靶向FOLR1的脫氧核酶增加鼻咽癌對紫杉醇敏感性研究
發(fā)布時間：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。盡管化療對改善鼻咽癌治療效果和提高生存率的問題上仍有爭論,但是臨床試驗結果顯示,放療結合化療能明顯改善中晚期鼻咽咽癌的預后,因此,化療仍然是鼻咽癌綜合治療重要手段之一,但是化療藥物的長期使用往往導致腫瘤細胞耐藥,從而導致化療的失敗,而且傳統(tǒng)化療由于缺乏特異性常帶來的較大毒副作用,嚴重影響了患者的生活質量,因此,研究如何逆轉腫瘤耐藥、消減化療所帶來的副反應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的較大毒副作用。葉酸受體α作為靶向治療的一個新靶點在鼻咽癌中的研究較少。本文以鼻咽癌為研究對象,探討葉酸受體α作為鼻咽咽癌靶向治療和耐藥逆轉的一個新靶點所具有的臨床意義,為開展鼻咽咽癌的靶向治療提供實驗依據(jù)。 第一章FOLR1在鼻咽癌中的異常表達及臨床意義 目的研究人正常鼻咽細胞與鼻咽癌細胞中葉酸受體α表達情況,探討其與腫瘤臨床分期及病理特征的關系。 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)的正常鼻咽上皮細胞NP69和三株鼻咽癌親本細胞(CNE1、HNE2、5-8F)及其鼻咽癌紫杉醇耐藥細胞(CNE-1/Taxol、HNE-2/Taxol、5-8F/Taxol)中葉酸受體α的表達情況,免疫組織化學法檢測72例鼻咽癌組織和10例正常鼻咽組織葉酸受體α的表達情況,分析鼻咽癌組織葉酸受體表達與臨床病理特征的關系。 結果三株鼻咽癌細胞葉酸受體α表達均為陽性,NP69正常鼻咽上皮細胞葉酸受體α表達為陰性。鼻咽癌組織葉酸受體α陽性表達率為86.11%(68/72),正常鼻咽黏膜組織中無葉酸受體α表達(0/10),葉酸受體α表達與患者性別、年齡因素無相關性；但臨床分期Ⅲ～Ⅳ期的病例葉酸受體α陽性表達明顯高于Ⅰ-Ⅱ期的病例。 結論正常鼻咽細胞和組織中均無葉酸受體α表達,而鼻咽癌細胞和組織中廣泛高表達葉酸受體α,并且與臨床分期成正相關,這為開展針對鼻咽癌的葉酸受體α的靶向治療奠定了基礎。 第二章設計、構建和篩選靶向FOLR1基因的脫氧核酶 目的根據(jù)FOLR1基因mRNA序列,設計、構建并篩選靶向特異FOLR1基因的10-23型脫氧核酶,通過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,為脫氧核酶應用于鼻咽癌基因治療及腫瘤耐藥逆轉提供實驗依據(jù)。 方法根據(jù)鼻咽癌FOLR1基因mRNA序列,利用計算機軟件RNAdraw1.1b2預測FOLR1mRNA的二級結構,通過脫氧核酶的設計原則選取脫氧核酶的催化和切割的位點,針對FOLR1基因設計靶向特異性10-23型脫氧核酶。利用熒光定量RT-PCR和Western blot技術分別在mRNA水平和蛋白水平驗證不同DRz對靶基因FOLR1表達的影響。 結果設計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)和一條陰性對照(ODNs),分別轉染鼻咽癌紫杉醇耐藥細胞CNE-1/Taxol,轉染24h后利用熒光定量RT-PCR和Western blot技術分別在mRNA水平和蛋白水平驗證了在不同DRz對靶基因FOLR1表達的影響,結果顯示DRzE對靶基因的抑制作用最強,ODNs實驗組不能對靶基因的表達產生抑制作用。 結論在mRNA水平和蛋白質水平DRzE對靶基因FOLR1抑制效果優(yōu)于其它脫氧核酶,這為后續(xù)的實驗奠定了基礎。 第三章脫氧核酶靶向抑制FOLR1基因表達后耐藥細胞對紫杉醇敏感性變化 目的通過脫氧核酶靶向抑制FOLR1基因表達后,觀察耐藥細胞對紫杉醇敏感性變化。 方法以CNE-1/Taxol為研究對象,利用DRzE脫氧核酶靶向抑制FOLR1基因表達后,通過CCK-8法觀察耐藥細胞對紫杉醇敏感性變化,同時通過Annexin流式細胞儀檢測細胞凋亡 結果脫氧核酶DRzE靶向抑制FOLR1基因表達后,CNE-1/Taxol組細胞對紫杉醇的敏感性明顯增強、其敏感性增加了約44%,而陰性對照組(ODNs組)細胞對紫杉醇的敏感性無明顯改變。脫氧核酶DRzE靶向抑制FOLR1基因表達后,能明顯增加紫杉醇(IC30:5ng/ml)誘導的細胞凋亡,其凋亡率從6.09±2.37%增加至23.19±2.01%。 結論靶向FOLR1的脫氧核酶DRzE能明顯增加鼻咽癌紫杉醇耐藥細胞對紫杉醇的敏感性。
[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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R739.63
本文編號：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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R739.63
【參考文獻】
相關期刊論文 前7條
1 張宏征;謝民強;張濤;陳帥君;;葉酸-順鉑納米藥物的制備及其對人鼻咽癌細胞的靶向治療作用[J];實用醫(yī)學雜志;2011年10期
2 趙家明,李明意,楊展,吳柱國,李震,張毅;脫氧核酶對鼻咽癌細胞端粒酶活性的抑制作用[J];華中科技大學學報(醫(yī)學版);2005年03期
3 曾宗淵,夏良平;鼻咽癌治療新進展[J];腫瘤學雜志;2003年01期
4 許瑞蓮,陳偉,葉建增,白樺,鄭瑾;紫杉醇與順鉑聯(lián)合治療晚期鼻咽癌50例分析[J];腫瘤學雜志;2004年04期
5 彭瑋丹,張杰,曹云新,惠宏襄,金明,王成濟;紫杉醇誘導食管癌細胞的細胞周期阻斷與細胞凋亡[J];中國藥理學通報;1998年05期
6 閔華慶,洪明晃,郭翔,錢朝南;鼻咽癌研究進展[J];中國腫瘤;1999年12期
7 黃騰波;鼻咽癌早期診斷研究進展[J];中國腫瘤;1997年07期
相關碩士學位論文 前1條
1 劉潔;葉酸靶向載順鉑磁性納米藥物治療喉癌的體外實驗研究[D];南方醫(yī)科大學;2011年
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