCR细胞生长及增殖的影响及罗格列酮逆转SGC7901/VCR细胞对MMC的耐药作用。结果 ROS对SGC7901/VCR及SGC7901细胞生长具有明显抑制作用,且其作用与ROS浓度呈时间-剂量依赖关系;40μmmol/L、80μmmol/L ROS逆转SGC7901/VCR细胞对MMC的耐药倍数(RI)分别为9.6、10.5,与增敏对照组作用相当(P>0.05);集落形成实验结果显示,ROS与MMC联用降低SGC7901/VCR细胞的集落形成率。结论罗格列酮能部分逆转多药耐药胃癌细胞株SGC7901/VCR对MMC的耐药。     

甲基莲心碱对耐长春新碱人胃癌细胞多药耐药性逆转机制的初步研究

目的探讨新的钙阻断剂甲基莲心碱(Nef)对耐长春新碱人胃癌细胞多药耐药性(MDR)的逆转作用及其机制。方法采用MTT比色法,检测药物的细胞毒作用;应用免疫细胞化学SP法及流式细胞术,检测Nef对人胃癌细胞Bcl-2蛋白表达的影响。结果 10μmol/L Nef对SGC7901及SGC7901/VCR无显著细胞毒作用;2.5、5、10μmol/L Nef能使VCR对SGC7901/VCR细胞的IC50从2.32μg/ml依次下降至0.340、0.128、0.053μg/ml,逆转倍数分别为6.8、18.1、43.8。当Nef浓度为10μmol/L时,逆转SGC7901/VCR多药耐药活性较VRP高(P<0.01);SGC7901/VCR细胞中Bcl-2蛋白表达水平较SGC7901细胞高,经Nef处理后,SGC7901/VCR细胞中Bcl-2蛋白表达水平明显下降,表明Nef能下调SGC7901/VCR细胞中Bcl-2蛋白表达水平。结论甲基莲心碱在体外能逆转耐长春新碱人胃癌细胞(SGC7901/VCR)的多药耐药性,其机制可能与下调Bcl-2蛋白表达水平有关。     

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。     

甲基莲心碱逆转人胃癌细胞株多药耐药性

目的:研究新的钙阻断剂甲基莲心碱(Nef)对耐长春新碱(VCR)人胃癌细胞多药耐药性(MDR)的逆转作用及其机制。方法:采用MTT比色法测定药物的细胞毒作用,SP法免疫细胞化学及流式细胞术检测Nef对人胃癌细胞多药耐药性相关蛋白(MRP)表达的影响。结果:①在10μmol/L的浓度下,Nef对SGC7901及SGC7901/VCR无显著细胞毒作用;②2.5、5、10μmol/LNef能使VCR对SGC7901/VCR细胞的IC50从2.32μg/ml依次下降至0.340、0.128、0.053μg/ml,逆转倍数分别为6.8、18.1、43.8。在浓度为10μmol/L时,逆转活性较VRP为高(P<0.01);③SGC7901/VCR细胞较SGC7901细胞高表达MRP,经Nef处理后,SGC7901/VCR细胞MRP表达明显下降。表明Nef能下调SGC7901/VCR细胞MRP的表达。结论:甲基莲心碱在体外能逆转耐长春新碱人胃癌细胞(SGC7901/VCR)的多药耐药性。其机制可能与下调MRP的表达有关。     

