Targeting of p38 Mitogen-activated Protein Kinases to Early Growth Response gene 1 （EGR-1） in the Human Paclitaxel-resistance Ovarian Carcinoma Cells

To investigate the relationship between the expression of early growth response gene 1 (EGR-1) and p38MAPK pathway in the paclitaxel resistance of ovarian carcinoma cells, the effect of p38MAPK inhibitor SB203580 on cell apoptosis was examined by using Hoechst 33258 staining. The intracellular Rh123 (Rhodamine 123) accumulation was detected by the flow cytometry (FCM). The 50% inhibition concentration (IC50) of paclitaxel for A2780/Taxol cells was determined by MTT method. Electrophoretic motility shift assay (EMSA) was employed to examine the EGR-1DNA binding activity. MDR1 and EGR-1 mRNA were assessed by RT-PCR. The expressed of p-gp, phos- phorylated p53 and p38 were detected by Western blotting. SB203580 could remarkably promote the apoptosis of A2780/Taxol cells, and the cell apoptosis was in a time-dependent manner. Cellular Rh123 accumulation was increased, and the IC50 of paclitaxel for A2780/Taxol cells was decreased significantly. A2780/Taxol cell line after SB203580 treatment was shown to have a significantly higher level of EGR-1 DNA binding activity. SB203580 down-regulated the activity of p38MAPK pathway, but up-regulated EGR-1 expression. SB203580 significantly increased the level of cellular phosphorylated p53 protein, but decreased the p-gp protein level and MDR1 mRNA level in A2780/Taxol cells. There existed a close relationship between p38MAPK pathway and the paclitaxel resistance of ovarian carcinoma cells. The expression of EGR-1 mediated by p38MAPK pathway plays a critical role in paclitaxel resistance of ovarian carcinoma cells.     

Reversal of Multi-Drug Resistance by Vector-Based-ShRNA-Mdr1 In Vitro and In Vivo

In order to investigate the effects of vector-based hairpin small interference RNA (shRNA) on the reversal of multi-drug resistance (mdr) of A2780/Taxol cells, a novel vector pEGFP-H1/mdr1 containing mdr1-shRNA targeting at position 2943-2963 of mdr1 was designed and synthesized.Subsequently, A2780/Taxol cells were transfected with pEGFP-H1/mdrl, and the expression of mdr1 mRNA and P-gp was detected by using RT-PCR and Western blot respectively. MTT was used to measure the 50% inhibition concentration (1C50) of Taxol to A2780/Taxol cells. The results showed that at the 24th and 48th h after transfection, the expression of mdr1 mRNA was decreased to (52.1±1.0)% and (0.01+1.7)%, and that of P-gp decreased to (88.3±2.1)% and 0%, respectively. At the 48th h after transfection, the relative reversal rate of A2780/Taxol cells to Taxol was 69.54%. In vivo, the nude mice xenografts were injected with pEGFP-H1/mdrl, and then administrated Taxol.The tumor volume in pEGFP-H1/mdr1-transfected group was significantly reduced as compared with that in blank control group or pEGFP-H1-transfected group (807.20±103.16 vs 1563.78±210.54 or 1480.78±241.24 mm3, both P<0.01). These results suggested that transfection of pEGFP-H1/mdr1 could efficiently down-regulate the expression of mdr1 mRNA and P-gp in A2780/Taxol cells, and effectively restore the sensitivity of A2780/Taxol cells to Taxol both in vitro and in vivo.     

