siRNA对SGC7901/VCR细胞mdr1基因沉默效果的影响因素分析

目的分析siRNA沉默人胃癌SGC7901/VCR细胞mdr1基因效果的相关因素。方法设计并体外转录合成4条靶向mdr1的siRNA(mdr1si326、mdr1si1513、mdr1si2631和mdr1si3071),转染SGC7901/VCR细胞,用RTPCR和免疫印迹检测mdr1mRNA和Pgp的表达、流式细胞仪检测细胞内阿霉素的蓄积和MTT法检测细胞对阿霉素的敏感性,综合这4方面结果评价4条siRNA的沉默效果情况;用分子生物学软分析siRNA沉默效果的影响因素。结果沉默效果最好的mdr1si326和较好的mdr1si2631靶序列编码Pgp的跨膜区而且自身无茎和袢;沉默效果较差的mdr1si3071和最差的mdr1si1513靶序列编码Pgp的胞内区,前者自身成茎和成袢。沉默效果最好的mdr1si326和较好的mdr1si2631靶序列在靶位点和靶位点外间有较少的碱基配对和氢键。siRNA的沉默效果与siRNA3’5’端3个碱基中的A/U数量、N1和N19、GC含量间无规律可循。结论siRNA沉默SGC7901/VCR细胞mdr1的效果与靶序列的结构关系密切。     

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。     

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。     

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。     

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。     

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。     

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。     

反义survivin核酸增强胃癌细胞对多西他赛敏感性及其逆转耐药机制的探讨

目的探讨反义survivin核酸(anti-pcDNA3-svv)对胃癌细胞系SGC7901的凋亡诱导和化疗增敏作用,及其与多药耐药基因(mdr-1)的相关性。方法采用电转染法转染胃癌细胞系SGC7901,并筛选阳性克隆。以Western blot法检测survivin蛋白的表达,用透射电镜观察转染后对SGC7901的凋亡诱导作用,RT-PCR检测mdr-1 mRNA的表达,MTT法检测转染细胞对多西他赛的敏感性。诱导SGC7901细胞耐药,检测耐药mdr-1和survivin的表达。结果转染anti-pcDNA3-svv的SGC7901细胞中,survivin蛋白表达较未转染组明显降低;透射电镜下,转染组细胞呈凋亡晚期变化;转染组的mdr-1 mRNA较未转染组明显降低,转染组与未转染组的MDR指数分别为0.196±0.013和3.126±O.019;转染组多西他赛的IC50值为(16.7±1.98)μg/L,而未转染组为(55.7±1.89)μg/L,差异有统计学意义。耐药SGC7901细胞的survivin mRNA表达随着mdr-1 mRNA表达增加而明显增强。结论反义survivin核酸可诱导SGC7901细胞凋亡,增强多西他赛化疗敏感性,其逆转耐药的机制与mdr-1低表达有关。     

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。     

Reversing Multidrug-Resistant by RNA Interference Through Silencing MDR1 Gene in Human Hepatocellular Carcinoma Cells Subline Bel-7402/ADM

Multidrug resistance (MDR) in hepatocellular carcinoma (HC) significantly impedes the effect of chemotherapy and is considered as a primary reason leading to its recurrences and metastasis. The aim of present study was to explore new molecular targets for the reversal of MDR in HC by screening the adriamycin (ADM)-induced, human MDR-resistant HC cell subline Bel-7402/ADM. Small interfering RNAs (siRNAs) of four (MDR1si326, MDR1si1513, MDR1si2631 and MDR1si3071) targeting MDR1 were designed and transfected into Bel-7402/ADM cell strains. The experiments involved the following: mRNA expression of MDR1 gene by RT-PCR, P-glycoprotein (P-gp) expression by Western blot, intracellular ADM accumulation flow cytometry, and IC₅₀ of ADM by a cytotoxic MTT assay. Four siRNAs reversed MDR in HC mediated by MDR1 to varying degrees. The expression level of MDR1 mRNA in cells of MDR1si326 or MDR1si2631 group (0.190?±?0.038 or 0.171?±?0.011) was more decreased. The expression level of P-gp in cells of MDR1si326 group was the lowest. The accumulation of ADM in cells of MDR1si326 or MDR1si2631 group (77.0?±?3.5 or 75.4?±?2.9) was more increased. The IC₅₀ of cells to ADM was lowest in MDR1si326 group (11.32?±?0.69 mg/L). Compared with other three siRNAs, MDR1si326 performed the optimal reversal effect of drug resistance in human HC Bel-7402/ADM.     

P13-K抑制剂LY294002逆转P-gP介导的白血病和胃癌细胞耐药

背景与目的：磷脂酰肌醇3-激酶／蛋白激酶B[phosphatidylinositol-3-kinase（P13-K）／proteinkinaseB（Akt）,P13-K／Aktl通路是调控细胞生存的重要信号转导通路之一。本研究旨在探讨P13-K抑制剂LY294002对P-gP过表达的人类白血病K562／DNR和胃癌SGC7901／ADR细胞多药耐药性的逆转作用。方法：将细胞分为单纯药物组和LY294002预处理组,单纯药物组分别以柔红霉素（daunorubicin,DNR）、阿霉素（adriamycin,ADR）、长春新碱（vincristine,VCR）和依托泊甙（etoposide,VP-16）处理,LY294002预处理组在加药前以LY294002进行预处理。用台盼蓝拒染法及MTT法检测药物敏感性及LY294002对细胞耐药性的影响。Westernblot检测K562／DNR和SGC7901／ADR细胞中P．gP及p-Akt的表达。流式细胞术检测细胞内药物浓度。结果：2．5μmol／LLY294002预处理显著降低DNR、ADR、VCR和VP-16对K562／DNR细胞的IC50,相对逆转效率分别为72．4％、64．9％、60．4％和52．8％。此外,LY294002部分逆转SGC7901／ADR对ADR的耐药性,相对逆转效率为31．0％。LY294002预处理可部分抑制p-Akt和P-gP的表达。随着处理时间的延长．K562／DNR、SGC7901／ADR细胞内DNR、ADR的蓄积效应有增强的趋势。结论：LY294002通过抑制P13-K／Akt信号转导通路,部分逆转P-gp介导的白血病和胃癌细胞的多药耐药。     

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。     

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.     

Shugoshin1 enhances multidrug resistance of gastric cancer cells by regulating MRP1, Bcl-2, and Bax genes

Multidrug resistance (MDR) is a major clinical obstacle in treatment of gastric cancer (GC) and it accounts for the majority of cancer-related mortalities. Shugoshin1 (SGO1) is an important player in appropriate chromosome segregation and is involved in tumorigenesis. In this study, we found endogenous SGO1 overexpression in the multidrug-resistant GC cell lines SGC7901/VCR and SGC7901/ADR compared with their parental cell line SGC7901. By enhancing expression of SGO1, sensitivity of SGC7901 cells to vincristine (VCR), adriamycin, 5-fluorouracil (5-FU), and cisplatin (CDDP) was significantly diminished. Silencing its expression resulted in enhanced sensitivity of SGC7901/VCR and SGC7901/ADR cells to these antitumor drugs. Additionally, we confirmed that SGO1 increased capacity of cells to enable adriamycin (ADR) efflux and inhibit drug-induced apoptosis by regulating MRP 1, Bcl-2, and Bax genes so as to confer a MDR phenotype to GC cells. In brief, these findings suggest that SGO1 promotes MDR of GC cells and may be useful as a novel therapeutic target for preventing or reversing MDR.     

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.     

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。