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。     

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.     

龙葵碱通过抑制Med19表达增加多柔比星耐药乳腺癌细胞对多柔比星的敏感性

目的 探讨龙葵碱（solanine）对乳腺癌细胞多柔比星耐药性的影响及其机制。方法 选用乳腺癌细胞MCF-7细胞和对阿霉素（adriamycin,ADM）或多柔比星耐药的乳腺癌细胞MCF-7/ADR细胞,转染pcDAN-Med19过表达中介体19(mediator 19,Med19),应用CCK-8法检测细胞增殖水平,qRT-PCR和Western blot检测细胞中Med19和凋亡相关Bcl-2、Bax及Cleaved-Caspase-3表达水平,TUNEL染色和流式细胞术分析细胞凋亡水平。结果 龙葵碱显著降低MCF-7/ADM细胞活力。Med19高表达于MCF-7/ADM细胞,龙葵碱显著抑制MCF-7/ADM细胞中Med19表达,龙葵碱对多柔比星处理的MCF-7/ADM细胞活力的降低、凋亡的促进、凋亡相关蛋白Bax和Cleaved-Caspase-3水平的上调及Bcl-2水平的下调可被过表达Med19明显抑制。结论 龙葵碱通过抑制Med19表达而降低乳腺癌细胞MCF-7/ADM对多柔比星的耐药性。     

白术抑制胃癌细胞SGC-7901增殖并促进其凋亡

目的观察白术对胃癌SGC-7901侧群细胞及非侧群细胞增殖及凋亡的影响。方法流式细胞仪分选SGC-7901中的侧群细胞;制备含白术药物血清;CCK-8法检测白术药物血清对细胞增殖能力的影响;流式细胞仪检测含白术药物血清对细胞凋亡的影响;Western blot检测药物血清对细胞中Bax及Bcl-2蛋白表达的影响;异种移植BALB/C小鼠成瘤实验观察白术对细胞体内成瘤能力影响;免疫组织化学检测白术对小鼠肿瘤组织中Bax及Bcl-2蛋白表达的影响。结果离体实验表明,含白术药物血清培养可明显降低SGC-7901侧群细胞的增殖率,同时可诱导其凋亡、分别上调和下调细胞中Bax和Bcl-2水平;在体实验显示,白术汤剂灌胃可显著抑制SGC-7901侧群细胞小鼠成瘤能力,分别上调和下调肿瘤组织中Bax和Bcl-2水平。非侧群细胞也有上述表现。结论白术可通过上调促凋亡蛋白Bax及下调抑凋亡蛋白Bcl-2从而促进胃癌SGC-7901侧群细胞及非侧群细胞凋亡,并抑制其增殖及体内成瘤能力。     

白血病耐药细胞系U937/ADR的建立及其生物学性状

目的：建立白血病耐药细胞系U937/ADR模型,并检测其多药耐药相关基因及其生物学性状的改变。方法：以大剂量阿霉素(IC50浓度),短时间(2h)暴露法诱导人白血病细胞系U937细胞的阿霉素耐药性。检测细胞的生长曲线,计算阿霉素耐药倍数,流式细胞术分析细胞周期分布;罗丹明123检测药物外排功能;荧光定量PCR（FQ-PCR）检测MDR1、MRP1、NF-Κb、Bcl-2、Bax mRNA水平变化;Western blot 检测Akt、p-Akt、P65、P-gp、MRP1和Bcl-2蛋白水平变化。结果：成功构建耐阿霉素U937/ADR细胞系,对阿霉素耐药指数为亲代U937细胞的11倍,U937/ADR群体倍增时间为43.6h,高于亲代细胞8.9h;流式细胞分析显示与U937细胞相比,U937/ADR的G0/G1期细胞增多,而G2/M期细胞减少。并对多种化疗药物产生交叉耐药性。罗丹明123外排试验显示,U937/ADR细胞外排明显增加。U937/ADR细胞MDR1、NF-Κb、Bcl-2 mRNA表达水平明显增加,P-gp及p-Akt、P65表达水平增加。结论：成功构建的U937/ADR细胞系其生物学特性明显不同与亲代U937细胞,对多种化疗药物产生多药耐药,高表达多药耐药蛋白P-gp,同时激活p-Akt及NF-Kb。     

