RNA干扰抑制表皮生长因子受体在鼻咽癌细胞中的表达对放射敏感性的影响

发布时间：2018-05-02 19:31

本文选题：短发夹RNA + 表皮生长因子受体　；参考：《南方医科大学》2010年硕士论文

【摘要】： 鼻咽癌(Nasopharyngeal carcinoma, NPC)是发生于鼻咽粘膜的一种恶性肿瘤,高发于我国南方及东南亚地区。尽管关于鼻咽癌的研究取得了极大的进展,但是其5年生存率仍徘徊于50%-60%。放射治疗一直是鼻咽癌治疗的首选方法,原因是多数鼻咽癌为低分化癌,对放射线的敏感性高,并且原发灶和颈部淋巴引流区域容易被包括在照射野内。鼻咽癌患者治疗失败的原因主要为局部复发或远处转移,严重影响鼻咽癌治疗生存率的提高。放射治疗是一个局部或区域治疗的手段,提高放射治疗的疗效不仅可以提高局部或区域控制率,同时也可影响生存及远地转移。放射治疗是一把双刃剑,在利用放射线治疗肿瘤的同时,不可避免地会造成正常组织一定程度的放射损伤。因此,如何增加肿瘤组织的放射敏感性是改善放射治疗疗效的关键。放射敏感性是组织对一定量射线的反应程度。肿瘤的放射敏感性与肿瘤组织的来源、分化程度、病理类型、瘤床、贫血、局部是否合并感染、生活指数等有关。放射增敏一直是放射肿瘤界最为活跃的研究领域。理想的放射增敏剂应该依据肿瘤细胞与正常细胞的差异,特异地作用于肿瘤细胞,从而达到在放射增敏的同时,对机体正常组织无毒或低毒的目的。如何提高放射治疗的疗效,提高放射敏感性一直困扰着肿瘤医师。表皮生长因子受体(Eepidermal Growth Factor Receptor, EGFR)是一个分子量为170kD的具有酪氨酸激酶活性的受体糖蛋白,广泛分布于正常的哺乳动物上皮细胞表面,平均每个细胞受体个数为5×104-10×104。EGFR通常与其相应的配体(EGF、TGF-α等)相结合,使受体自身磷酸化,可激活下游多种信号转导通路,从而引起胞内一系列信号的级联放大及传递,最终影响核内基因的表达,导致细胞的增殖与分化。继Hendler等首次检测出非小细胞肺癌细胞上有EGFR超表达以后,人们相继在多种实体瘤如乳腺癌、结直肠癌、头颈部肿瘤、脑瘤、卵巢癌、膀胱癌、肾癌中均检到EGFR的超表达或扩增。以EGFR为靶点的分子靶向治疗的放射增敏作用的理论基础包括：1.EGFR能够抑制放射线引起的肿瘤细胞凋亡；2.放射治疗诱发的肿瘤细胞再增殖依赖于EGFR;3.放疗通过激活EGFR而使其下游基因高表达,加速细胞的增殖；4.放疗通过激活EGFR可能诱导肿瘤血管的生成；5.EGFR可能促进肿瘤细胞因放疗引起的潜在致死性损伤和亚致死性损伤的修复,从而引起细胞的放射抗拒。据报道在鼻咽癌患者瘤体中,EGFR阳性表达率高达80%-100%,其中超过25%的肿瘤细胞EGFR阳性表达患者表现为低分化、局部控制率低下和远处转移,并且肿瘤分化程度越低EGFR阳性表达率越高；伴有颈淋巴结转移者更高。临床数据已经显示EGFR的单克隆抗体cetuximab联合不同化疗方案治疗头颈部肿瘤具有可行性和有效性,但是由于个体差异而导致的治疗失败以及药物耐受性的形成,仍然需要我们去寻求以EGFR为靶点的进一步治疗方法。 RNA干扰(RNAi, RNA interference)作为一种实用的分子生物学技术近年来发展非常迅速，，siRNA介导的瞬时或稳定的基因沉默有高度的特异性并且无副作用。在实用性上利用RNAi不仅能提供一种经济、快捷、高效的抑制基因表达的技术手段,而且有可能在基因功能测定和基因治疗等方面开辟一条新思路。近年来,裸鼠已成为医学生物学研究领域中不可缺少的实验动物模型。人体肿瘤移植于免疫缺陷动物用于研究其对药物的敏感性有很大的帮助。移植于裸鼠的人类恶性肿瘤具有以下特征：1.被移植肿瘤仍保持原有组织学构造或各种机能。2.将人癌组织的组织培养物移种于裸鼠时,能重现已在组织培养中消失的原有的癌结构。3.几乎未发现被移植肿瘤的转移。本研究采用体外化学合成RNA干扰EGFR基因表达载体shRNA-EGFR向人鼻咽癌细胞裸鼠移植瘤模型的瘤内注射后行瘤体放射治疗,观察瘤体对放射敏感性的影响,以期为肿瘤基因放射治疗提供实验数据和理论依据。