靶向FOLR1的脱氧核酶增加鼻咽癌对紫杉醇敏感性研究
发布时间：2018-08-11 12:18
【摘要】：鼻咽癌是由多种因素致病的恶性上皮癌。中国南方是鼻咽癌的高发区。目前以放射治疗为主,辅以化疗和中医药治疗,是鼻咽癌主要的治疗手段,但是鼻咽癌的5年生存率仍徘徊在50%左右。尽管化疗对改善鼻咽癌治疗效果和提高生存率的问题上仍有争论,但是临床试验结果显示,放疗结合化疗能明显改善中晚期鼻咽咽癌的预后,因此,化疗仍然是鼻咽癌综合治疗重要手段之一,但是化疗药物的长期使用往往导致肿瘤细胞耐药,从而导致化疗的失败,而且传统化疗由于缺乏特异性常带来的较大毒副作用,严重影响了患者的生活质量,因此,研究如何逆转肿瘤耐药、消减化疗所带来的副反应具有重要的临床意义。靶向治疗是肿瘤治疗的较好方法之一,它避免了传统化疗由于缺乏特异性而带来的较大毒副作用。叶酸受体α作为靶向治疗的一个新靶点在鼻咽癌中的研究较少。本文以鼻咽癌为研究对象,探讨叶酸受体α作为鼻咽咽癌靶向治疗和耐药逆转的一个新靶点所具有的临床意义,为开展鼻咽咽癌的靶向治疗提供实验依据。 第一章FOLR1在鼻咽癌中的异常表达及临床意义 目的研究人正常鼻咽细胞与鼻咽癌细胞中叶酸受体α表达情况,探讨其与肿瘤临床分期及病理特征的关系。 方法分别以荧光定量RT-PCR,激光共聚焦、Western-blot方法观察体外培养的正常鼻咽上皮细胞NP69和三株鼻咽癌亲本细胞(CNE1、HNE2、5-8F)及其鼻咽癌紫杉醇耐药细胞(CNE-1/Taxol、HNE-2/Taxol、5-8F/Taxol)中叶酸受体α的表达情况,免疫组织化学法检测72例鼻咽癌组织和10例正常鼻咽组织叶酸受体α的表达情况,分析鼻咽癌组织叶酸受体表达与临床病理特征的关系。 结果三株鼻咽癌细胞叶酸受体α表达均为阳性,NP69正常鼻咽上皮细胞叶酸受体α表达为阴性。鼻咽癌组织叶酸受体α阳性表达率为86.11%(68/72),正常鼻咽黏膜组织中无叶酸受体α表达(0/10),叶酸受体α表达与患者性别、年龄因素无相关性；但临床分期Ⅲ～Ⅳ期的病例叶酸受体α阳性表达明显高于Ⅰ-Ⅱ期的病例。 结论正常鼻咽细胞和组织中均无叶酸受体α表达,而鼻咽癌细胞和组织中广泛高表达叶酸受体α,并且与临床分期成正相关,这为开展针对鼻咽癌的叶酸受体α的靶向治疗奠定了基础。 第二章设计、构建和筛选靶向FOLR1基因的脱氧核酶 目的根据FOLR1基因mRNA序列,设计、构建并筛选靶向特异FOLR1基因的10-23型脱氧核酶,通过脱氧核酶沉默人鼻咽癌FOLR1基因mRNA,从而降低FOLR1靶基因的蛋白表达,为脱氧核酶应用于鼻咽癌基因治疗及肿瘤耐药逆转提供实验依据。 方法根据鼻咽癌FOLR1基因mRNA序列,利用计算机软件RNAdraw1.1b2预测FOLR1mRNA的二级结构,通过脱氧核酶的设计原则选取脱氧核酶的催化和切割的位点,针对FOLR1基因设计靶向特异性10-23型脱氧核酶。利用荧光定量RT-PCR和Western blot技术分别在mRNA水平和蛋白水平验证不同DRz对靶基因FOLR1表达的影响。 结果设计合成了五条靶向特异FOLR1基因的10-23型脱氧核酶(DRzA、 DRzB、DRzC、DRzD、DRzE)和一条阴性对照(ODNs),分别转染鼻咽癌紫杉醇耐药细胞CNE-1/Taxol,转染24h后利用荧光定量RT-PCR和Western blot技术分别在mRNA水平和蛋白水平验证了在不同DRz对靶基因FOLR1表达的影响,结果显示DRzE对靶基因的抑制作用最强,ODNs实验组不能对靶基因的表达产生抑制作用。 结论在mRNA水平和蛋白质水平DRzE对靶基因FOLR1抑制效果优于其它脱氧核酶,这为后续的实验奠定了基础。 第三章脱氧核酶靶向抑制FOLR1基因表达后耐药细胞对紫杉醇敏感性变化 目的通过脱氧核酶靶向抑制FOLR1基因表达后,观察耐药细胞对紫杉醇敏感性变化。 方法以CNE-1/Taxol为研究对象,利用DRzE脱氧核酶靶向抑制FOLR1基因表达后,通过CCK-8法观察耐药细胞对紫杉醇敏感性变化,同时通过Annexin流式细胞仪检测细胞凋亡 结果脱氧核酶DRzE靶向抑制FOLR1基因表达后,CNE-1/Taxol组细胞对紫杉醇的敏感性明显增强、其敏感性增加了约44%,而阴性对照组(ODNs组)细胞对紫杉醇的敏感性无明显改变。脱氧核酶DRzE靶向抑制FOLR1基因表达后,能明显增加紫杉醇(IC30:5ng/ml)诱导的细胞凋亡,其凋亡率从6.09±2.37%增加至23.19±2.01%。 结论靶向FOLR1的脱氧核酶DRzE能明显增加鼻咽癌紫杉醇耐药细胞对紫杉醇的敏感性。
[Abstract]:Nasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma caused by a variety of factors. Southern China is a high incidence area of NPC. At present, radiotherapy, chemotherapy and traditional Chinese medicine are the main treatment methods for NPC, but the 5-year survival rate of NPC is still around 50%. There is still controversy on this issue, but clinical trials show that radiotherapy combined