【作者】 缪叶；

【导师】 吴军正；

【作者基本信息】 第四军医大学 ， 口腔基础医学， 2013， 博士

【摘要】 粘液表皮样癌，是人类涎腺恶性肿瘤当中一个很常见的类型。根据以往的报道，此恶性肿瘤占所有涎腺恶性肿瘤的百分之三十以上，占颌面部所有肿瘤的5%到10%。而临床上对于此种肿瘤的治疗方法还是以外科手术为主。化疗对于此种肿瘤的效果并不是非常理想，主要障碍主要在于用药之后产生的多药耐药（Multidrugresistance，MDR）。解决了多药耐药的问题，可以为治疗这种恶性肿瘤提供一个新的有效的治疗途径。MDR是指由于使用一种药物而对多种药物产生耐药性，降低了临床抗肿瘤药物的有效性。目前研究最广泛的耐药机制是细胞将药物泵出的膜转运蛋白。p-糖蛋白（p-glycoprotein，P-gp）、多药耐药相关蛋白（Multidrug resistance-associated protein，MRP）是与耐药相关的两个主要蛋白。细胞的生物学功能大部分是通过信号通路实现，根据对于信号通路的研究，可以反过来揭示细胞的生物学特性。通过阻断这些和疾病相关的信号通路，可以达到研究和治疗的目的。我们在前期研究中建立了MC3/5-FU的耐药细胞系，可以和MC3细胞系进行对照。设计多条shRNA片段构建表达载体DNA,对体外培养的人粘液表皮样癌耐药细胞MC3/5-FU进行稳定转染，检测转染效率、检测细胞MDRl基因的mRNA的表达，筛选出更加有效的shRNA片段进行后续研究。检测RNA干扰后细胞耐药逆转率。检测MC3/5-FU和MC3之间信号通路活性差异，寻找耐药相关通路。抑制各通路活性后检测耐药逆转率以及生物学特性。通过抑制各通路活性后检测MDR1表达，并且RNAi沉默MDR1后检测各通路活性，从而推导各耐药相关通路与MDR1之间关系。检测RNAi与抑制各通路表达的抑制剂共同干预MC3/5-FU后耐药逆转率。RNAi沉默MRP1后检测MDR1以及信号通路表达，推测MRP1参与粘液表皮样癌耐药机制。主要研究内容及结果第一部分RNAi逆转MC3/5-FU耐药性用MTT法检测MC3/5-FU耐药倍数，对于诱导药物5-FU耐药倍数为16.7达到高度耐药。针对MDR1基因根据siRNA设计原则设计3条shRNA并分别用脂质体转染MC3/5-FU细胞，real time-PCR检测，片段1沉默效果最佳，并筛选出片段1的稳定转染细胞株，RT-PCR检测细胞细胞MDR1基因表达，测定转染后细胞株耐药倍数及耐药逆转率。转染后细胞株MDR1基因表达量下降，MC3/5-FU细胞对5-FU、BLM、VCR和VBL耐药逆转率分别为达到70.6%、40.8%、48.6%和58.8%。第二部分抑制信号通路逆转MC3/5-FU耐药性通过western blot方法找出MC3/5-FU细胞和MC3细胞之间差异的耐药相关细胞信号蛋白，MC3/5-FU细胞中NF-κB、Nrf2-ARE、MAPK/ERK活性明显高于MC3细胞。用抑制剂抑制NF-κB、Nrf2-ARE和MAPK/ERK后MC3/5-FU细胞对5-FU的耐药逆转率分别为67.7%、43.4%和54.3%。。用MTT法测定抑制后MC3/5-FU生长曲线，软琼脂克隆实验计算各种干预后细胞克隆数，可见各种干预后Nrf2-ARE组生长最快，NF-κB和MAPK/ERK组最慢。第三部分耐药相关信号通路在粘液表皮样癌中与MDR1基因之间的关系用RT-PCR和western blot.检测耐药相关信号通路在粘液表皮样癌中与MDR1基因之间的关系。 NF-κB、Nrf2-ARE和MAPK/ERK通路活性被抑制后MC3/5-FU细胞MDR1mRNA表达降低，NF-κB通路效果最明显。RNAi沉默MC3/5-FU中MDR1的表达后可见各通路活性未发生明显变化。抑制剂与RNAi共同作用后检测耐药逆转率与RNAi组无明显差异。第四部分MRP1基因引起耐药的机制用免疫组化实验检测MEC临床手术标本发现MRP1基因在MEC中表达，且与MDR1基因的表达关系密切。用RNAi沉默MRP1基因后RT-PCR检测发现MDR1基因表达被抑制。沉默MRP1基因后western blot方法检测Nrf2-ARE通路的活性被明显抑制。结论：1MDR1基因的表达是粘液表皮样癌细胞产生多药耐药的一个重要因素。2用RNAi的方法沉默MDR1基因可以有效逆转粘液表皮样癌细胞的耐药性。3NF-κB、Nrf2-ARE、MAPK/ERK参与粘液表皮样癌细胞的耐药过程，通过抑制这些通路可以逆转粘液表皮样癌细胞的耐药性。4NF-κB通路在粘液表皮样癌细胞中与MDR1基因的表达关系密切。5推测在粘液表皮样癌细胞中，MRP1可以激活Nrf2-ARE从而促进MDR1基因表达，导致细胞耐药。而不是传统认为的膜蛋白药泵作用。更多还原