目的：构建EGFR shRNA表达载体,完成转染和鉴定；检测转染后细胞EGFR的表达。方法：1.除转染EGFR干扰质粒外,还以lipofectimne2000组和转染无义质粒组作为对照,实验分为4组：对照组(未加任何处理)、Lipofectamine组、shNC组和shRNA-EGFR组；2.Real-time PCR检测转染前后及转染不同质粒后细胞EGFR mRNA的表达；3.Western blot检测转染不同质粒后EGFR蛋白质的表达。结果：1.成功地构建了shRNA-EGFR表达质粒并且得到了较高的转染率。2. Real-time PCR检测CNE1、CNE2细胞中EGFR mRNA的表达：CNE1细胞中转染shRNA-EGFR组与对照组、Lipofectamine组和shNC组比较EGFR mRNA的表达显著降低。统计学分析：CNE1细胞转染shRNA-EGFR组与转染shNC组比较有统计学差异(P0.001)；转染shRNA-EGFR组与lipofectimne2000组比较也有统计学差异(P0.001)；转染shRNA-EGFR组：转染后48小时与转染前(即CNE1)比较有统计学差异(P0.001)。CNE2细胞中转染shRNA-EGFR组与对照组、Lipofectamine组和shNC组比较EGFR mRNA的表达显著降低。统计学分析：CNE2细胞转染shRNA-EGFR组与转染shNC组比较有统计学差异(P0.001)；转染shRNA-EGFR组与lipofectimne2000组比较也有统计学差异(P0.001)；转染shRNA-EGFR组：转染后48小时与转染前(即CNE2)比较有统计学差异(P0.001)。3.Western blotting结果示CNE1、CNE2细胞转染shRNA-EGFR后与其他处理组相比蛋白质的表达明显降低。结论：成功地构建了shRNA-EGFR表达载体,shRNA-EGFR对CNE1、CNE2细胞中EGFR的表达具有明显的抑制效果,为进一步在裸鼠人鼻咽癌细胞移植瘤中的应用提供实验依据。目的：研究RNA干扰EGFR后鼻咽癌裸鼠移植瘤放射敏感性的变化。方法：1.用CNE1、CNE2细胞株建立裸鼠鼻咽癌移植瘤模型；2.待肿瘤长径达到6mm-8mm时,将32个符合条件的瘤体随机分为4组,每组8个：对照组(未加任何处理)、单纯放射治疗组(瘤体局部照射20Gy)、shRNA-EGFR治疗组(瘤内注射shRNA-EGFR质粒)和shRNA-EGFR联合放射治疗组(瘤内注射shRNA-EGFR质粒24h后瘤体局部照射20Gy)。3.测量各组裸鼠瘤体的体积和瘤体质量,观察各试验组之间的差异。结果：1.成功地构建了裸鼠鼻咽癌移植瘤的模型；2.裸鼠移植瘤体积变化：shRNA-EGFR联合放射治疗组与对照组、单纯放射治疗组和shRNA-EGFR治疗组相比,均有显著性差异(P0.01)。各试验组裸鼠移植瘤剥离后称质量：shRNA-EGFR联合放射治疗组与对照组、单纯放射治疗组、shRNA-EGFR治疗组相比,均有显著性差异(P0.01)。结论：应用X线放射治疗裸鼠移植瘤,在经过shRNA-EGFR表达载体抑制裸鼠移植瘤细胞中EGFR的表达后,能够显著抑制瘤体生长。shRNA-EGFR表达载体增加了肿瘤对放射治疗的敏感性。
[Abstract]:Nasopharyngeal carcinoma (NPC) is a malignant tumor occurring in the nasopharyngeal mucosa, which is high in South and Southeast Asia. Although the study of nasopharyngeal carcinoma has made great progress, its 5 year survival rate still lingers in 50%-60%.
