【作者】 俞艳萍；

【导师】 孔维佳；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉科， 2010， 博士

【摘要】 目的：探讨ABCG2在鼻咽癌活检标本及鼻咽癌细胞系CNE-2中的表达及其与鼻咽癌化疗中的多药耐药性的关系。材料和方法：鼻咽癌活检标本取自30位鼻咽癌患者,对照组粘膜是20份鼻咽炎性组织,取自20位慢性鼻咽炎患者的鼻咽部活检组织。采用实时定量PCR(Real-time PCR)检测ABCG2 mRNA的表达,用免疫组化的方法检测ABCG2蛋白的表达。结果：鼻咽癌活检组织中,ABCG2在mRNA水平和蛋白水平的表达均高于鼻咽炎性组织中的表达(t=5.4060,P=0.0124),而且在伴有淋巴结转移的患者中,其表达显著增高(t=5.4547,P=0.014)。我们用低浓度的托泊替康(0.75μg/ml)和盐酸米托蒽醌(0.6μg/ml)分别处理CNE-2细胞24小时,48小时和72小时。Real-time PCR的结果表明在托泊替康或者盐酸米托蒽醌处理之后,ABCG2 mRNA表达增高,并与处理时间正相关(r=0.9857,P=0.0143)。结论：ABCG2在鼻咽癌中相对高表达,与淋巴结转移相关,很可能是淋巴结转移的一个分子标志,而且ABCG2可能参与了鼻咽癌化疗中的多药耐药。目的：探讨ABCG2在Mitoxantrone处理CNE-2细胞过程中的作用方法：用不同浓度Mitoxantrone (0,10 nM,20 nM,40 nM,60 nM,80 nM)处理CNE-2细胞24h,48h和72h后,用MTT法分析加入ABCG2特异性抑制剂FTC前后其IC50的变化,并用Realtime-PCR和Western Blot检测Mitoxantrone处理前后ABCG2mRNA和蛋白的表达。结果：在5μM FTC存在时,CNE-2细胞24h,48h和72h的IC50都减少约30%。说明当ABCG2的作用被抑制后,CNE-2细胞对Mitoxantrone更敏感。通过用Realtime-PCR发现Mitoxantrone可以诱导ABCG2 m RNA的表达,并且有药物浓度依赖性,在24h,48h和72h的相关系数分别是r2=0.9520,P=0.0009；r2=0.8826,P=0.0054 and r2=0.9351,p=0.0016,也与作用的时间正相关,Mitoxantrone10nM,20 nM,40 nM,60 nM,80 nM时,相关系数分别是r2=0.9505,P=0.0251；r2=0.9650,P=0.0177；r2=0.9923,P=0.0039；r2=0.9671,P=0.0166;r2=0.9672,P=0.0155。WB检测蛋白的表达与Real-time PCR的结果相一致。结论：用Mitoxantrone处理CNE-2细胞时,细胞膜上表达的ABCG2可能将细胞内的Mitoxantrone转运出去以保护细胞,这也可能是ABCG2参与多药耐药性的机制。目的：构建靶向人ABCG2基因的pGC-silencer-U6/Neo/GFP/ABCG2真核表达质粒,运用RNA干扰下调ABCG2基因在鼻咽癌细胞系CNE-2中的表达,并筛选出其基因沉默效果最明显的shRNA质粒表达载体。方法：针对ABCG2基因的mRNA序列设计,分别构建3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经大肠杆菌扩增,酶切、PCR、测序鉴定,用脂质体法转染鼻咽癌CNE-2细胞,用real-time PCR和western blot检测ABCG2基因在mRNA和蛋白水平检测抑制效果。结果：经测序证实,成功构建pGC-silencer-U6/Neo/GFP/ABCG2真核表达质粒。重组质粒转染CNE-2细胞后,ABCG2基因的mRNA水平及蛋白水平明显下调,其中以1号重组质粒效应最强,mRNA水平下调75%,蛋白水平下调68%。结论：成功构建以人ABCG2为靶向的pGC-silencer-U6/Neo/GFP/ABCG2的重组质粒。其对鼻咽癌CNE-2细胞中ABCG2的表达具有显著抑制效应,为进一步研究ABCG2在CNE-2细胞中的功能和鼻咽癌的基因治疗奠定了基础.目的：观察沉默ABCG2基因的表达后,CNE-2细胞对Mitoxantrone敏感性的变化,探讨ABCG2在鼻咽癌细胞系CNE-2耐药中的作用机制。方法：建立转染pGC-silencer-U6/Neo/GFP/ABCG2真核质粒和转染control质粒的稳定单克隆细胞,用不同浓度的Mitoxantrone (0,10 nM,20 nM,40 nM,60nM,80 nM)处理CNE-2, CNE-2/siRNA-ABCG2和CNE-2/control细胞24h,48h和72h,用MTT实验分析三个时间点,每种细胞的IC50；并用35.3 nMMitoxantrone(24h时CNE-2细胞IC50的浓度)处理CNE-2, CNE-2/siRNA-ABCG2和CNE-2/control细胞24h,用CCK-8和流式细胞检测分析各种细胞的增殖情况和细胞周期的变化。结果：在24h,48h和72h三个时间点,CNE-2/siRNA-ABCG2细胞的IC50s均比CNE-2细胞要低(t=11.9137,P=0.0003)；用35.3 nM Mitoxantrone (24h时CNE-2细胞IC50的浓度)作用CNE-2, CNE-2/siRNA-ABCG2和CNE-2/control细胞,每天用CCK-8检测细胞生长情况,连续检测十天,绘制生长曲线,发现CNE-2／siRNA-ABCG2细胞的增殖与CNE-2细胞相比,明显减慢(t=2.2614,P=0.036)；35.3nM Mitoxantrone处理之后,CNE-2细胞中凋亡细胞的比例从8.70%增加到20.91%,G1期细胞的比例从37.22%增加到40.43%,G2／M期细胞比例从33.76%下降到17.54%, CNE-2/siRNA-ABCG2细胞中凋亡细胞的比例由8.32%增加到29.73%,G1期细胞的比例由39.94%增加到41.15%,G2／M期细胞比例由30.47%下降到6.7%。结论：转染pGC-silencer-U6/Neo/GFP/ABCG2真核质粒的CNE-2/siRNA-ABCG2细胞对Mitoxantrone的敏感性增加。目的：探讨ABCG2在Mitoxantrone处理鼻咽癌裸鼠移植瘤过程中的作用方法：建立鼻咽癌裸鼠移植瘤模型,用10nM Mitoxantrone分别在CNE-2,CNE-2／siRNA-ABCG2以及CNE-2/control细胞形成的移植瘤的体部和基底部多点微量注射,注射等体积的PBS作为对照组,记录肿瘤体积的增长曲线,并在4周后处死裸鼠,取出移植瘤,用TUNEL检测肿瘤的细胞原位凋亡率。