稳定过表达XAF1基因对A2780卵巢癌细胞生物学功能的影响

目的 构建稳定过表达XAF1基因A2780卵巢癌细胞株,并观察XAF1基因对卵巢癌细胞增殖、凋亡、细胞周期及对紫杉醇敏感性的影响。方法 分别将质粒pcDNA3.1（+）和pcDNA3.1（+）-XAF1转染至卵巢癌细胞A2780,通过质粒抗性标记（遗传霉素G418）筛选得到阴性对照细胞株（A2780/Negative control, A2780/NC）和稳定表达XAF1的细胞株（A2780/XAF1）;通过行细胞克隆形成实验和CCK8实验检测细胞增殖及对紫杉醇的敏感性,流式细胞仪检测细胞周期及细胞凋亡。结果 成功构建了稳定表达XAF1的卵巢癌细胞A2780/XAF1,细胞形态无明显变化;与A2780/NC相比,A2780/XAF1克隆形成能力能力更低（P= 0.0016）,细胞贴壁后第1天和第3天增殖活性更低（P=0.009,0.0035）,两组细胞的细胞周期分布差异存在统计学意义（P< 0.0001）,两两比较结果显示A2780/XAF1组G2-M期细胞百分比显著增加（P<0.001）。在无凋亡刺激、无血清培养及紫杉醇诱导下,A2780/XAF1的凋亡率均比A2780/NC更高（P<0.001）;在不同紫杉醇浓度作用下,A2780/XAF1的增殖活性均显著低于A2780/NC（P<0.001）,且A2780/XAF1的紫杉醇半数抑制浓度显著低于A2780/NC。结论 成功构建稳定表达XAF1的卵巢癌细胞A2780/XAF1;XAF1调控卵巢癌细胞A2780的增殖、凋亡及细胞周期,并增加了卵巢癌对紫杉醇的敏感性。     

RNA干扰技术沉默MDR1基因逆转大肠癌耐药细胞株LoVo/5-Fu的耐药性

目的  探讨多药耐药（MDR1）基因沉默对大肠癌耐药细胞系LoVo/5-Fu耐药性的逆转作用。方法  构建靶向MDR1的短发夹RNA(EASY-shRNA-MDR1)干扰质粒转染LoVo/5 Fu,实时荧光定量PCR（Realtime-PCR）在mRNA水平检测抑制效果,噻唑蓝（MTT）法检测细胞对5-Fu的敏感性,流式细胞仪检测细胞凋亡情况。结果  与未转染组相比,EASY-shRNA-MDR1组细胞MDR1mRNA表达明显下调（P<0.05）;细胞对5-Fu的IC50及RI显著降低（P<0.05）,敏感性的相对逆转率为74.7%;凋亡率明显升高(P<0.05)。结论  EASY-shRNA-MDR1可有效抑制大肠癌耐药细胞中MDR1的表达,进而降低了大肠癌耐药株对5-Fu的耐药性。     

能量可控陡脉冲联合H-ras干扰性RNA治疗卵巢癌裸鼠皮下移植瘤的实验研究

目的　利用陡脉冲电场的可逆性电穿孔电转基因,观察陡脉冲治疗仪联合基因治疗对肿瘤生长的影响。方 法　构建抗H- ras基因表达的小发夹状RNA重组质粒载体(pshRNA H- ras);建立卵巢癌荷瘤裸鼠模型,光、电镜观察陡脉 冲对肿瘤组织的病理学改变,荧光报告质粒观察陡脉冲电场的可逆性电穿孔;实验分为4组:对照组、陡脉冲治疗组、陡脉 冲加基因单次干扰治疗组和加基因重复干扰治疗组,记录治疗后21d各荷瘤鼠肿瘤的体积和体质量。结果　陡脉冲对肿 瘤组织有明显的破坏作用,肿瘤边缘残存有活性的癌细胞;各治疗组肿瘤体积与体质量明显低于对照组,差异具有极显著 性(P<0.01),其中,陡脉冲加基因重复干扰治疗组肿瘤体积和重量显著低于仅用陡脉冲治疗组(P<0.01)。结论　陡脉 冲是一种新型有效的治癌工具,联合基因治疗能更有效地抑制肿瘤生长。     

