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。     

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。     

冬凌草活性部位逆转SGC7901/ADR细胞多药耐药性的体外研究北大核心CSCD

以SGC7901和SGC7901/ADR为细胞模型,检测了冬凌草活性部位与化疗药物联用后,对SGC7901/ADR耐药性的逆转效应;冬凌草活性部位处理细胞后,检测耐药细胞内阿霉素的蓄积变化、耐药细胞P-糖蛋白(P-gp)的表达水平以及mdr1基因的表达变化。结果显示,冬凌草氯仿部位和乙酸乙酯部位可以有效提高化疗药物阿霉素在SGC7901/ADR细胞内的蓄积,降低P-gp的表达,降低mdr1基因的转录。冬凌草逆转胃癌耐药细胞SGC7901/ADR多药耐药性的活性部位是冬凌草氯仿部位和乙酸乙酯部位,其逆转作用与抑制P-gp的表达相关。     

冬凌草活性部位逆转SGC7901/ADR细胞多药耐药性的体外研究

以SGC7901和SGC7901/ADR为细胞模型,检测了冬凌草活性部位与化疗药物联用后,对SGC7901/ADR耐药性的逆转效应;冬凌草活性部位处理细胞后,检测耐药细胞内阿霉素的蓄积变化、耐药细胞P-糖蛋白(P-gp)的表达水平以及mdr1基因的表达变化。结果显示,冬凌草氯仿部位和乙酸乙酯部位可以有效提高化疗药物阿霉素在SGC7901/ADR细胞内的蓄积,降低P-gp的表达,降低mdr1基因的转录。冬凌草逆转胃癌耐药细胞SGC7901/ADR多药耐药性的活性部位是冬凌草氯仿部位和乙酸乙酯部位,其逆转作用与抑制P-gp的表达相关。     

高温逆转人胃癌细胞SGC7901多药耐药性的初步研究

目的:观察高温对人胃癌耐药细胞多药耐药性的逆转作用.方法:对人胃癌耐药细胞株SGC7901/ADM予高温43℃处理,用MTT试验检测高温对ADM、5-FU、DDP、TAX作用下细胞生存率和IC50,并以人胃癌敏感细胞株SG-C7901为对照.结果:实验发现人胃癌耐药株sGc7901/ADM细胞除了对诱导耐药的ADM耐受,对CDDP、5-FU、TAX也有交叉耐药.加温至43℃,耐药株SGC7901/ADM细胞的耐药指数明显下降,而且对ADM组、CDDP组、TAX组人胃癌耐药株SGC7901/ADM细胞耐药逆转倍数分别为3.77、2.24、6.25,但对5-FU组SGC7901/ADM细胞的耐药逆转指数较低,为1.11.结论:高温可以增加耐药株SGC7901/ADM细胞对化疗药物ADM、DDP、TAX的敏感性,一定程度逆转细胞的多药耐药性.     

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.     

胃癌多药耐药相关microRNA的筛选和鉴定

目的:研究胃癌多药耐药相关microRNA并对其进行鉴定、靶基因预测和预测靶基因的生物信息学分析。方法:运用microRNA芯片对胃癌多药耐药细胞SGC7901/ADR和其亲本细胞SGC7901进行microRNA表达谱分析;采用实时定量PCR的方法对差异表达的miRNA进行验证;再运用生物信息学方法对差异表达的miRNA进行靶基因预测;再对预测的靶基因进行GO和KEGG通路分析。结果:与SGC7901相比SGC7901/ADR表达上调超过2倍的miRNA有6个,表达下调超过2倍的有11个。实时定量PCR对共同差异表达的microRNA进行验证显示与芯片结果的一致性。对这17个差异表达的miRNA进行靶基因预测,再对预测得到的靶基因进行GO和KEGG通路分析显示预测的靶基因参与了肿瘤相关通路、MAPK通路、Focal Adhesion通路等。结论:我们初步筛选得到了胃癌多药耐药相关miRNA并对其进行了生物信息学分析,为进一步地探索miRNA在胃癌多药耐药中的作用及其分子机制奠定了基础。     