胃癌多药耐药逆转短肽GMBP42的活性鉴定

目的:鉴定短肽GMBP42与胃癌多药耐药细胞(SGC7901/VCR和SGC7901/ADR)的结合特异性及其多药耐药逆转活性。方法:体外培养胃癌亲本细胞SGC7901及多药耐药细胞(SGC7901/VCR和SGC7901/ADR)。免疫细胞化学染色技术鉴定短肽GMBP42与多药耐药细胞的结合特异性。体外药物敏感实验测定短肽GMBP42对多药耐药细胞化疗药物敏感性的影响。结果:免疫细胞化学染色结果显示:短肽GMBP42能够与胃癌多药耐药细胞(SGC7901/VCR和SGC7901/ADR)结合,亚细胞定位于核周胞浆及胞膜,而与亲本细胞无明显结合。体外药物敏感试验结果显示:与对照组相比,短肽GMBP42处理组胃癌多药耐药细胞(SGC7901/VCR和SGC7901/ADR)对化疗药物的IC50值及细胞存活率均降低(P<0.05)。结论:短肽GMBP42能特异结合于多种胃癌多药耐药细胞,短肽GMBP42可提高胃癌多药耐药细胞对阿霉素的敏感性,部分逆转多药耐药细胞对阿霉素的耐药表型。     

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。     

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.     

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。     

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。     

Overexpression of ZNRD1 promotes multidrug-resistant phenotype of gastric cancer cells through upregulation of P-glycoprotein

ZNRD1, a new zinc ribbon gene, has been previously identified as an upregulated gene in a multidrug-resistant gastric cancer cell line SGC7901/VCR comparing to its parental cell SGC7901 by subtractive hybridization and RT-PCR. The antisense nucleic acid for ZNRD1 could enhance adriamycin accumulation in SGC7901/VCR cells and sensitize SGC7901/VCR cells to vincristine. The present study aims to explore the role of ZNRD1 in multidrug resistance in gastric cancer cells. Upregulation of ZNRD1 protein in SGC7901/VCR cells was confirmed by Western blot and immunocytochmical staining. ZNRD1 was genetically overexpressed in SGC7901 cells by gene transfection. It was found that overexpression of ZNRD1 could sensitize SGC7901 cells to P-glycoprotein (P-gp)-related anticancer drugs (vincristine, adriamycin, etoposide) but not to P-gp-nonrelated drugs (5-fluorouracil and cisplatin), which was accompanied with significantly decreased adriamycin accumulation and retention and increased adriamycin releasing in SGC7901 cells. Verapamil, an inhibitor for P-gp, could reverse the effects of ZNRD1 on drug sensitivity and drug accumulation in SGC7901 cells to a great extent. Western blot and Northern blot revealed that overexpression of ZNRD1 could upregulate P-gp at both protein and mRNA levels. Together, these results suggest that overexpression of ZNRD1 could promote multidrug-resistant phenotype of gastric cancer cells through upregulation of P-gp.     

CIAPIN1 confers multidrug resistance by upregulating the expression of MDR-1 and MRP-1 in gastric cancer cells

In a previous study, we observed that cytokine-induced apoptosis inhibitor 1 (CIAPIN1), a newly identified apoptosis inhibitor, was upregulated at the mRNA level in a multidrug-resistant gastric cancer cell line SGC7901/VCR. The aim of this study was to explore the role of CIAPIN1 in the development of multidrug resistance (MDR) in gastric cancer cells. Upregulation of CIAPIN1 in MDR gastric cancer cells was confirmed by semiquantitative RT-PCR and Western blotting. Using cDNA transfection and RNA interference, we successfully established stable transfectants with upregulation (i.e., SGC7901-pCIAPIN1) or downregulation (i.e., SGC7901-pSiCIAPIN1 and SGC7901/ADR-pSiCIAPIN1) of CIAPIN1 expression, respectively. In vitro drug sensitivity assay demonstrated that overexpression of CIAPIN1 conferred MDR in SGC7901 cells whereas downregulation of CIAPIN1 sensitized SGC7901 and SGC7901/ADR cells to anticancer drugs. CIAPIN1 protected both SGC7901 and SGC7901/ADR cells from ADR-induced apoptosis and reduced intracellular accumulation and retention of adriamycin. Moreover, expression of P-glycoprotein (P-gp or MDR-1, a product of MDR-1 gene) and MDR-related protein-1 (MRP-1) was upregulated by CIAPIN1. In addition, Western blotting revealed that CIAPIN1 decreased the expression of Bcl-2, Bax and p53. Therefore, it is concluded that CIAPIN1 confers MDR in gastric cancer cells, likely by upregulating MDR-1 and MRP-1.     