with chemotherapy can significantly improve the prognosis of advanced nasopharyngeal carcinoma. Therefore, chemotherapy is still one of the important means of comprehensive treatment of nasopharyngeal carcinoma. However, long-term use of chemotherapy drugs often leads to drug resistance of tumor cells, leading to the failure of chemotherapy, and traditional chemotherapy due to lack of. Targeted therapy is one of the better methods for cancer treatment, which avoids the serious side effects of traditional chemotherapy due to lack of specificity. Folate receptor alpha as a new target for targeting therapy in nasopharyngeal carcinoma is seldom studied. In this paper, the clinical significance of folate receptor alpha as a new target for targeting therapy and reversal of drug resistance in nasopharyngeal carcinoma is discussed, which provides experimental basis for targeting therapy of nasopharyngeal carcinoma.
Chapter 1 abnormal expression of FOLR1 in nasopharyngeal carcinoma and its clinical significance
Objective To investigate the expression of folate receptor alpha in normal nasopharyngeal cells and nasopharyngeal carcinoma cells and its relationship with clinical stage and pathological characteristics of the tumor.
Methods The expression of folate receptor alpha in normal nasopharyngeal epithelial cells NP69 and three strains of nasopharyngeal carcinoma parental cells (CNE1, HNE2, 5-8F) and their paclitaxel-resistant cells (CNE-1/Taxol, HNE-2/Taxol, 5-8F/Taxol) were detected by fluorescence quantitative RT-PCR, laser confocal and Western-blot, respectively. The expression of folate receptor alpha in nasopharyngeal carcinoma tissues and 10 normal nasopharyngeal tissues was analyzed.
Results The expression of folate receptor alpha was positive in all three nasopharyngeal carcinoma cells and negative in NP69 normal nasopharyngeal epithelial cells. However, the positive expression of folate receptor alpha in stage III-IV was significantly higher than that in stage I-II.
Conclusion There is no expression of folate receptor alpha in normal nasopharyngeal cells and tissues, and the expression of folate receptor alpha is highly expressed in nasopharyngeal carcinoma cells and tissues, which is positively correlated with clinical stage.
The second chapter designs, constructs and screens the deoxy ribozyme targeting FOLR1 gene.