Radiotherapy is the first choice for the treatment of nasopharyngeal carcinoma because most nasopharyngeal carcinoma is a low differentiated carcinoma with high sensitivity to radiation, and the primary and cervical lymphatic drainage areas are easily included in the field. The main cause of the failure of nasopharyngeal cancer patients is local relapse or distant metastasis, which seriously affects the survival of nasopharyngeal carcinoma for the treatment of survival. Increase in rate. Radiotherapy is a local or regional treatment. Improving the efficacy of radiation therapy can not only improve local or regional control rate, but also affect survival and distant metastasis.
Radiation therapy is a double-edged sword. It is unavoidable to cause a certain degree of radiation damage to normal tissues while using radiation to treat tumors. Therefore, how to increase the radiosensitivity of tumor tissue is the key to improve the therapeutic effect of radiation therapy. Sensibility is related to the origin of the tumor tissue, the degree of differentiation, the pathological type, the tumor bed, anaemia, the local combination of infection and the life index. Radiosensitization has been the most active research field in the radiological field. The ideal radiosensitizer should be based on the difference between the tumor cells and the normal cells and specifically act on the tumor cells, thus achieving a specific effect on the tumor cells. At the same time of radiosensitization, it is nontoxic or low toxic to the normal tissue of the body. How to improve the curative effect of radiotherapy and improve the radiosensitivity have been puzzling the oncologist.
The epidermal growth factor receptor (Eepidermal Growth Factor Receptor, EGFR) is a receptor glycoprotein with a molecular weight of 170kD, with tyrosine kinase activity. It is widely distributed in normal mammalian epithelial cells. The average number of each cell receptor is 5 * 104-10 x 104.EGFR, which is usually combined with its corresponding ligand (EGF, TGF- alpha). The phosphorylation of the receptor itself can activate a variety of downstream signal transduction pathways, thereby causing cascade amplification and transmission of a series of intracellular signals, and ultimately affecting the expression of genes in the nucleus, resulting in cell proliferation and differentiation. After the first detection of EGFR overexpression on non small cell lung cancer cells, people have been in a variety of solid tumors, such as Hendler. Over expression or amplification of EGFR was detected in breast cancer, colorectal cancer, head and neck tumors, brain tumors, ovarian cancer, bladder cancer, and renal cell carcinoma.
The theoretical basis for the radiosensitization of molecular targeted therapy targeting EGFR includes: 1.EGFR can inhibit the apoptosis of tumor cells induced by radiation; 2. the proliferation of tumor cells induced by radiation therapy depends on EGFR; 3. the downstream genes are highly expressed by activating EGFR and the proliferation of the cells is accelerated by activating EGFR; 4. radiation is activated by EGF R may induce the formation of tumor vessels, and 5.EGFR may promote the potential fatal injury and the repair of sublethal injury caused by radiotherapy, which may cause the radiation resistance of the cells.
It is reported that the positive expression rate of EGFR is as high as 80%-100% in the tumor body of patients with nasopharyngeal carcinoma, of which more than 25% of the tumor cells with EGFR positive expression are characterized by low differentiation, low local control rate and distant metastasis, and the higher the EGFR positive expression rate is, the higher the tumor differentiation degree, the higher the positive rate of the cervical lymph node metastasis. The clinical data have shown EGFR The monoclonal antibody cetuximab combined with different chemotherapy regimens is feasible and effective in the treatment of head and neck tumors. However, the failure of treatment and the formation of drug tolerance due to individual differences still require us to seek further treatment with EGFR as a target.
RNA interference (RNAi, RNA interference), as a practical molecular biology technology, has developed very rapidly in recent years. SiRNA mediated transient or stable gene silencing has high specificity and no side effects. In practice, the use of RNAi can not only provide an economic, fast and efficient technique for inhibiting gene expression, but also can be used as a technical means of inhibiting gene expression. It may open up a new idea in gene function determination and gene therapy.
In recent years, nude mice have become an indispensable experimental animal model in the field of medical biology. Human tumor transplantation in immune deficient animals is of great help to the study of drug sensitivity. Human malignant tumors transplanted in nude mice have the following characteristics: 1. the transplanted tumor remains the original histology structure or various kinds of machines. When the tissue culture of human cancer tissue is moved to nude mice by.2., the original cancerous structure,.3., which has disappeared in tissue culture, has been found to have hardly found the metastasis of the transplanted tumor.