XIAP基因与耐紫杉醇卵巢癌细胞A2780/Taxol耐药关系的研究

目的 探讨X连锁凋亡抑制蛋白（XIAP）基因与卵巢癌对紫杉醇耐药的相关性。方法 以5、10、20、40、80、160、320 ng/mL的紫杉醇处理卵巢癌紫杉醇敏感细胞A2780和耐药细胞A2780/T,噻唑蓝（MTT）比色法检测各组细胞的抑制率（IR）, 逆转录实时荧光定量PCR（RT-qPCR）、蛋白质印记（Western blot）检测紫杉醇100 ng/mL处理A2780和A2780/T细胞后XIAP基因和蛋白的表达;将A2780/T细胞分为空转染组（转染空载体质粒）、小干扰RNA（siRNA）-XIAP（siRNA-XIAP）组（转染siRNA-XIAP质粒）、非特异性转染组（转染非特异性质粒）、空白对照组（不转染质粒）,RT-qPCR和Western blot检测各组细胞XIAP基因和蛋白的表达,并加入含不同质量浓度的紫杉醇（0、1 000、1 500、2 000、2 500 ng/mL）,流式细胞仪检测细胞凋亡率。结果 不同质量浓度紫杉醇处理后,A2780细胞IR随紫杉醇质量浓度的增加而增高（P<0.05）,A2780/T细胞IR无明显变化。紫杉醇100 ng/mL处理A2780和A2780/T细胞后,A2780细胞XIAP mRNA的表达低于紫杉醇未处理组（P<0.05）,A2780/T细胞中XIAP mRNA的表达与紫杉醇未处理组差异无统计学意义(P>0.05),但A2780/T细胞XIAP mRNA和蛋白表达均高于A2780紫杉醇处理组（P均<0.05）;siRNA-XIAP组XIAP mRNA和蛋白的表达均低于其他各组（P<0.05）,在紫杉醇2 000 ng/mL及2 500 ng/mL作用下,siRNA-XIAP组凋亡率高于其他各组（P<0.05）。结论 卵巢癌对紫杉醇耐药与XIAP基因的高表达有关,特异性的siRNA可通过降低XIAP的表达,促进细胞凋亡,增加耐药癌细胞对紫杉醇的敏感性。     

The Relationship between p38MAPK and Apoptosis during Paclitaxel Resistance of Ovarian Cancer Cells

To investigate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and cell apoptosis during the paclitaxel resistance of ovarian carcinoma cell lines, flow cytometry (FCM) and PI staining were employed to determine the effect of p38MAPK inhibitor SB203580 on the apoptosis of A2780/Taxol cells, a drug-resistant human ovarian carcinoma cell line. p38MAPK protein expression in SB203580-treated cells was immunochemically measured. The 50% inhibition concentration (IC50) of paclitaxel on A2780/Taxol cells was determined by MTT assay. MDR-1 mRNA, and expression of p38MAPK and phospho-p53 protein were detected by RT-PCR and West- ern blotting, respectively. The apoptosis rate of A2780/Taxol cells was (19.7±1.04)% 24 h after SB203580 treatment. A significant difference in apoptosis rate was found among experiment group, control group and untreated group (P<0.05). The relative reversal rate of A2780/Taxol cells to pacli- taxel was (57.18±2.01)%. As compared with the control group and the untreated group, p38MAPK protein and MDR-1 mRNA in SB203580-treated cells was substantially decreased. The expression of p53 protein was significantly increased. It is concluded that p38MAPK pathway is related to pacli- taxel resistance of ovarian carcinoma, and blockade of this pathway can promote the apoptosis of the drug-resistant cells and reverse the drug-resistance. Moreover, p38MAPK-mediated apoptosis in pa- clitaxel-resistant ovarian carcinoma cells depends on the activation of p53.     

MDR1 and MDR3 Genes and Drug Resistance to Cisplatin of Ovarian Cancer Cells

To investigate the relationship between MDR1 and MDR3 gene and drug resistance to cisplatin of ovarian cancer cells. Two siRNAs (MDR1, MDR3) which specifically targeted MDR1 and MDR3 genes were transfered into A2780/DDP cells. Then double staining with Annexin- V-FITC/PI was used to detect cell apoptosis by the flow cytometry (FCM). A2780/DDP cell viability was determined by MTT. MDR1 and MDR3 mRNA were assessed by RT-PCR. Caspase-3 protein was detected by Western blotting. Transfection of MDR1 and MDR3 siRNA into A2780/DDP cells failed to reverse the drug-resistance of A2780/DDP cells to cisplatin (P>0.05). No significant differ- ence in the apoptosis efficiency was observed between the MDR1 and MDR3 siRNA, pSuppressor- Neo vector transfection cells and untreated cells (P>0.05). In the presence of cisplatin of different concentrations, the viability of A2780/DDP cells was not significantly decreased after the transfection. No changes in MDR1 and MDR3 mRNA were found in MDR1 and MDR3 siRNA-transfected A2780/DDP cells. As compared with pSuppressorNeo and untreated groups, no significant difference existed in the expression of MDR1 and MDR3 mRNA (P>0.05). The expression of caspase-3 protein in MDR1 and MDR3 siRNA transfected A2780/DDP cells was not significantly increased. It is con- cluded that multidrug resistance induced by cisplatin in ovarian carcinoma cell lines is not due to overexpression of MDR1 and MDR3 gene. The drug resistance of ovarian carcinoma cells to cisplatin is not mediated by P-glycoprotein.     