幽门螺杆菌毒素蛋白CagA诱导的蛋白的差异表达分析及其基因在胃癌组织中的表达

为了研究胃癌细胞中幽门螺杆菌(Hp)毒素蛋白CagA诱导的蛋白差异表达及其基因在人胃癌组织中的表达,用Hp感染胃癌细胞系SGC 7901和AGS及用含CagA基因的表达载体稳定转染SGC 7901细胞, 构建3组实验模型.提取各组细胞的总蛋白进行双向凝胶电泳,筛选3组重叠的差异表达蛋白质斑点进行质谱鉴定.共获得135个差异表达的蛋白质,其中上调蛋白质73个,下调蛋白质62个. 鉴定出10个差异表达蛋白质, 其中有6个差异表达蛋白是首次发现,它们主要参与细胞的能量代谢和信号转导等.最后定量检测了这10个差异表达蛋白基因在人胃癌组织中的表达, 发现有4个基因高表达和1个基因低表达. 本结果将为研究幽门螺杆菌感染引起胃癌的分子机制提供新的线索.     

至真方对人大肠癌细胞株HCT8/VCR多药耐药的逆转作用及P-gp表达的影响

目的:研究至真方对大肠癌耐药细胞株HCT-8/VCR的逆转作用及P糖蛋白(P-gp)表达的影响.方法iMTT法测定至真方含药血清对HCT-8/VCR细胞株的逆转作用,并以流式细胞术测定P-gP的表达.结果:经至真方含药血清作用72h后,HCT-8/VCR细胞株对化疗的耐药性显著下降,对长春新碱逆转指数为0.937±0.093,其逆转倍数大于13.6倍,与维拉帕米比较无统计学差异(P>0.05).HCT-8/VCR细胞的P-gp表达显著高于HCT-8(P<0.01),经至真方含药血清作用后p-gP表达显著降低(P<0.01).结论:至真方可以逆转人大肠癌耐药细胞株HCT-8/VCR的多药耐药性,其机制可能与其下调P-gp的表达有关.     

Cetuximab抑制胃癌SGC7901/ADR细胞的增殖并增加其化疗敏感性

目的:探讨EGFR在胃癌耐药细胞中的表达及作用.方法:采用Western blot和免疫组化检测EGFR蛋白在胃癌耐药细胞系及组织标本中的表达,WST-1检测EGFR特异性抗体Cetuximab对胃癌耐药细胞增殖及化疗敏感性的影响.结果:SGC7901/ADR胃癌耐药细胞系中EGFR的表达明显高于亲本细胞,多数化疗后进展的胃癌组织EGFR的表达高于化疗前组织,针对EGFR的单克隆抗体Cetuximab可抑制SGC7901/ADR细胞的增殖,并增加其对部分化疗药物的敏感性.结论:EGFR过表达是胃癌细胞化疗耐药的分子机制之一,Cetuximab可逆转EGFR高表达耐药细胞的耐药表型.     

Proteome analysis of multidrug resistance in vincristine-resistant human gastric cancer cell line SGC7901/VCR

In order to elucidate the mechanisms of multidrug resistance (MDR) of vincristine-resistant human gastric carcinoma cell line SGC7901/VCR, 2-DE was used to separate the total proteins of SGC7901/VCR and its parental cell line SGC7901. PDQuest software was applied to analyze 2-DE images, and the differential protein spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Then the differential expressional levels of partially identified proteins were determined by Western blot analysis and real-time RT-PCR. Furthermore, the association of heat shock protein (HSP27), one of the highly expressed proteins in sgc7901/vcr, with MDR was analyzed using antisense inhibition of HSP27. In this study, the well-resolved, reproducible 2-DE patterns of SGC7901/VCR and SGC7901 were established, and yielded about 1100 protein-spots each. All the 24 differential proteins between the two cell lines were identified, and the differential expression levels of the partial proteins were confirmed. The suppression of HSP27 expression by HSP27 antisense oligonucleotides could enhance vincristine chemosensitivity in sgc7901/vcr and induce the cells to exhibit apoptotic morphological features after vincristine treatment. The differentially expressed proteins could be divided into six groups based on their functions: calcium-binding proteins, chaperones, proteins involved in drug detoxification or repair of DNA damage, metabolic enzymes, proteins related to cellular structure, and proteins relative to signal transduction, some of which may contribute to MDR of human gastric carcinoma cell line SGC7901/VCR. These data will be valuable for further study of the mechanisms of MDR in human gastric cancer.     