Objective To construct and screen the 10-23 type deoxyribozyme targeting the specific FOLR1 gene according to the FOLR1 gene mRNA sequence, and to silence the FOLR1 gene mRNA of human nasopharyngeal carcinoma (NPC) by deoxyribozyme, thereby reducing the expression of FOLR1 target gene, providing experimental basis for the application of deoxyribozyme in gene therapy of NPC and reversal of drug resistance.
Methods According to the FOLR1 gene mRNA sequence of nasopharyngeal carcinoma, the secondary structure of FOLR1 mRNA was predicted by using computer software RNA draw1.1b2. The catalytic and cleavage sites of deoxyribozyme were selected according to the design principle of deoxyribozyme, and the specific 10-23 type deoxyribozyme was designed for FOLR1 gene. MRNA level and protein level verified the effect of different DRz on the FOLR1 expression of target gene.
Results Five specific FOLR1 gene-targeted 10-23 deoxyribozymes (DRzA, DRzB, DRzC, DRzD, DRzE) and one negative control (ODNs) were synthesized and transfected into NPC drug-resistant cell line CNE-1/Taxol. After 24 hours of transfection, fluorescence quantitative RT-PCR and Western blot were used to verify the different DRz pairs at mRNA and protein levels, respectively. The results showed that DRzE had the strongest inhibitory effect on FOLR1 expression, and ODNs could not inhibit FOLR1 expression.
Conclusion The inhibitory effect of DRzE on target gene FOLR1 at mRNA and protein levels is superior to that of other deoxyribozymes, which lays a foundation for further experiments.
CHAPTER III CHANGES IN SENSITIVITY OF RESISTANT CELLS TO PAclitaxel AFTER DEOXYRIDASE TARGETIVE INHIBITION OF FOLR1 GENE EXPRESSION
Objective To observe the changes of sensitivity of drug-resistant cells to paclitaxel after targeted inhibition of FOLR1 gene expression by deoxyribozyme.
Methods CNE-1/Taxol was used as the research object. After the FOLR1 gene expression was inhibited by DRzE deoxyribozyme targeting, the sensitivity of drug-resistant cells to paclitaxel was observed by CCK-8 method, and apoptosis was detected by Annexin flow cytometry.
Results The sensitivity of CNE-1/Taxol cells to paclitaxel was increased by 44% after targeting inhibition of FOLR1 gene expression by deoxyribozyme DRzE, while the sensitivity of ODNs cells to paclitaxel did not change significantly. After targeting inhibition of FOLR1 gene expression by deoxyribozyme DRzE, the sensitivity of CNE-1/Taxol cells to paclitaxel increased significantly (IC30:5ng/ml). The rate of apoptosis was increased from 6.09 + 2.37% to 23.19 + 2.01%..
Conclusion Deoxyribozyme DRzE targeting FOLR1 can significantly increase the sensitivity of paclitaxel-resistant nasopharyngeal carcinoma cells to paclitaxel.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63
本文编号：2176972
[Abstract]:Nasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma caused by a variety of factors. Southern China is a high incidence area of NPC. At present, radiotherapy, chemotherapy and traditional Chinese medicine are the main treatment methods for NPC, but the 5-year survival rate of NPC is still around 50%. There is still controversy on this issue, but clinical trials show that radiotherapy combined with chemotherapy can significantly improve the prognosis of advanced nasopharyngeal carcinoma. Therefore, chemotherapy is still one of the important means of comprehensive treatment of nasopharyngeal carcinoma. However, long-term use of chemotherapy drugs often leads to drug resistance of tumor cells, leading to the failure of chemotherapy, and traditional chemotherapy due to lack of. Targeted therapy is one of the better methods for cancer treatment, which avoids the serious side effects of traditional chemotherapy due to lack of specificity. Folate receptor alpha as a new target for targeting therapy in nasopharyngeal carcinoma is seldom studied. In this paper, the clinical significance of folate receptor alpha as a new target for targeting therapy and reversal of drug resistance in nasopharyngeal carcinoma is discussed, which provides experimental basis for targeting therapy of nasopharyngeal carcinoma.