MDR1 and MDR3 genes and drug resistance to cisplatin of ovarian cancer cells

Summary To investigate the relationship between MDR1 and MDR3 gene and drug resistance to cisplatin of ovarian cancer cells. Two siRNAs (MDR1, MDR3) which specifically targeted MDR1 and MDR3 genes were transfered into A2780/DDP cells. Then double staining with Annexin-V-FITC/PI was used to detect cell apoptosis by the flow cytometry (FCM). A2780/DDP cell viability was determined by MTT. MDR1 and MDR3 mRNA were assessed by RT-PCR. Caspase-3 protein was detected by Western blotting. Transfection of MDR1 and MDR3 siRNA into A2780/DDP cells failed to reverse the drug-resistance of A2780/DDP cells to cisplatin (P>0.05). No significant difference in the apoptosis efficiency was observed between the MDR1 and MDR3 siRNA, pSuppressorNeo vector transfection cells and untreated cells (P>0.05). In the presence of cisplatin of different concentrations, the viability of A2780/DDP cells was not significantly decreased after the transfection. No changes in MDR1 and MDR3 mRNA were found in MDR1 and MDR3 siRNA-transfected A2780/DDP cells. As compared with pSuppressorNeo and untreated groups, no significant difference existed in the expression of MDR1 and MDR3 mRNA (P>0.05). The expression of caspase-3 protein in MDR1 and MDR3 siRNA transfected A2780/DDP cells was not significantly increased. It is concluded that multidrug resistance induced by cisplatin in ovarian carcinoma cell lines is not due to overexpression of MDR1 and MDR3 gene. The drug resistance of ovarian carcinoma cells to cisplatin is not mediated by P-glycoprotein.     

转录因子CTCF对人肝癌干细胞及细胞增殖的影响

目的 探讨CCCTC结合因子 （CTCF）蛋白对人肝癌细胞 （HepG2）的肿瘤干细胞的影响以及对HepG2和CNE1 （鼻咽癌细胞系）细胞生存能力的影响。方法 构建pEGFP-N1/CTCF、CTCF shRNA和GFP-shRNA质粒,转染至HepG2和CNE1细胞后,以RT-PCR及Western blot 检测CTCF mRNA和蛋白的表达。采用流式细胞术检测转染CTCF-shRNA质粒后48 h的HepG2细胞中携带表面抗原CD90肿瘤干细胞的比例,以转染GFP-shRNA的HepG2细胞和野生型HepG2细胞为对照。采用MTT方法分别检测转染CTCF过表达重组质粒及CTCF-shRNA质粒后24、48、72 h HepG2和CNE1细胞的生存能力。 结果 pEGFP-N1/CTCF转染后增加HepG2和CNE1细胞中CTCF mRNA和蛋白的表达（与pEGFP-N1相比,P<0.05）,CTCF-shRNA转染则降低表达（GFP-shRNA相比,P<0.05）。流式细胞仪检测结果显示,CD90 +细胞的检出率在转染CTCF-shRNA质粒细胞 〔(1.733 0±0.417 7)%〕高于野生型HepG2细胞 〔(0.575 0±0.062 9)%〕及转染对照GFP-shRNA质粒细胞 〔(0.350 0±0.086 6)%〕 （P<0.05）;MTT实验结果显示CTCF的表达改变对HepG2和CNE1 细胞的生存能力的影响不明显 （P>0.05）。结论 CTCF可抑制人肝癌干细胞生成,对HepG2和CNE1细胞生长无明显影响。     

Effect of spindle checkpoint on Akt2-mediated paclitaxel-resistance in A2780 ovarian cancer cells

