靶向FOLR1的脫氧核酶增加鼻咽癌對(duì)紫杉醇敏感性研究
發(fā)布時(shí)間：2018-08-11 12:18
【摘要】：鼻咽癌是由多種因素致病的惡性上皮癌。中國(guó)南方是鼻咽癌的高發(fā)區(qū)。目前以放射治療為主,輔以化療和中醫(yī)藥治療,是鼻咽癌主要的治療手段,但是鼻咽癌的5年生存率仍徘徊在50%左右。盡管化療對(duì)改善鼻咽癌治療效果和提高生存率的問(wèn)題上仍有爭(zhēng)論,但是臨床試驗(yàn)結(jié)果顯示,放療結(jié)合化療能明顯改善中晚期鼻咽咽癌的預(yù)后,因此,化療仍然是鼻咽癌綜合治療重要手段之一,但是化療藥物的長(zhǎng)期使用往往導(dǎo)致腫瘤細(xì)胞耐藥,從而導(dǎo)致化療的失敗,而且傳統(tǒng)化療由于缺乏特異性常帶來(lái)的較大毒副作用,嚴(yán)重影響了患者的生活質(zhì)量,因此,研究如何逆轉(zhuǎn)腫瘤耐藥、消減化療所帶來(lái)的副反應(yīng)具有重要的臨床意義。靶向治療是腫瘤治療的較好方法之一,它避免了傳統(tǒng)化療由于缺乏特異性而帶來(lái)的較大毒副作用。葉酸受體α作為靶向治療的一個(gè)新靶點(diǎn)在鼻咽癌中的研究較少。本文以鼻咽癌為研究對(duì)象,探討葉酸受體α作為鼻咽咽癌靶向治療和耐藥逆轉(zhuǎn)的一個(gè)新靶點(diǎn)所具有的臨床意義,為開(kāi)展鼻咽咽癌的靶向治療提供實(shí)驗(yàn)依據(jù)。 第一章FOLR1在鼻咽癌中的異常表達(dá)及臨床意義 目的研究人正常鼻咽細(xì)胞與鼻咽癌細(xì)胞中葉酸受體α表達(dá)情況,探討其與腫瘤臨床分期及病理特征的關(guān)系。 方法分別以熒光定量RT-PCR,激光共聚焦、Western-blot方法觀察體外培養(yǎng)的正常鼻咽上皮細(xì)胞NP69和三株鼻咽癌親本細(xì)胞(CNE1、HNE2、5-8F)及其鼻咽癌紫杉醇耐藥細(xì)胞(CNE-1/Taxol、HNE-2/Taxol、5-8F/Taxol)中葉酸受體α的表達(dá)情況,免疫組織化學(xué)法檢測(cè)72例鼻咽癌組織和10例正常鼻咽組織葉酸受體α的表達(dá)情況,分析鼻咽癌組織葉酸受體表達(dá)與臨床病理特征的關(guān)系。 結(jié)果三株鼻咽癌細(xì)胞葉酸受體α表達(dá)均為陽(yáng)性,NP69正常鼻咽上皮細(xì)胞葉酸受體α表達(dá)為陰性。鼻咽癌組織葉酸受體α陽(yáng)性表達(dá)率為86.11%(68/72),正常鼻咽黏膜組織中無(wú)葉酸受體α表達(dá)(0/10),葉酸受體α表達(dá)與患者性別、年齡因素?zé)o相關(guān)性；但臨床分期Ⅲ～Ⅳ期的病例葉酸受體α陽(yáng)性表達(dá)明顯高于Ⅰ-Ⅱ期的病例。 結(jié)論正常鼻咽細(xì)胞和組織中均無(wú)葉酸受體α表達(dá),而鼻咽癌細(xì)胞和組織中廣泛高表達(dá)葉酸受體α,并且與臨床分期成正相關(guān),這為開(kāi)展針對(duì)鼻咽癌的葉酸受體α的靶向治療奠定了基礎(chǔ)。 第二章設(shè)計(jì)、構(gòu)建和篩選靶向FOLR1基因的脫氧核酶 目的根據(jù)FOLR1基因mRNA序列,設(shè)計(jì)、構(gòu)建并篩選靶向特異FOLR1基因的10-23型脫氧核酶,通過(guò)脫氧核酶沉默人鼻咽癌FOLR1基因mRNA,從而降低FOLR1靶基因的蛋白表達(dá),為脫氧核酶應(yīng)用于鼻咽癌基因治療及腫瘤耐藥逆轉(zhuǎn)提供實(shí)驗(yàn)依據(jù)。 