【作者】 彭小伟；

【导师】 谭国林；

【作者基本信息】 中南大学 ， 耳鼻咽喉科， 2010， 博士

【摘要】 第一章顺铂逆转鼻咽癌紫杉醇耐药细胞系的耐药性目的：比较顺铂对鼻咽癌细胞CNE-1及其紫杉醇耐药细胞系CNE-1/taxol生长抑制率的差异,观察顺铂对鼻咽癌紫杉醇耐药细胞系CNE-1/taxol耐药性的影响。方法：以CNE-1和CNE-1/taxol为实验对象,通过集落形成试验检测顺铂对CNE-1和CNE-1/taxol的敏感性；运用流式细胞仪检测顺铂对细胞凋亡的影响；等效剂量分析紫杉醇和顺铂联合作用两种细胞产生的效用。结果：紫杉醇处理CNE-1和CNE-1/taxol后,其IC50值分别为1.33±0.05nmol/L和11.24±0.51nmol/L,显示CNE-1/taxol的耐药指数为8.43；不同剂量(300nmol/L、600nmol/L、900nmol/L、1200nmol/L和1500nmol/L)的顺铂作用于两种细胞后,通过集落形成实验发现顺铂作用于CNE-1/taxol的IC50值为591.1±23.2 nmol/L,而作用于CNE-1的IC50值为1269.6±52.2 nmol/L,显示出CNE-1/taxol对顺铂的敏感性显著高于CNE-1对顺铂的敏感性(p<0.01)；低剂量顺铂(IC20)预处理细胞后,紫杉醇对CNE-1/taxol生长抑制的IC50值为5.34±0.29nmol/L,耐药指数下降为4.02,其敏感性显著增加(p<0.01),而CNE-1对紫杉醇的敏感性没有因顺铂的预处理发生明显变化(P>0.05)；当紫杉醇和顺铂联合作用CNE-1/taxol, CI值均小于1,表明两药具有协同作用。不同剂量顺铂(300-1500nmol/L)作用于两种细胞后,运用流式细胞仪检测其凋亡率,发现5个浓度顺铂使耐药细胞(CNE-1/taxol)的凋亡率显著高于亲本细胞(CNE-1)的凋亡率(P<0.01)。结论：紫杉醇耐药细胞系CNE-1/taxol对顺铂的敏感性高于亲本细胞CNE-1对顺铂的敏感性；顺铂能够逆转CNE-1/taxol对紫杉醇的耐药性。第二章应用基因芯片技术筛选鼻咽癌紫杉醇耐药及耐药逆转相关基因的表达谱目的：应用基因芯片技术筛选鼻咽癌紫杉醇耐药及耐药逆转相关基因的表达谱,试图发现与鼻咽癌紫杉醇耐药及耐药相关的基因。方法：使用Affymetrix HG-U133 Plus 2.0基因芯片对紫杉醇和顺铂分别处理前后的CNE-1及CNE-1/taxol细胞进行大规模的基因差异表达的检测,并进行数据分析。结果：经过多重分析与筛选,297个基因可能与紫杉醇耐药及耐药逆转相关。在耐药细胞系下调、而顺铂使之上调的基因有133个,这些基因涉及到了血管形成、凋亡、细胞生长等方面；在耐药细胞系上调、而顺铂使之下调的基因164个,主要涉及类固醇的合成和代谢,以及细胞的生长和凋亡等方面。其中改变超过5倍以上的基因有TSP1、LAMC2、PSG7、NRG1、KRT14、AKAP12、G0S2、TMEM40、MYEOV、SERPINA3、CFI、MAOA、MYOM2、METTL7A、FOS、NOV、ABP1。通过对已知耐药相关基因的分析,结果显示：1.具有药物转运作用的ATP结合盒家族中MDR1基因在各样本中都没有出现阳性表达；出现差异表达的家族成员中,有个8基因表达上调,而顺铂处理没有使这8个基因的表达下降。2.P450家族中CYP1A1在亲本细胞中不表达,在耐药细胞中出现了较强的阳性表达,经紫杉醇处理后,其表达进一步大幅下调,顺铂处理后其表达下调。3.肿瘤坏死因子家族中在耐药细胞系表达下调的有TNFAIP1、3、6、8和肿瘤坏死因子受体TNFRSF10B、12A、21。其中经顺铂处理后出现逆向表达增强的基因有TNFAIP1、3和TNFRSF12A、214. Caspase家族除CASP4在耐药细胞中表达下调,经顺铂处理后,其表达进一步下调,而在紫杉醇作用后,表达出现上调。CASP6在耐药细胞中表达上调,经顺铂处理后,其表达出现了下调,其余的Caspase家族成员的表达变化都不明显或者不表达。5.与紫杉醇耐药相关的β-微管蛋白同型中除β-微管蛋白Ⅱ出现差异表达外,β-微管蛋白Ⅲ、Ⅵ在各样本中表达差异不明显(p<0.05),β-微管蛋白Ⅰ、Ⅳ在6个样本中都没有出现阳性表达6.紫杉醇耐药基因TXR1在耐药细胞中表达上调,经紫杉醇和顺铂处理后,其表达变化均不明显。凝血酶敏感蛋白TSP1基因在耐药细胞中表达下调,经紫杉醇处理后,表达进一步下调,但是,经顺铂作用后,其表达出现了明显的上调。结论：17个显著变化的基因可能与鼻咽癌紫杉醇耐药及耐药逆转相关,如TSP1、LAMC2等。目前认为与紫杉醇耐药相关基因中的ATP-结合盒转运体家族、β微管蛋白都不是CNE-1/taxol耐药性产生过程中的关键因素；凋亡相关基因Caspase家族表达与CNE-1/taxol耐药性无关,而肿瘤坏死因子家族的部分基因可能参与了鼻咽癌紫杉醇耐药；CYP1A1的表达改变可能与CNE-1/taxol的耐药及耐药逆转有一定的相关性。第三章TXR1/TSP1信号通路与鼻咽癌紫杉醇耐药及耐药逆转的关系目的：探讨TXR1/TSP1信号通路在鼻咽癌紫杉醇耐药及耐药逆转中的作用。方法：通过RT-PCR、免疫荧光和western blot,在mRNA及蛋白质水平对基因的表达进行验证,同时,运用不同的药物剂量处理CNE-1和CNE-1/taxol,观察TSP1的表达变化。结果：通过RT-PCR试验,发现TXR1在CNE-1/taxol的表达大约是CNE-1的7倍；而TSP1在mRNA和蛋白质水平分别下调了8.9和5.6倍；经591.1 nmol/L (IC50)顺铂作用24h后,CNE-1/taxol中TSP1在mRNA水平表达上调了8.7倍,经300 nmol/L-1500 nmol/L顺铂作用后,随着药物浓度的增加,TSP1蛋白的表达随之上调；然而,两种细胞经顺铂作用后,TXR1的表达都没有出现明显的改变。结论：CNE-1/taxol耐药性的产生可能与TXR1的表达有关,并且是通过下调TSP1的表达实现的；顺铂可能是通过上调TXR1的下游基因TSP1来逆转CNE-1/taxol的耐药性的。更多还原