Recent evidence has suggested that Akt2 plays an important role in the protection of cells from paclitaxel （PTX）-induced apoptosis and control of the cell cycle. In addition, some scholars suggested that the PTX sensitivity depends on a functional spindle assembly checkpoint. In the present study, we investigated the role of the Akt2/Bubl cross-talking in apoptosis and cell cycle after exposure of the A2780 ovarian cancer cells to paclitaxel （PTX）. Recombinant expression plasmid WT-Akt2 was transfected into A2780 cells by lipofectamine2000, and then the expression level of Akt2 gene was detected by using RT-PCR and Western blotting. Cell apoptosis and cell cycle were detected by flow cytometry and Hoechst 33342 staining after treatment with PTX. Moreover, we compared the expression level of Bubl in different groups by Western blotting. Our study showed that up-regulation of Akt2 contributed to A2780 ovarian cancer cells overriding PTX-induced G2/M arrest, and inhibited Bubl expression. Our findings might shed light on the molecular mechanism of PTX-induced resistance in ovarian cancer and help develop novel anti-neoplastic strategies.     

The Relationship between p38MAPK and Apoptosis during Paclitaxei Resistance of Ovarian Cancer Cells

To investigate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and cell apoptosis during the paclitaxel resistance of ovarian carcinoma cell lines, flow cytometry (FCM) and PI staining were employed to determine the effect of p38MAPK inhibitor SB203580 on the apoptosis of A2780/Taxol cells, a drug-resistant human ovarian carcinoma cell line. p38MAPK protein expression in SB203580-treated cells was immunochemically measured. The 50% inhibition concentration (IC(50)) of paclitaxel on A2780/Taxol cells was determined by MTT assay. MDR-1 mRNA, and expression of p38MAPK and phospho-p53 protein were detected by RT-PCR and Western blotting, respectively. The apoptosis rate of A2780/Taxol cells was (19.7+/-1.04)% 24 h after SB203580 treatment. A significant difference in apoptosis rate was found among experiment group, control group and untreated group (P<0.05). The relative reversal rate of A2780/Taxol cells to paclitaxel was (57.18+/-2.01)%. As compared with the control group and the untreated group, p38MAPK protein and MDR-1 mRNA in SB203580-treated cells was substantially decreased. The expression of p53 protein was significantly increased. It is concluded that p38MAPK pathway is related to paclitaxel resistance of ovarian carcinoma, and blockade of this pathway can promote the apoptosis of the drug-resistant cells and reverse the drug-resistance. Moreover, p38MAPK-mediated apoptosis in paclitaxel-resistant ovarian carcinoma cells depends on the activation of p53.     

人突变CD59基因在卵巢癌细胞表达及其生物活性检测

目的 研究野生型CD59与突变型CD59在卵巢癌细胞A2780表面的抗补体活性.方法 取突变型CD59质粒、野生型CD59质粒分别转染A2780细胞,G418 筛选稳定表达细胞克隆;酶免疫法及RT-PCR方法检测细胞表面CD59蛋白及mRNA的表达;制备抗CD59多克隆抗体,MTT法观察CD59蛋白的抗补体活性.结果 野生型及突变型CD59质粒扩增成功,并成功建立了稳定转染野生型和突变型CD59的A2780细胞株;与野生型CD59 相比,突变型CD59失去对补体的抑制功能,与未转染A2780表面CD59表达无显著差异.结论 CD59的W40位点对其功能具有重要作用,封闭该位点能够提高补体溶细胞作用,有望应用于肿瘤治疗.     

miR-98前体转染卵巢癌细胞的效率检测及增殖抑制检测

目的 pre-hsa-miR-98转染卵巢癌SKOV3、HO8910和A2780细胞,观察转染率并研究转染后对卵巢癌细胞的增殖抑制作用,为卵巢癌新治疗方法研究提供实验依据。方法利用构建的pre-hsa-miR-98质粒HmiR和连接随机序列的质粒CmiR转染人卵巢癌细胞SKOV3、HO8910和A2780细胞,荧光显微镜观察和流式细胞仪计数计算转染率,MTT法检测转染对卵巢癌细胞的增殖抑制作用。结果 HmiR转染后24 h,荧光显微镜下观察,SKOV3、HO8910、A2780细胞的转染率分别为61.50%、68.59%、63.40%;流式细胞术计数,转染HmiR后SKOV3、HO8910、A2780细胞的转染率分别为57.93%、66.73%、58.59%。转染HmiR后,MTT法检测SKOV3、HO8910、A2780细胞的增殖抑制率随转染时间延长而增加,72 h达到49.54%、53.25%、46.09%。结论三种卵巢癌细胞的转染率均接近或超过60%,HmiR转染后可抑制卵巢癌细胞增殖,其抑制作用随转染时间延长而增强。     