Clinical significance and correlation of miR-200c and P-gp expression in gastric cancer and the effects on multidrug resistance

BackgroundPoor prognosis is common in gastric cancer patients due to multidrug resistance (MDR)-induced recurrence and metastasis. In the present study, we investigated the expression of microRNA (miR)-200c in gastric cancer tissues and cell lines and its relationship with the expression of the drug resistant gene ABCB1, which encodes P-glycoprotein (P-gp).MethodsThe basic characteristics of 102 patients with gastric cancer were reviewed. Real time-polymerase chain reaction (PCR), immunohistochemistry, and Western blot were employed to detect the expression levels of miR-200c and P-gp in gastric carcinoma tissues and cell lines. The correlation of miR-200c messenger RNA (mRNA) level with clinicopathological characteristics and P-gp protein expression were analyzed. SGC7901/vincristine (VCR) cells were transfected with miR-200c mimics or a specific small interfering RNA (siRNA) targeting the ABCB1 gene. The methyl thiazolyl tetrazolium (MTT) assay and flow cytometry were used to determine the role of miR-200c and ABCB1 on the viability and apoptosis of gastric carcinoma cell lines.ResultsThe level of miR-200c in carcinoma tissues was significantly lower than that in adjacent tissues, and the expression level of P-gp in carcinoma tissues was obviously higher than that in adjacent tissues (P<0.01, P=0.029). The expression levels of miR-200c and P-gp were associated with the malignant characteristics of gastric cancer, and patients with high expression of miR-200c or negative expression of P-gp had a better prognosis (P=0.006, P=0.022). MiR-200c negatively regulated the ABCB1 gene in gastric cancer cell lines. MiR-200c overexpression and ABCB1 down-regulation increased the sensitivity of SGC7901/VCR cells to VCR and reversed MDR by promoting cell apoptosis.ConclusionsThe expression level of miR-200c decreases in gastric carcinoma tissues and drug-resistant gastric cancer SGC7901/VCR cells. Overexpression of miR-200c may enhance the sensitivity of SGC7901/VCR cells to VCR by regulating the expression of P-gp.     

胃癌耐药细胞特异性结合小肽对多药耐药的影响

目的 研究环九肽（SY1）与胃癌耐药细胞（SGC7901／VCR）的结合特性及其对胃癌耐药性的逆转作用。方法 将细胞分为SGC7901和SGC7901／VCR2组培养。以无关噬菌体、阴性噬菌体为对照组,用免疫荧光技术检测含有SY1的阳性噬菌体与SGC7901／VCR的结合特性;通过体外药敏试验（MTT）分析含有SY1的阳性噬菌体和化学合成的SY1对SGC7901／VCR耐药性的改变;应用流式细胞仪检测在SY1作用下SGC7901／VCR细胞内阿霉素（ADM）的蓄积和储留。结果 （1）免疫荧光分析的结果显示,含有SY1的阳性噬菌体能够结合于SGC7901／VCR的膜表面,而不与亲本细胞SGC7901结合,无关噬菌体和阴性噬菌体均不与SGC7901／VCR结合,表明SY1可与SGC7901／VCR特异性结合。（2）MTT试验结果显示,在含有SY1的阳性噬菌体及化学合成的SY1的作用下,SGC7901／VCR的存活率显著降低（P〈0．05）,其对长春新碱的耐药性降低。（3）在化学合成的SY1作用下,SGC7901／VCR细胞内ADM的储留蓄积高于对照组（P〈0．05）。结论 利用环七肽库差减筛选得到的环九肽SY1不仅能与SGC7901／VCR特异性结合,而且可部分逆转其对长春新碱的耐药性。这可能为胃癌多药耐药的逆转提供新思路。