【Abstract】 Chapter One Reversal of the taxol-resistant phenotype by cisplatin in nasopharyngeal carcinomaObject:To compare the difference of cell growth inhibition between taxol-resistance NPC cells and its parental cells treated by cisplatin, and observe if cisplatin reverse the taxol-resistance phenotype in NPC.Method:A taxol-resistant NPC cell line(CNE-1/taxol) and its parental cell line (CNE-1) were cultured in normal condition. The sensitivity of both CNE-1 and CNE-1/taxol to cisplatin was detected using the colony formation assay. Apoptotic death was measured by flow cytometry. The CI-isobologram was used to analyze the drug combination assays of taxol and cisplatin.Result:The IC50 value of CNE-1 and CNE-1/taxol was respectively 1.33±0.05nmol/L,11.24±0.51nmol/L when both cell lines treated by taxol. The index of CNE-1/taxol was 8.43. The IC50 value of CNE-1/taxol and CNE-1 was respectively 591.1±23.2 nmol/L and 1269.6±52.2 nmol/L when both cell lines treated by different doses of cisplatin (300nmol/L、600nmol/L、900nmol/L、1200nmol/L and 1500nmol/L). The sensitivity to cisplatin in CNE-1/taxol was significantly higher than in CNE-1 (p<0.01). When the cells were pretreated with low-dose cisplatin (IC20), the growth inhibition rates of taxol in CNE-1 cells remained unchanged, however, the rate significantly increased in CNE-1/taxol cells(p<0.01), its IC50 value was changed to 5.34±0.29nmol/L. The coadministration of taxol and cisplatin produced the synergistic effect in taxol-resistant NPC cells, but only additive effects in parental NPC cells. The apoptotic rate was significantly higher in CNE-1/taxol than in CNE-1 cells when both cell lines were treated by different doses of cisplatin (P＜0.01)Conclusion:CNE-1/taxol cells were more sensitive to treatment with cisplatin than its parental cells, CNE-1.Cisplatin reversed the taxol-resistant phenotype in NPC cells. Chapter Two Screening the differential expression of taxol-resistance related genes of nasopharyngeal carcinoma by cDNA microarrayObject:To screen the profiling of gene expression related to taxol resistance and reversal of taxol resistance, and search for genes related to taxol-resistance phenotype.Method:To detect the differential expression of CNE-1/taxol and CNE-1 treated by taxol and cisplatin using cDNA microarray.Result:297 genes was traped in our designed criteria through multiple steps. Some of these genes should be taxol-resistant genes.133 genes were down-expressed in CNE-1/taxol, and its expression level could be restored by cisplatin.164 genes were overexpressed in CNE-1/taxol, and its expression level could also be recovered by cisplatin. These genes involved in angiogenesis, cell growth, cell metabolism, apoptosis, etc.17 genes were screened out when the fold change must be more than 5, including TSP1、LAMC2、PSG7、NRG1、KRT14、AKAP12、G0S2、TMEM40、MYEOV、SERPINA3、CFI、MAOA、MYOM2、METTL7A、FOS、NOV、ABP1.1.Through Analyzing documented drug-resistant genes, MDR1 expression was not detected in all NPC samples. Eight ATP-binding cassette transporters that highly expressed in paclitaxel-resistance nasopharyngeal carcinoma cells were detected. However, the expression of these