结果：60只裸鼠无一死亡,体重均匀增加,成瘤率100%。在CNE-2组,CNE-2／siRNA-ABCG2组和CNE-2/control组中,注射Mitoxantrone组的肿瘤体积的增长均比相应的注射PBS组要慢；CNE-2组和CNE-2/control组之间,注射Mitoxantrone组和注射PBS组的肿瘤体积的增长均无明显差异(t=0.9678,P=0.3460);在CNE-2组,CNE-2/siRNA-ABCG2组和CNE-2/control组中,三组注射PBS的裸鼠肿瘤体积增长均无明显差异(P>0.05)；在CNE-2组和CNE-2/siRNA-ABCG2组中,用Mitoxantrone处理后,CNE-2/siRNA-ABCG2组肿瘤体积增长明显比CNE-2组要慢(t=2.6255,P=0.0172)。对取出的移植瘤进行原位TUNEL检测,发现各组的凋亡率各不相同。在CNE-2/siRNA-ABCG2组,凋亡率为29.45±5.29%,在CNE-2组,凋亡率为18.51±4.33%,而在CNE-2/control组的凋亡率为12.13±6.04%。在CNE-2组,CNE-2/siRNA-ABCG2组和CNE-2/control组三组中,用PBS处理的肿瘤凋亡无明显差异。结论：抑制了ABCG2基因的表达后,CNE-2移植瘤对Mitoxantrone的敏感性增强,ABCG2的表达是鼻咽癌耐药的重要原因。更多还原

【Abstract】 Objective:This study investigated the expression of ABCG2 in nasopharyngeal carcinoma biopsy samples and CNE-2 cell line and its relationship with the multidrug resistance in nasopharyngeal carcinoma chemotherapy.Material and Methods:The tumor biopsy samples were from 30 patients with nasopharyngeal carcinoma, and the tissues in control group were chronic inflammatory biopsy tissues from 20 patients with chronic nasopharyngitis. ABCG2 mRNA expression was detected by Real-time polymerase chain reaction (Real-time PCR) and ABCG2 protein expression was evaluated by immunohistochemical analysis.Results:It was found that the expression levels of ABCG2 mRNA and protein were higher in nasopharyngeal carcinoma tissues than in the chronic inflammatory tissues (t=5.4060, P=0.0124), and it was markedly increased in patients with lymph node metastasis (t=5.4547, P= 0.014). And then we chose low concentrating of Topotecan (0.75μg/ml) and Mitoxantone (0.6μg/ml) to treat CNE-2 cells for 24,48 and 72 h respectively. The results of real-time PCR showed that the ABCG2 mRNA expression was increased in a time-dependent manner after treatment of CNE-2 cells with Topotecan or Mitoxantone (r= 0.9857, P= 0.0143).Conclusion:The expression of ABCG2 is higher in nasopharyngeal carcinoma, which is related with the N staging. ABCG2 may be a marker of lymph node metastasis in nasopharyngeal carcinoma. And ABCG2 is potentially involved in Topotecan and Mitoxantrone resistance in nasopharyngeal carcinoma chemotherapy. Objective:This study investigated the function of ABCG2 in Mitoxantrone treatment in CNE-2 cells.Methods:ABCG2 function was determined by measuring the IC50s of CNE-2 cells for different concentrations of Mitoxantrone (0,10 nM,20 nM,40 nM,60 nM,80 nM) in the presence and absence of the specific ABCG2 inhibitor fumitremorgin C (FTC) for 24h,48h and 72h. The expression of ABCG2 before and after Mitoxantrone treatment was detected by the real-time polymerase chain reaction (Real-time PCR) and western blot at both mRNA and protein levels in CNE-2 cells.Results:We found that the IC50s of CNE-2 cells for Mitoxantrone were reduced by approximately one-third of that without FTC. Mitoxantrone could