Identification of proteins responsible for the development of adriamycin resistance in human gastric cancer cells using comparative proteomics analysis

Resistance to anticancer drugs is a major obstacle in the effective treatment of tumors. To understand the mechanisms responsible for multidrug resistance (MDR), a proteomic approach was used to identify proteins that were expressed in different levels by the adriamycinresistant human gastric cancer cell line, SGC7901/ADR, and its parental cell line, SGC7901. Two-dimensional gel electrophoresis (2-DE) and image analysis was used to determine which protein spots were expressed in different levels by the two cell lines. These spots were then partially identified using ESI-Q-TOF mass spectrometry, and the differential expressional levels of the partially identified proteins were then determined by western blot analysis and real-time RT-PCR. Additionally, the association of Nucleophosmin (NPM1), a protein that was highly expressed by SGC7901/ADR, with MDR was analyzed using siRNA. As a result of this study, well-resolved, reproducible 2-DE patterns of SGC7901/ADR and SGC7901 were established, and 16 proteins that may play a role in the development of thermoresistance were identified. Additionally, suppression of NPM1 expression was found to enhance adriamycin chemosensitivity in SGC7901/ADR. These results provide a fundamental basis for the elucidation of the molecular mechanism of MDR, which may assist in the treatment of gastric cancer.     

Ribosomal proteins S13 and L23 promote multidrug resistance in gastric cancer cells by suppressing drug-induced apoptosis

Ribosomal proteins (RP) S13 and RPL23 were previously identified as two upregulated genes in a multidrug-resistant gastric cancer cell line SGC7901/VCR compared to its parental cell SGC7901 by differential display PCR. The aim of this study was to explore the roles of RPS13 and RPL23 in multidrug resistance (MDR) in gastric cancer cells. RPS13 and RPL23 were genetically overexpressed in SGC7901 cells, respectively. Either RPS13 or RPL23 enhanced resistance of SGC7901 cells to vincristine, adriamycin, and 5-fludrouracil. RPL23 also enhanced resistance of SGC7901 cells to cisplatin. Overexpression of either RPS13 or RPL23 did not alter the population doubling time, [³H]leucine incorporation, and intracellular adriamycin accumulation of SGC7901 cells. However, either RPS13 or RPL23 could protect SGC7901 cells from undergoing vincristine-induced apoptosis. Western blot analysis revealed that both RPS13 and RPL23 significantly increased the expression level of Bcl-2 and Bcl-2/Bax ratio in SGC7901 cells. In addition, overexpression of RPL23 enhanced glutathione S-transferase (GST) activity and intracellular glutathione content in SGC7901 cells. Together, this work demonstrates that either RPS13 or RPL23 can promote MDR in gastric cancer cells by suppressing drug-induced apoptosis, and that RPL23 may also promote MDR in gastric cancer cells through regulation of glutathione S-transferase-mediated drug-detoxifying system.     