In this study, in vitro chemical synthesis of RNA interfering EGFR gene expression vector shRNA-EGFR was injected into the tumor model of nude mice with nasopharyngeal carcinoma cells after intratumoral radiation therapy, and the effects of tumor body on radiosensitivity were observed in order to provide experimental data and theoretical basis for tumor gene radiotherapy.
Objective: to construct the expression vector of EGFR shRNA, to complete the transfection and identification, and to detect the expression of EGFR after transfection.
Methods: 1. in addition to transfection of EGFR plasmid, lipofectimne2000 group and transfected non sense plasmid group were also used as control. The experiment was divided into 4 groups: control group (without any treatment), Lipofectamine group, shNC group and shRNA-EGFR group; 2.Real-time PCR was used to detect the expression of EGFR mRNA after transfection and transfection of different plasmids; 3.Western blot detection. Expression of EGFR protein after transfection of different plasmids.
Results: 1. the expression plasmid of shRNA-EGFR was successfully constructed and the high transfection rate was obtained by.2. Real-time PCR. The expression of EGFR mRNA in CNE2 cells: the expression of shRNA-EGFR group and the control group in CNE1 cells, Lipofectamine group and shNC group. There was a statistically significant difference between the group and the transfected shNC group (P0.001), and there was a statistical difference between the transfected shRNA-EGFR group and the lipofectimne2000 group (P0.001), and the transfected shRNA-EGFR group: 48 hours after transfection and before transfection (CNE1), there was a statistically significant difference (P0.001) in.CNE2 cells transfected to the shRNA-EGFR group and the control group, Lipofectamine group and shNC group. The expression of EGFR mRNA was significantly reduced. Statistical analysis: there was a statistically significant difference between the transfected shRNA-EGFR group and the transfected shNC group (P0.001), and there was a statistical difference (P0.001) in the transfected shRNA-EGFR group with the lipofectimne2000 group (P0.001), and the transfected shRNA-EGFR group: 48 hours after transfection, there was a statistically significant difference between the transfected and before the transfection (i.e. CNE2). P0.001).3.Western blotting results showed that CNE1 and CNE2 cells transfected shRNA-EGFR significantly reduced protein expression compared with other treatment groups.
Conclusion: the expression vector of shRNA-EGFR has been successfully constructed, and shRNA-EGFR has obvious inhibitory effect on the expression of EGFR in CNE1 and CNE2 cells. It provides an experimental basis for further application of the human nasopharyngeal carcinoma cell xenografts in nude mice.
Objective: To study the radiosensitivity of nasopharyngeal carcinoma (NPC) xenografts in nude mice after EGFR interference with RNA.
Methods: 1. CNE1 and CNE2 cell lines were used to establish nude mice model of nasopharyngeal carcinoma transplantation tumor; 2. when the length of the tumor reached 6mm-8mm, 32 conforming tumor bodies were randomly divided into 4 groups, 8 groups in each group: the control group (without any treatment), the simple radiotherapy group (the tumor body locally irradiated 20Gy), the shRNA-EGFR treatment group (intratumor injected shRNA-EGFR plasmid) and shRNA-E GFR combined with radiation therapy group (intratumoral injection of shRNA-EGFR plasmid 24h after local irradiation of 20Gy).3. was used to measure the volume and mass of tumor body of nude mice, and the difference between the experimental groups was observed.
Results: 1. the model of nude mice nasopharyngeal carcinoma transplantation tumor was successfully constructed; 2. the volume changes of xenograft tumor in nude mice: shRNA-EGFR combined with radiation therapy group and control group, compared with shRNA-EGFR treatment group, there were significant differences (P0.01). The mass of xenograft in nude mice in the experimental group: shRNA-EGFR combined with radiotherapy group. Compared with the control group, radiotherapy alone group and shRNA-EGFR treatment group, there was significant difference (P0.01).
Conclusion: the use of shRNA-EGFR expression vector to inhibit the expression of EGFR in nude mice transplanted tumor cells can significantly inhibit the growth of.ShRNA-EGFR expression vector of the tumor and increase the sensitivity of tumor to radiation therapy after the use of X-ray radiation therapy in nude mice.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.63

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