induce ABCG2 mRNA expression, which was dose-dependent at 24h,48h and72h (r2=0.9520, P=0.0009; r2=0.8826, P=0.0054 and r2=0.9351, P=0.0016) and was also exposure time-dependent at 10 nM,20 nM,40 nM,60 nM and 80 nM(r2=0.9505, P=0.0251; r2=0.9650, P=0.0177; r2=0.9923, P=0.0039; r2=0.9671, P=0.0166; r2=0.9672, P=0.0155). The protein expression of ABCG2 was determined by Western Blot and found to consistent with the Real-time PCR data.Conclusion:We speculated that ABCG2 would transport mitoxantrone out to protect the cells in Mitoxantrone treatment in CNE-2 cells and ABCG2 is potentially involved in Mitoxantrone resistance in nasopharyngeal carcinoma chemotherapy. Objective; To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human ABCG2 gene, to assay the expression of ABCG2 in a human nasopharyngeal carcinoma (NPC) cell line, CEN-2 cell line, after transfected with recombinant plasmids, and to detect the RNAi effect of shRNA.Methods:Targeting ABCG2 gene sequence, three plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and then identified by restriction digestion, PCR and sequencing. The vectors were transfected into CEN-2 cells. ABCG2 expression was assayed by real-time quantitative PCR and western blot.Results:The successful construction of pGC-silencer-U6/Neo/GFP/ABCG2 was confirmed by DNA sequencing. Transfection of shRNA plasmids significantly down-regulated ABCG2 expression in CEN-2 cells at both mRNA level and protein level. Recombinant plasmid 1 had the strongest effect, with an inhibition ratio of 75% at the mRNA level and 68% at the protein level, which showed a significant difference from plasmids 2 and 3 (P<0.05).Conclusion:pGC-silencer-U6/Neo/GFP/ABCG2 has been successfully constructed and it can down-regulate ABCG2 expression after transfected into CEN-2 cells, which could facilitate further studies of ABCG2 functions CEN-2 cell line and its application in NPC gene therapy. Objective:To investigate the mechanism of multidrug resistaance in nasopharyngeal carcinoma by detecting the sensitivity to Mitoxantrone of CNE-2/siRNA-ABCG2 cells after inhibition of ABCG2 by RNAi technology.Methods:pGC-silencer-U6/Neo/GFP/ABCG2 and control plasmids were transfected into CNE-2 cells separately and then stable clones were isolated named as CNE-2/ siRNA-ABCG2 cells and CNE-2/control cells, respectively. The sensitivity to Mitoxantrone of CNE-2, CNE-2/siRNA-ABCG2 and CNE-2/control cells were evaluated by measuring the IC50s of these cells for different concentrations of Mitoxantrone (0,10 nM,20 nM,40 nM,60 nM,80 nM). Then CNE-2, CNE-2/ siRNA-ABCG2 and CNE-2/control cells were exposed to 35.3 nM Mitoxantrone, which is the IC50 of CNE-2 cells at 24h, and their growth and cell cycle were detected by CCK-8 and PI staining.Results:For all three time points, the IC50s of CNE-2/siRNA-ABCG2 for Mitoxantrone were significantly lower than that