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。     

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。     

华蟾素诱导人胃癌SGC-7901细胞凋亡

本实验旨在探讨华蟾素诱导人胃癌SGC-7901细胞凋亡的作用及其作用机制。采用不同浓度的华蟾素作用胃癌SGC-7901细胞48 h后,MTT法检测细胞活性;光学、荧光显微镜、流式细胞术检测细胞凋亡情况。Real Time RT-PCR和Western Blot分别检测Bax、Bcl-2基因m RNA和蛋白表达水平。结果显示,华蟾素对胃癌SGC-7901细胞增殖具有抑制作用且呈剂量依赖性关系,华蟾素处理7901细胞48 h的IC50值为35.67μg/m L,凋亡率为(5.01±1.69)%。显微镜下观察细胞呈明显凋亡现象,线粒体膜电位(ΔΨm)显著下降(p0.05),细胞阻滞于G1期;Bcl-2的表达下调,Bax的表达明显增加(p0.05,p0.01)。提示华蟾素可能通过上调Bax基因,下调Bcl-2基因诱导人胃癌SGC-7901细胞凋亡。     

选择性环氧化酶-2抑制剂对乳腺癌细胞的化疗增敏作用

目的:观察选择性环氧化酶-2(COX-2)的抑制剂塞来昔布对表柔比星抗乳腺癌MCF-7细胞增殖和诱导凋亡作用以及探讨其机制.方法:应用四甲基偶氮唑蓝(MTT)比色法分析塞来昔布联用表柔比星对MCF-7细胞的生长抑制作用,流式细胞术检测细胞的凋亡,western blotting检测凋亡相关蛋白Bcl-2、Bax、caspase-3的表达.结果:10μmol/l的cxlecoxib和10μg/l表柔比星联用细胞抑制率和早期凋亡率均显著增高,并引起caspase-3上调及裂解激活,bcl-2下调,bax则变化不大.结论:塞来昔布对表柔比星抗乳腺癌MCF-7细胞有协同作用,其诱导凋亡与caspase-3激活和Bcl-2表达下调有关.     

应用蛋白质组学技术筛选胃癌耐药相关蛋白质

胃癌多药耐药性是临床胃癌化疗失败最主要的原因之一,但其分子机制仍然不太清楚.为了寻找新的胃癌耐药相关的蛋白质,揭示胃癌多药耐药的分子机制,以胃癌细胞SGC7901和长春新碱诱导的耐药胃癌细胞SGC7901/VCR为研究对象,应用二维凝胶电泳(two-dimensionalelectrophoresis,2-DE)技术分离两种细胞的总蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)及电喷雾电离串联质谱(electrosprayionizationtandemmassspectrometry,ESI-Q-TOF)对差异表达的蛋白质点进行鉴定,蛋白质印迹和实时RT-PCR验证部分差异蛋白质在两株细胞中的表达水平,反义核酸转染技术分析HSP27(heatshockprotein27,HSP27)高表达与SGC7901/VCR耐药的相关性.得到了分辨率较高、重复性较好的两株细胞系的二维凝胶电泳图谱,质谱分析共鉴定了24个差异蛋白质点,蛋白质印迹和实时RT-PCR验证了部分差异蛋白的表达水平,反义寡核苷酸抑制HSP27表达能增加SGC7901/VCR对长春新碱的敏感性.研究结果不仅提示这些差异蛋白质如HSP27,Sorcin等可能与胃癌的多药耐药相关,而且为揭示胃癌细胞的多药耐药性产生机制提供了线索.     

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.     

Depression of MAD2 inhibits apoptosis of gastric cancer cells by upregulating Bcl-2 and interfering mitochondrion pathway

Mitotic arrest deficient 2 (MAD2) is an essential component of the mitotic spindle checkpoint pathway. It was previously shown to be associated with drug resistance of tumor cells. To further explore the roles of MAD2 in responses of gastric cancer cells to chemotherapy drugs, we constructed the siRNA vectors of MAD2 and transfected them into gastric cancer SGC7901 cells to inhibit expression of MAD2. MTT assay showed that the downregulation of MAD2 increased the resistance of SGC7901 cells to spindle inhibitors and DNA damaging agents. The apoptosis rates of gastric cancer cells transfected with MAD2-siRNA were 10.7% and 10%, respectively, after treated by 1.0microg/ml VCR and cisplatin. In contrast, the apoptosis rates of SGC7901 and SGC7901/psilencer3.1 induced by VCR were 43.2%, 38.7%; and that induced by cispaltin were 34.1%, 31.4%. The ratio of Bcl-2 to Bax was much higher in the MAD2-siRNA transfectants compared with the SGC7901/psilencer. In SGC7901/psilencer, cytochrome c and cleaved caspase 3 protein levels increased along with the exposure time increased. However, these protein levels of SGC7901/MAD2-siRNA had no changes during the drug treatment. These results indicate that down regulation of MAD2 could promote the drug resistance of gastric cancer cells and inhibit anticancer drugs induced-apoptosis by upregulating Bcl-2 and interfering the mitochondrion apoptosis pathway.     

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.     

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。