本文選題：短發(fā)夾RNA + 表皮生長因子受體��； 參考：《南方醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 鼻咽癌(Nasopharyngeal carcinoma, NPC)是發(fā)生于鼻咽粘膜的一種惡性腫瘤,高發(fā)于我國南方及東南亞地區(qū)。盡管關(guān)于鼻咽癌的研究取得了極大的進展,但是其5年生存率仍徘徊于50%-60%。 放射治療一直是鼻咽癌治療的首選方法,原因是多數(shù)鼻咽癌為低分化癌,對放射線的敏感性高,并且原發(fā)灶和頸部淋巴引流區(qū)域容易被包括在照射野內(nèi)。鼻咽癌患者治療失敗的原因主要為局部復(fù)發(fā)或遠處轉(zhuǎn)移,嚴重影響鼻咽癌治療生存率的提高。放射治療是一個局部或區(qū)域治療的手段,提高放射治療的療效不僅可以提高局部或區(qū)域控制率,同時也可影響生存及遠地轉(zhuǎn)移。 放射治療是一把雙刃劍,在利用放射線治療腫瘤的同時,不可避免地會造成正常組織一定程度的放射損傷。因此,如何增加腫瘤組織的放射敏感性是改善放射治療療效的關(guān)鍵。放射敏感性是組織對一定量射線的反應(yīng)程度。腫瘤的放射敏感性與腫瘤組織的來源、分化程度、病理類型、瘤床、貧血、局部是否合并感染、生活指數(shù)等有關(guān)。放射增敏一直是放射腫瘤界最為活躍的研究領(lǐng)域。理想的放射增敏劑應(yīng)該依據(jù)腫瘤細胞與正常細胞的差異,特異地作用于腫瘤細胞,從而達到在放射增敏的同時,對機體正常組織無毒或低毒的目的。如何提高放射治療的療效,提高放射敏感性一直困擾著腫瘤醫(yī)師。 表皮生長因子受體(Eepidermal Growth Factor Receptor, EGFR)是一個分子量為170kD的具有酪氨酸激酶活性的受體糖蛋白,廣泛分布于正常的哺乳動物上皮細胞表面,平均每個細胞受體個數(shù)為5×104-10×104。EGFR通常與其相應(yīng)的配體(EGF、TGF-α等)相結(jié)合,使受體自身磷酸化,可激活下游多種信號轉(zhuǎn)導(dǎo)通路,從而引起胞內(nèi)一系列信號的級聯(lián)放大及傳遞,最終影響核內(nèi)基因的表達,導(dǎo)致細胞的增殖與分化。繼Hendler等首次檢測出非小細胞肺癌細胞上有EGFR超表達以后,人們相繼在多種實體瘤如乳腺癌、結(jié)直腸癌、頭頸部腫瘤、腦瘤、卵巢癌、膀胱癌、腎癌中均檢到EGFR的超表達或擴增。 以EGFR為靶點的分子靶向治療的放射增敏作用的理論基礎(chǔ)包括：1.EGFR能夠抑制放射線引起的腫瘤細胞凋亡；2.放射治療誘發(fā)的腫瘤細胞再增殖依賴于EGFR;3.放療通過激活EGFR而使其下游基因高表達,加速細胞的增殖；4.放療通過激活EGFR可能誘導(dǎo)腫瘤血管的生成；5.EGFR可能促進腫瘤細胞因放療引起的潛在致死性損傷和亞致死性損傷的修復(fù),從而引起細胞的放射抗拒。 據(jù)報道在鼻咽癌患者瘤體中,EGFR陽性表達率高達80%-100%,其中超過25%的腫瘤細胞EGFR陽性表達患者表現(xiàn)為低分化、局部控制率低下和遠處轉(zhuǎn)移,并且腫瘤分化程度越低EGFR陽性表達率越高；伴有頸淋巴結(jié)轉(zhuǎn)移者更高。臨床數(shù)據(jù)已經(jīng)顯示EGFR的單克隆抗體cetuximab聯(lián)合不同化療方案治療頭頸部腫瘤具有可行性和有效性,但是由于個體差異而導(dǎo)致的治療失敗以及藥物耐受性的形成,仍然需要我們?nèi)で笠訣GFR為靶點的進一步治療方法。 RNA干擾(RNAi, RNA interference)作為一種實用的分子生物學(xué)技術(shù)近年來發(fā)展非常迅速，，siRNA介導(dǎo)的瞬時或穩(wěn)定的基因沉默有高度的特異性并且無副作用。在實用性上利用RNAi不僅能提供一種經(jīng)濟、快捷、高效的抑制基因表達的技術(shù)手段,而且有可能在基因功能測定和基因治療等方面開辟一條新思路。 近年來,裸鼠已成為醫(yī)學(xué)生物學(xué)研究領(lǐng)域中不可缺少的實驗動物模型。人體腫瘤移植于免疫缺陷動物用于研究其對藥物的敏感性有很大的幫助。移植于裸鼠的人類惡性腫瘤具有以下特征：1.被移植腫瘤仍保持原有組織學(xué)構(gòu)造或各種機能。2.將人癌組織的組織培養(yǎng)物移種于裸鼠時,能重現(xiàn)已在組織培養(yǎng)中消失的原有的癌結(jié)構(gòu)。3.幾乎未發(fā)現(xiàn)被移植腫瘤的轉(zhuǎn)移。 本研究采用體外化學(xué)合成RNA干擾EGFR基因表達載體shRNA-EGFR向人鼻咽癌細胞裸鼠移植瘤模型的瘤內(nèi)注射后行瘤體放射治療,觀察瘤體對放射敏感性的影響,以期為腫瘤基因放射治療提供實驗數(shù)據(jù)和理論依據(jù)。 