genes were not increased in CNE-1/taxol cells after treated by IC50 taxol.2.CYP1A1 of P450 family members was not expressed in CNE-1, but significantly increased expression was found in CNE-1/taxol and these increased expression could be restored by cisplatin.3.The expression level of 4 genes and 3 receptor genes of tumor necrosis factor family members were decresed in CNE-1/taxol, and restored by cisplatin. These genes included TNFAIP1、3、6、8 and TNFRSF10B、12A、21。4.The expression of CASP4 was decreased in CNE-1/taxol, but not changed by cisplatin. The other genes of Caspase family members were not significantly changed. 5.The expression ofβ-tubulin I and IV were not detected in all samples. No differential expression was found in P-tubulin III and VI when both CNE/taxol and CNE-1 cells treated or untreated by taxol and cisplatin(p>0.05). The expression ofβ-tubulin II were down-regulated in CNE-1/taxol.6.The expression of TXR1 was higher in CNE-1/taxol than in CNE-1. TSP1 was obviously down-regulated in CNE-1/taxol compared with CNE-1, and a more significant down-regulation of TSP1 was found when CNE-1/taxol treated by taxol. However, it was greatly up-regulation after treated by cisplatin in CNE-1/taxol.Conclusion:17 Significant changed genes in CNE-1/taxol may relate to taxol resistance and reversal of taxol resistance in NPC cells, such as TSP1, LAMC2, etc. ATP-binding cassette transporters (including MDR1) were not associated with taxol resistance of NPC, and P-tubulin isotype were not key genes in drug resistance. Caspase family members of apoptotic-related genes were nothing to do with CNE-1/taxol resistance. CYP1A1 may play a role of taxol resistance and reversal of taxol resistance in NPC cells.Chapter Three The role of TXR1/TSP1 in taxol resistance and reversal of taxol resistance in NPC cellsObject:To understand the role of TXR1/TSP1 in taxol resistance and reversal of taxol resistance in NPC cells.Method:Both CNE-1/taxol and CNE-1 cells were treated by different doses of taxol and cisplatin. The mRNA expression of genes was determined by RT-PCR, protein western of genes was detected by immunofluorescence and western blot.Result:Approximate 7-fold increase of TXR1 mRNA expression and 8.9-fold decrease of TSP1 mRNA expression were observed in taxol-resistant cells compared to their parental cells. An 8.7-fold increase in TSP1 mRNA expression was observed in CNE-1/taxol cells exposed to 590 nM cisplatin for 24 hours. An increase in TSP1 protein expression was obtained in a dose-dependent manner after CNE-1/taxol cells were exposed to cisplatin. However, there was no change in TXR1 mRNA expression after both CNE-1 and CNE-1/taxol cells were exposed to cisplatin.Conclusion:The TXR1/TSP1 regulation pathway may relate to taxol resistance in NPC cells. It was determined that cisplatin reverses drug resistance through the up-regulatin of TSP1 downstream of TXR1.更多还原