Reversal of Adriamycin Resistance in Human Mammary Cancer Cells by Small Interfering RNA of MDR1 and MDR3 Genes

Resistance to antineoplastics is still the major cause of the failure of chemotherapy in cancer patients[1]. RNA interference (RNAi) is a conserved cellular mechanism in which double-stranded RNA silences the correspond-ing homologous cellular gene effectively and specificity. RNAi has a great potential of application in the reversal of drug resistance of tumor[2]. MDR1 and MDR3 are the two ATP binding cassette transporter genes. MDR3 is the second member of the human p-gp family next …     

RNA干扰靶向沉默FLIP基因对卵巢癌细胞A2780生物学特性的影响及生长抑制作用

目的探讨靶向FLIP基因的小干扰RNA（siRNA）对人卵巢癌细胞A2780生物学特性的影响及生长抑制作用。方法设计并体外化学合成FLIP序列特异性双链RNA，在脂质体（LipofectamineTM2000）介导下转染人卵巢癌细胞株A2780。采用半定量RT-PCR和Western blot法检测FLIP siRNAs转染前后A2780细胞FLIP基因mRNA和蛋白表达的变化,并筛选出抑制作用最强的FLIP-siRNA。采用四甲基偶氮唑盐（MTT）法、Annexin-V-PI双染法流式细胞术（FCM）分别检测FLIP-siRNA转染对A2780细胞的生长抑制作用和对凋亡的影响。结果特异性FLIP-siRNAs片段能有效降低A2780细胞中FLIP的 mRNA和蛋白水平（P＜0.01）,最大抑制率分别为77.4％和66.1％；转染FLIP-siRNA后，A2780细胞的生长活力明显下降（P＜0.05），RNA干扰组的细胞凋亡也显著增加（P＜0.05）。结论靶向FLIP基因的siRNAs可在转录和翻译水平抑制FLIP基因表达；并可抑制卵巢癌A2780细胞生长，促进其凋亡。     

Effect of Mad2 on paclitaxel-induced cell death in ovarian cancer cells

In this article, the status of spindle assembly checkpoint and the alteration of its major component, Mad2 protein level were examined in A2780 and SKOV3 ovarian cancer cell lines.Recombinant eukaryotic expression plasmid pEGFP-Mad2 was transfected into paclitaxel-resistant SKOV3 cells and Mad2 protein was knocked down by Mad2-specific siRNA in paclitaxel-sensitive A2780 cells.Then the expression level of Mad2 gene was detected by Western blotting.Flow cytometry revealed that SKOV3 cells were not fully arrested in G2/M phase in contrast to A2780 cells in the presence of paclitaxel.However, paclitaxel sensitivity assay showed that sensitivity to paclitaxel was reversed after the transfection in both cell lines in terms of number of cells arrested at G2/M phase and the expression of Bcl-2 was significantly changed.These results suggest that weakened spindle checkpoint with reduced expression of Mad2 is associated with resistance to paclitaxel in ovarian cells and Bcl-2 may be involved in this process.     

NDY1shRNA真核表达质粒构建及其在卵巢癌A2780细胞中的稳定表达

目的：构建NDY1 shRNA真核表达载体,并获得稳定表达shNDY1质粒的卵巢癌A2780细胞。方法根据Gen‐Bank数据库提供的NDY1基因核苷酸序列,设计并合成靶向干扰NDY1基因的小发夹RNA（shRNA）序列,插入表达载体获得重组质粒pGPU6／GFP／Neo‐shNDY1。重组质粒测序鉴定正确后,脂质体介导转染A2780细胞,经G418筛选及有限稀释法获得稳定转染细胞,采用实时定量聚合酶链反应法（RT‐qPCR）和蛋白免疫印迹法分别检测A2780稳定转染细胞NDY1 mRNA及蛋白表达。结果重组质粒测序正确,转染shNDY1后,A2780细胞mRNA及蛋白表达水平分别下降（72．89±4．83）％及（55．85±4．84）％,与对照组相比差异有统计学意义（ P＜0．05）。结论成功构建 pGPU6／GFP／Neo‐shNDY1真核表达质粒,并获得shNDY1稳定转染的卵巢癌A2780细胞,为细胞水平研究NDY1与卵巢癌的关系奠定实验基础。     