方法根據(jù)鼻咽癌FOLR1基因mRNA序列,利用計(jì)算機(jī)軟件RNAdraw1.1b2預(yù)測(cè)FOLR1mRNA的二級(jí)結(jié)構(gòu),通過(guò)脫氧核酶的設(shè)計(jì)原則選取脫氧核酶的催化和切割的位點(diǎn),針對(duì)FOLR1基因設(shè)計(jì)靶向特異性10-23型脫氧核酶。利用熒光定量RT-PCR和Western blot技術(shù)分別在mRNA水平和蛋白水平驗(yàn)證不同DRz對(duì)靶基因FOLR1表達(dá)的影響。 結(jié)果設(shè)計(jì)合成了五條靶向特異FOLR1基因的10-23型脫氧核酶(DRzA、 DRzB、DRzC、DRzD、DRzE)和一條陰性對(duì)照(ODNs),分別轉(zhuǎn)染鼻咽癌紫杉醇耐藥細(xì)胞CNE-1/Taxol,轉(zhuǎn)染24h后利用熒光定量RT-PCR和Western blot技術(shù)分別在mRNA水平和蛋白水平驗(yàn)證了在不同DRz對(duì)靶基因FOLR1表達(dá)的影響,結(jié)果顯示DRzE對(duì)靶基因的抑制作用最強(qiáng),ODNs實(shí)驗(yàn)組不能對(duì)靶基因的表達(dá)產(chǎn)生抑制作用。 結(jié)論在mRNA水平和蛋白質(zhì)水平DRzE對(duì)靶基因FOLR1抑制效果優(yōu)于其它脫氧核酶,這為后續(xù)的實(shí)驗(yàn)奠定了基礎(chǔ)。 第三章脫氧核酶靶向抑制FOLR1基因表達(dá)后耐藥細(xì)胞對(duì)紫杉醇敏感性變化 目的通過(guò)脫氧核酶靶向抑制FOLR1基因表達(dá)后,觀察耐藥細(xì)胞對(duì)紫杉醇敏感性變化。 方法以CNE-1/Taxol為研究對(duì)象,利用DRzE脫氧核酶靶向抑制FOLR1基因表達(dá)后,通過(guò)CCK-8法觀察耐藥細(xì)胞對(duì)紫杉醇敏感性變化,同時(shí)通過(guò)Annexin流式細(xì)胞儀檢測(cè)細(xì)胞凋亡 結(jié)果脫氧核酶DRzE靶向抑制FOLR1基因表達(dá)后,CNE-1/Taxol組細(xì)胞對(duì)紫杉醇的敏感性明顯增強(qiáng)、其敏感性增加了約44%,而陰性對(duì)照組(ODNs組)細(xì)胞對(duì)紫杉醇的敏感性無(wú)明顯改變。脫氧核酶DRzE靶向抑制FOLR1基因表達(dá)后,能明顯增加紫杉醇(IC30:5ng/ml)誘導(dǎo)的細(xì)胞凋亡,其凋亡率從6.09±2.37%增加至23.19±2.01%。 結(jié)論靶向FOLR1的脫氧核酶DRzE能明顯增加鼻咽癌紫杉醇耐藥細(xì)胞對(duì)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63
本文編號(hào)：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63
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