Chapter 1 abnormal expression of FOLR1 in nasopharyngeal carcinoma and its clinical significance
Objective To investigate the expression of folate receptor alpha in normal nasopharyngeal cells and nasopharyngeal carcinoma cells and its relationship with clinical stage and pathological characteristics of the tumor.
Methods The expression of folate receptor alpha in normal nasopharyngeal epithelial cells NP69 and three strains of nasopharyngeal carcinoma parental cells (CNE1, HNE2, 5-8F) and their paclitaxel-resistant cells (CNE-1/Taxol, HNE-2/Taxol, 5-8F/Taxol) were detected by fluorescence quantitative RT-PCR, laser confocal and Western-blot, respectively. The expression of folate receptor alpha in nasopharyngeal carcinoma tissues and 10 normal nasopharyngeal tissues was analyzed.
Results The expression of folate receptor alpha was positive in all three nasopharyngeal carcinoma cells and negative in NP69 normal nasopharyngeal epithelial cells. However, the positive expression of folate receptor alpha in stage III-IV was significantly higher than that in stage I-II.
Conclusion There is no expression of folate receptor alpha in normal nasopharyngeal cells and tissues, and the expression of folate receptor alpha is highly expressed in nasopharyngeal carcinoma cells and tissues, which is positively correlated with clinical stage.
The second chapter designs, constructs and screens the deoxy ribozyme targeting FOLR1 gene.
Objective To construct and screen the 10-23 type deoxyribozyme targeting the specific FOLR1 gene according to the FOLR1 gene mRNA sequence, and to silence the FOLR1 gene mRNA of human nasopharyngeal carcinoma (NPC) by deoxyribozyme, thereby reducing the expression of FOLR1 target gene, providing experimental basis for the application of deoxyribozyme in gene therapy of NPC and reversal of drug resistance.
Methods According to the FOLR1 gene mRNA sequence of nasopharyngeal carcinoma, the secondary structure of FOLR1 mRNA was predicted by using computer software RNA draw1.1b2. The catalytic and cleavage sites of deoxyribozyme were selected according to the design principle of deoxyribozyme, and the specific 10-23 type deoxyribozyme was designed for FOLR1 gene. MRNA level and protein level verified the effect of different DRz on the FOLR1 expression of target gene.
Results Five specific FOLR1 gene-targeted 10-23 deoxyribozymes (DRzA, DRzB, DRzC, DRzD, DRzE) and one negative control (ODNs) were synthesized and transfected into NPC drug-resistant cell line CNE-1/Taxol. After 24 hours of transfection, fluorescence quantitative RT-PCR and Western blot were used to verify the different DRz pairs at mRNA and protein levels, respectively. The results showed that DRzE had the strongest inhibitory effect on FOLR1 expression, and ODNs could not inhibit FOLR1 expression.
Conclusion The inhibitory effect of DRzE on target gene FOLR1 at mRNA and protein levels is superior to that of other deoxyribozymes, which lays a foundation for further experiments.
CHAPTER III CHANGES IN SENSITIVITY OF RESISTANT CELLS TO PAclitaxel AFTER DEOXYRIDASE TARGETIVE INHIBITION OF FOLR1 GENE EXPRESSION
Objective To observe the changes of sensitivity of drug-resistant cells to paclitaxel after targeted inhibition of FOLR1 gene expression by deoxyribozyme.
Methods CNE-1/Taxol was used as the research object. After the FOLR1 gene expression was inhibited by DRzE deoxyribozyme targeting, the sensitivity of drug-resistant cells to paclitaxel was observed by CCK-8 method, and apoptosis was detected by Annexin flow cytometry.
Results The sensitivity of CNE-1/Taxol cells to paclitaxel was increased by 44% after targeting inhibition of FOLR1 gene expression by deoxyribozyme DRzE, while the sensitivity of ODNs cells to paclitaxel did not change significantly. After targeting inhibition of FOLR1 gene expression by deoxyribozyme DRzE, the sensitivity of CNE-1/Taxol cells to paclitaxel increased significantly (IC30:5ng/ml). The rate of apoptosis was increased from 6.09 + 2.37% to 23.19 + 2.01%..
Conclusion Deoxyribozyme DRzE targeting FOLR1 can significantly increase the sensitivity of paclitaxel-resistant nasopharyngeal carcinoma cells to paclitaxel.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63
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