of untransfected CNE-2 cells (t=11.9137, P=0.0003). We examined the growth rate of these cells for 10 days, and found that the proliferation of CNE-2/siRNA-ABCG2 cells was significantly slower than that of CNE-2 cells (t=2.2614, P=0.0364) with Mitoxantrone treatment. After treated with Mitoxantrone, proportion of apoptosis cells was increased from 8.70% to 20.91%, proportion of G1 stage cells were increased from 37.22% to 40.43%, whereas G2/M stages cells were reduced from 33.76% to 17.54% in CNE-2 cells, however in CNE-2/siRNA-ABCG2 cells, proportion of apoptosis cells was increased from 8.32% to 29.73%, proportion of G1 stage cells were increased from 39.94% to 41.15%, whereas G2/M stages cells were reduced from 30.47% to 6.70%. Conclusion:The sensitivity of CNE-2 cells to Mitoxantrone was increased after transfected with pGC-silencer-U6/Neo/GFP/ABCG2 plasmid Objective:To investigate the sensitivity to Mitoxantrone of CNE-2 cells after inhibition of ABCG2 by RNAi technology in nude miceMethods:Created the model of human nasopharyngeal carcinoma xnograft in nude mice. The base of the tumors and the tumors themselves CNE-2, CNE-2/ siRNA-ABCG2 or CNE-2/control cells formed were micro-multi-point injected of Mitoxantrone (10n M,20μl) per week for 4 weeks. An equal amount of PBS (20μ1) was injected as control. The growth curves of tumors were recorded. Then tumors were isolated from nude mice after 4weeks. The apoptotic indexes of these tumors were detected using the in situ TUNEL method.Results:No one died in experimental nude mice, and their body weight continued to grow. Tumor formation rate reached 100%. the increase of volume in control groups was faster than that in experimental groups in all of CNE-2 group, CNE-2/ siRNA-ABCG2 group and CNE-2/control group (as described in Materials and methods); (2)between the group injected with CNE-2/control cells and the group injected with untreated CNE-2 cells, there was no significant difference in tumor growth in their experimental groups as well as their control groups(t=0.9678,P=0.3460); (3)in these three groups, the growth of tumors was no significant difference among their control groups (p>0.05); (4) injected with Mitoxantrone, the growth of CNE-2/siRNA-ABCG2 cells formed tumors was significantly suppressed compared with that of untreated CNE-2 cells formed tumors (t=2.6255,P=0.0172). Using the in situ TUNEL method, we found that treated with Mitoxantrone, the apoptotic index was 29.45±5.29% for the tumors CNE-2/ siRNA-ABCG2 cells formed,18.51±4.33% for the tumors untreated CNE-2 cells formed, and 12.13±6.04% for the tumors CNE-2/control cells formed, respectively. Injected with PBS, the apoptotic indexes for the three groups were no significant difference.Conclusion:The sensitivity to Mitoxantrone of CNE-2 cells after inhibition of ABCG2 by RNAi technology in nude mice was increased.更多还原