【Abstract】 Mucoepidermoid carcinoma(MEC) is a common malignacy in salivary glands，itaccounts for about30%of malignant tumors in salivary glands and for5%to10%ofmalignant tumors in maxillofacial region in Chinese population. Anyhow,the main way ofits therapy is still surgery because chemotherapy is not very efficient to the tumor. The mainobstacle is the Multidrug resistance (MDR) effect after medication. Solving the problemof MDR may provide a new efficient way to treat MEC.MDR reduces the effectiveness of chemotherapy. MDR is characterized by crossresistance to antineoplastic drugs, and the members of the ATP-binding cassette (ABC)transporter superfamily, such as ABCB1(MDR1) and ABCC1(MRP1), which play themost importent role in MDR.Most biological functions of cells work by signal pathway. The bionomics of cellscan be revealed by studying the signal pathway. Interrupting these signal pathways relatedto illness may creat a new way to study and therapy.In previous study we established multi drug resistant cell line MC3/5-FU from theparental cell line MC3. Therefor, in this study, we designed several pieces of shRNA,constructed MDR1-shRNA expression vector and silenced MDR1in MC3/5-FU cells withstable transfection. Then we detected the reverse rate of MDR by RNAi. Furthermore, wedetected the NF-κB,Nrf2-ARE and MAPK/ERK signal pathways between MC3/5-FU andMC3to find the MDR related ones. We used RNAi and signal pathway depressor todeduce the relationship between MDR1,MRP1, signal pathways and multidrug resistancein mucoepidermoid carcinoma. The study includs four parts as follows.1Reversion of MDR in MC3/5-FU by RNAi.The plasmid expressing shRNA homologous to MDR1mRNA was constructed withshRNA expressing vector pRNAT-U-6.1/Neo based on the principle of siRNA design.Thecombinant plasmid was transfected stably into MC3/5-FU Cells using Lipofectamine2000.The shRNA efficiency was detected by real time-PCR analysis. The result showedMDR1-shRNA could effectively block the expression of MDR1in MC3/5-FU cells.RT-PCR analysis showed the expression of MDR1was silenced after stable transfection.The resistance index of MC3/5-FU was detected by MTT assay. The result showed thatthe multidrug resistance rates of MC3/5-FU cells to5-FU, BLM, VCR and VBL werereversed by70.6%,40.8%,48.6%and58.8%respectively.2Reversion of MDR in MC3/5-FU by inhibiting signal pathway.The expression of signal pathways between MC3/5-FU and MC3cells were detectedby western blot analysis. The results show that the activity of NF-κB,Nrf2-ARE andMAPK/ERK in MC3/5-FU was much higher than in MC3. The reversal of MDR wasdetected after the signal pathways was inhibited by the inhibitors. Inhibition of NF-κB，Nrf2-ARE and MAPK/ERK reversed MDR of MC3/5-FU cells to5-FU by67.7%,43.4%and54.3%respectively. MTT assay and soft agar cloning experiments showed thatinhibition of NF-κB resulted in proliferation inhibition of MC3/5-FU cells.3The relationship between signal pathway and MDR1gene inmucoepidermoid carcinoma.The relationship between signal pathway and MDR1gene in MC3/5-FU cell wereinvestigated by RT-PCR and western blot. Inhibition of NF-κB， Nrf2-ARE andMAPK/ERK inhibited MDR1mRNA expression in MC3/5-FU cells, inhibition of NF-κBshowed the strongest effects.While silence of MDR1gene by RNAi did not change theexpression of NF-κB，Nrf2-ARE and MAPK/ERK activity in MC3/5-FU cells.4The function of MRP1in MDR MRP1protein was found in surgical samples of MEC by immunohistochemistryexperiment. RT-PCR analysis showed the expression of MDR1in MC3/5-FU cells wasinhibited after MRP1was silenced by RNAi. The activity of each signal pathway wasdetected by western blot after MRP1was silenced by RNAi. The resuit showed onlyNrf2-ARE was inhibited.Conclusion1MDR1is an important factor of multi-drug resistance in mucoepidermoid carcinoma.2Silenc of MDR1by RNAi is effective in the reversal of MDR in mucoepidermoidcarcinoma.3NF-κB,Nrf2-ARE and MAPK/ERK signal pathways take part in the MDR inmucoepidermoid carcinoma.4NF-κB pathway has close relationship with the expression of MDR1in mucoepidermoidcarcinoma5MRP1may enhance the expression of MDR1by activating Nrf2-ARE rather than bymembrane protein as drug pump.更多还原