目的：構(gòu)建EGFR shRNA表達載體,完成轉(zhuǎn)染和鑒定；檢測轉(zhuǎn)染后細胞EGFR的表達。 方法：1.除轉(zhuǎn)染EGFR干擾質(zhì)粒外,還以lipofectimne2000組和轉(zhuǎn)染無義質(zhì)粒組作為對照,實驗分為4組：對照組(未加任何處理)、Lipofectamine組、shNC組和shRNA-EGFR組；2.Real-time PCR檢測轉(zhuǎn)染前后及轉(zhuǎn)染不同質(zhì)粒后細胞EGFR mRNA的表達；3.Western blot檢測轉(zhuǎn)染不同質(zhì)粒后EGFR蛋白質(zhì)的表達。 結(jié)果：1.成功地構(gòu)建了shRNA-EGFR表達質(zhì)粒并且得到了較高的轉(zhuǎn)染率。2. Real-time PCR檢測CNE1、CNE2細胞中EGFR mRNA的表達：CNE1細胞中轉(zhuǎn)染shRNA-EGFR組與對照組、Lipofectamine組和shNC組比較EGFR mRNA的表達顯著降低。統(tǒng)計學(xué)分析：CNE1細胞轉(zhuǎn)染shRNA-EGFR組與轉(zhuǎn)染shNC組比較有統(tǒng)計學(xué)差異(P0.001)；轉(zhuǎn)染shRNA-EGFR組與lipofectimne2000組比較也有統(tǒng)計學(xué)差異(P0.001)；轉(zhuǎn)染shRNA-EGFR組：轉(zhuǎn)染后48小時與轉(zhuǎn)染前(即CNE1)比較有統(tǒng)計學(xué)差異(P0.001)。CNE2細胞中轉(zhuǎn)染shRNA-EGFR組與對照組、Lipofectamine組和shNC組比較EGFR mRNA的表達顯著降低。統(tǒng)計學(xué)分析：CNE2細胞轉(zhuǎn)染shRNA-EGFR組與轉(zhuǎn)染shNC組比較有統(tǒng)計學(xué)差異(P0.001)；轉(zhuǎn)染shRNA-EGFR組與lipofectimne2000組比較也有統(tǒng)計學(xué)差異(P0.001)；轉(zhuǎn)染shRNA-EGFR組：轉(zhuǎn)染后48小時與轉(zhuǎn)染前(即CNE2)比較有統(tǒng)計學(xué)差異(P0.001)。3.Western blotting結(jié)果示CNE1、CNE2細胞轉(zhuǎn)染shRNA-EGFR后與其他處理組相比蛋白質(zhì)的表達明顯降低。 結(jié)論：成功地構(gòu)建了shRNA-EGFR表達載體,shRNA-EGFR對CNE1、CNE2細胞中EGFR的表達具有明顯的抑制效果,為進一步在裸鼠人鼻咽癌細胞移植瘤中的應(yīng)用提供實驗依據(jù)。 目的：研究RNA干擾EGFR后鼻咽癌裸鼠移植瘤放射敏感性的變化。 方法：1.用CNE1、CNE2細胞株建立裸鼠鼻咽癌移植瘤模型；2.待腫瘤長徑達到6mm-8mm時,將32個符合條件的瘤體隨機分為4組,每組8個：對照組(未加任何處理)、單純放射治療組(瘤體局部照射20Gy)、shRNA-EGFR治療組(瘤內(nèi)注射shRNA-EGFR質(zhì)粒)和shRNA-EGFR聯(lián)合放射治療組(瘤內(nèi)注射shRNA-EGFR質(zhì)粒24h后瘤體局部照射20Gy)。3.測量各組裸鼠瘤體的體積和瘤體質(zhì)量,觀察各試驗組之間的差異。 結(jié)果：1.成功地構(gòu)建了裸鼠鼻咽癌移植瘤的模型；2.裸鼠移植瘤體積變化：shRNA-EGFR聯(lián)合放射治療組與對照組、單純放射治療組和shRNA-EGFR治療組相比,均有顯著性差異(P0.01)。各試驗組裸鼠移植瘤剝離后稱質(zhì)量：shRNA-EGFR聯(lián)合放射治療組與對照組、單純放射治療組、shRNA-EGFR治療組相比,均有顯著性差異(P0.01)。 結(jié)論：應(yīng)用X線放射治療裸鼠移植瘤,在經(jīng)過shRNA-EGFR表達載體抑制裸鼠移植瘤細胞中EGFR的表達后,能夠顯著抑制瘤體生長。shRNA-EGFR表達載體增加了腫瘤對放射治療的敏感性。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R739.63

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相關(guān)期刊論文 前1條

1 翁德勝;吳正蓉;王爽;丁彥青;;RNAi抑制鼻咽癌細胞表皮生長因子受體表達對細胞生長的影響[J];南方醫(yī)科大學(xué)學(xué)報;2006年01期

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