Reversal of multidrug resistance of vincristine-resistant gastric adenocarcinoma cells through up-regulation of DARPP-32

Here we investigated the roles of DARPP-32 in multidrug resistance (MDR) of gastric cancer cells and the possible underlying mechanisms. We constructed the eukaryotic expression vector of DARPP-32 and transfected it into human vincristine-resistant gastric adenocarcinoma cell line SGC7901/VCR. Up-regulation of DARPP-32 could significantly enhance the sensitivity of SGC7901/VCR cells towards vincristine, adriamycin, 5-fluorouracil and cisplatin, and could decrease the capacity of cells to efflux adriamycin. What's more, the results of subrenal capsule assay confirmed that DARPP-32 might play a certain role in MDR of gastric cancer. DARPP-32 could significantly down-regulate the expression of P-gp and zinc ribbon domain-containing 1 (ZNRD1), but not alter the expression of multidrug resistance-associated protein (MRP) or the glutathione S-transferase (GST). DARPP-32 could also significantly decrease the anti-apoptotic activity of SGC7901/VCR cells. Further study of the biological functions of DARPP-32 might be helpful for understanding the mechanisms of MDR in gastric cancer.     

Sunitinib Reverse Multidrug Resistance in Gastric Cancer Cells by Modulating Stat3 and Inhibiting P-gp Function

Sunitinib, a small-molecule multi-targeted tyrosine kinase inhibitor, has been applied in phase II clinical trial as second-line treatment for advanced gastric cancer. In this study, we determined the effect of Sunitinib on the multidrug resistance in gastric cancer cells selected by vincristine. Our results showed that Sunitinib significantly enhanced the cytotoxicity of adriamycin, vincristine, etoposide, 5-Fluorouracil, and cisplatin in multidrug-resistant gastric cancer cells (SGC7901/VCR). Sunitinib significantly increased the intracellular accumulation and retention of rhodamine 123 in the SGC7901/VCR cells. However, Sunitinib, at a concentration that reverses MDR, had no significant effect on P-gp protein or mRNA expression levels. In addition, the present study revealed that Sunitinib inhibited Stat3 and down-regulated Bcl-2 in SGC7901/VCR cells, which might also contribute to the reversal of MDR. In conclusion, Sunitinib reverses multidrug resistance in gastric cancer cells by inhibiting P-gp transporter function and modulating Stat3 and Bcl-2. Further study with Sunitinib may be helpful for developing combination therapeutic strategy or circumventing gastric cancer MDR to other conventional anti-cancer drugs.     

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.     

内源性活性氧水平及锰型超氧化物歧化酶的表达与胃癌细胞分化、增殖相关

检测了不同分化的胃癌细胞株内MnSOD基因的表达及胞内活性氧限（ROS)的水平。同时通过基因转染观察上调或下调MnSOD基因表达对SGC790l胃癌细胞胞内ROS水平及增殖能力的影响。用电穿孔法将人反义和正义MnSOD cDNA真核表达载体pHβA—SOD(-)／pHβA—SOD（ )转入790l细胞,用含G418的RPMIl640培养基筛选稳定表达克隆。然后用RT-PCR鉴定MnDSOD基因的表达。同时用RT-PCR方法检测正常胃粘膜组织及MKN-28、SGC790l、BGC823、HGC-27四株高、中、低、未分化胃癌细胞株内的MnSoD的mRNA表达。利用DCFH-DA荧光染色方法检测不同分化胃癌细胞株及790l转染细胞株内的ROS水平。四唑蓝比色法(MTT)绘制MKN-28、SGC790l、BGC823、HGC-27四株不同分化胃癌细胞及正义、反义、空载MnSOD转染790l细胞的生长曲线。发现不同分化胃癌细胞内的MnSOD普遍呈低表达且与分化程度平行,不同分化胃癌细胞株胞内ROS水平随着MnSOD表达的下调逐步上升,细胞增殖加快。较之MnSOD空载790l,正义、反义MnSOD转染的790l细胞中该基因的表达出现明显上调及下调,胞内ROS水平较对照细胞也相应有显著降低和升高。正义株增殖受抑,反义株增殖加快。表明胃癌细胞内MnSOD的表达与肿瘤的分化程度呈负相关。可通过改变胞内RoS水平改变MnSOD基因的表达,从而调节胃癌细胞的生长。