土贝母皂苷甲增强人卵巢癌A2780/DDP细胞对顺铂的敏感性

目的探讨土贝母皂苷甲(tubeimoside 1,TBMS1)促进人卵巢癌A2780/DDP细胞对顺铂(cisplatin,DDP)的增敏作用。方法采用MTT法分别检测顺铂和TBMS1对A2780/DDP细胞作用的半抑制浓度值(IC50),将实验分为对照组:只加等量生理盐水;顺铂组:给予6μmol/L顺铂处理;联合用药Ⅰ组:6μmol/L顺铂+3μmol/L TBMS1;联合用药Ⅱ组:6μmol/L顺铂+6μmol/L TBMS1。按上述方法处理细胞后,MTT法检测细胞活力;电泳法检测细胞内DNA损伤;流式细胞技术检测细胞凋亡及周期;Western blot分析周期蛋白A(Cyclin A)和周期蛋白D1(Cyclin D1)的表达。结果 TBMS1(3、6μmol/L)与顺铂(6μmol/L)联用使人卵巢癌A2780/DDP细胞对顺铂的敏感性增强;细胞的DNA损伤随TBMS1浓度的增加而明显增多;细胞周期被阻滞在S期,联合用药Ⅰ和Ⅱ组S期所占比例分别为(51.23±3.53)%、(77.34±2.80)%;细胞凋亡率为(5.19±0.78)%、(7.11±1.06)%;Cyclin A的表达为1.89±0.28及2.91±0.43,CyclinD1为0.66±0.09及0.28±0.05(P<0.05,P<0.01)。结论 TBMS1显著增加顺铂对A2780/DDP细胞增殖抑制作用,Cyclin A、Cyclin D1可能参与其调节。     

下调FoxM1通过激活JNK/线粒体通路增加人鼻咽癌细胞对紫杉醇的敏感性

目的 探讨特异性小干扰RNA(small interfering RNA,siRNA) 沉默转录因子叉头框M1(FoxM1) 对人鼻咽癌HONE-1细胞增殖、凋亡、紫杉醇敏感性的影响及可能的分子机制.方法 采用siRNA靶向沉默鼻咽癌HONE-1细胞FoxM1表达并通过RT-PCR、实时定量PCR、Western blot检测转染后FoxM1的表达水平.siRNA转染后细胞的增殖和对紫杉醇的敏感性采用MTT法检测,流式细胞仪检测细胞周期分布,Annexin V-FITC /PI双染法检测细胞凋亡率.Western blot检测Ki67、CyclinE1、多药耐药基因1(multidrug resistance 1,MDR1) 及c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK) 、p-JNK、 c-Jun、p-c-Jun、凋亡相关蛋白Bax、Bcl-2的表达水平.结果 转染后FoxM1 mRNA及蛋白的表达均被下调(P＜0. 01) .siRNA转染组细胞增殖速率减缓(P＜0. 05),Ki67表达降低(P＜0. 05) .siRNA转染组G1期细胞比例增加(P＜0. 01),S期比例减低(P＜0. 05),Cyclin E1低表达(P＜0. 01) .同时, siRNA +紫杉醇组凋亡率明显高于阴性及空白对照+ 紫杉醇组(P＜0. 01) .siRNA转染组细胞MDR1水平下降(P＜0. 05),对紫杉醇的敏感性较两对照组明显增强(P＜0. 01) .siRNA + 紫杉醇组与阴性对照+ 紫杉醇组相比p-JNK1、p-c-Jun、Bax表达上调(P＜0. 01),Bcl-2表达下调(P＜0. 01) .结论 特异性siRNA沉默FoxM1表达可以影响JNK/线粒体通路活性,抑制鼻咽癌HONE-1细胞增殖,促进其凋亡,提高其对紫杉醇的敏感性.