本文選題：喉癌　切入點：IL-24　出處：《濟南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的喉癌是耳鼻咽喉科常見的惡性腫瘤,約90%以上是鱗狀細胞癌。喉癌的發(fā)病率在近幾年有上升的趨勢,這與診斷水平的提高、生活習(xí)慣改變以及空氣污染都有關(guān)系。統(tǒng)計數(shù)據(jù)顯示:喉癌的發(fā)病率在城市高于農(nóng)村,在重工業(yè)發(fā)達的城市高于輕工業(yè)發(fā)達城市。手術(shù)治療是喉癌最常用的治療手段,隨著頭頸外科的發(fā)展,喉外科手術(shù)不斷完善,患者的5年生存率及生存質(zhì)量明顯提高。但仍有30%-40%的患者,在經(jīng)過根治手術(shù)和放化療治療后復(fù)發(fā)或轉(zhuǎn)移。當(dāng)出現(xiàn)復(fù)發(fā)和/或遠處轉(zhuǎn)移時患者對放化療多不敏感,3年生存率僅為10%。因此如何進一步提高喉癌的療效成為當(dāng)務(wù)之急。隨著分子生物學(xué)和基因工程的飛速發(fā)展,基因免疫治療日益受到重視,顯示出良好的發(fā)展前景。 白細胞介素24,又名黑色素瘤分化相關(guān)抗原7(melanoma differentiation associated antigen7,MDA-7),它主要由CD3~+T淋巴細胞和單核細胞表達,為分泌型蛋白,屬于IL-10家族成員。研究顯示IL-24是目前發(fā)現(xiàn)唯一一種既具有抑制腫瘤細胞生長和血管形成又具有免疫刺激作用的細胞因子。這種抑制作用并不依賴p53、Rb和p16等抑癌基因,對正常細胞幾乎無影響�，F(xiàn)已成為腫瘤治療研究熱點。 mda-7/IL-24對多種腫瘤細胞有抑制作用,但尚不清楚它對喉癌細胞是否具有相似的作用。為此我們對其進行了克隆,通過腺病毒介導(dǎo)進行體外過量表達,觀察對喉癌細胞Hep2增殖的影響并對作用機制進行初步探討。具體工作如下: 方法: 1.利用PHA刺激培養(yǎng)人外周血淋巴細胞使其表達IL-24基因,采用RT-PCR方法得到mda-7/IL-24編碼的cDNA。構(gòu)建T克隆載體PEasy-T1 Simple-IL-24。 2.從T克隆載體PEasy-T1 Simple-IL-24中大量擴增mda-7/IL-24,酶切回收目的片段插入到pAdTrack-CMV穿梭質(zhì)粒中,與pAdEasy骨架質(zhì)粒在E.coliBJ5183中進行同源重組為腺病毒載體質(zhì)粒,線形化后轉(zhuǎn)染293A細胞進行包裝。TCID_(50)方法測得病毒效價、PCR和Western-blot鑒定重組腺病毒Ad-mda-7/IL-24。3. Ad-mda-7/IL-24感染喉癌細胞及相關(guān)生物學(xué)檢測:Ad-mda-7/IL-24和Adv分別感染人喉癌細胞Hep2,MTT觀察Ad-mda-7/IL-24對人喉癌細胞Hep2的增殖影響,RT-PCR法檢測感染后相關(guān)基因mRNA表達情況,Western-blot檢測感染后相關(guān)蛋白表達。 結(jié)果: 1.通過RT-PCR從PHA刺激培養(yǎng)的人外周血淋巴細胞中克隆出mda-7/ IL-24編碼cDNA。構(gòu)建T克隆載體PEasy-T1 Simple-IL-24成功。經(jīng)酶切和測序驗證為正確的IL-24 cDNA。 2.經(jīng)基因重組技術(shù)獲得腺病毒表達相關(guān)的一系列載體,穿梭質(zhì)粒pAdTrack- CMV-mda-7/IL-24、含目的基因的腺病毒質(zhì)粒pAd-mda-7/IL-24和空載體腺病毒質(zhì)粒pAd,經(jīng)酶切和測序驗證正確。成功構(gòu)建了腺病毒表達系列載體。質(zhì)粒線性化后經(jīng)脂質(zhì)體法轉(zhuǎn)染293A細胞后,能夠在熒光顯微鏡下發(fā)出綠色熒光,RT-PCR檢測出IL-24 mRNA的表達,Western Blot鑒定有目的蛋白IL-24的表達。 3.Ad-mda-7/IL-24感染Hep2細胞后對Hep2細胞的增殖活性有較強抑制作用;凋亡相關(guān)分子BAX、Caspase-3表達有明顯增強,BCL-2表達則下降。 結(jié)論: 腫瘤治療的主要目的就是抑制腫瘤的生長和改善患者的預(yù)后,基礎(chǔ)研究的首要任務(wù)則為闡明腫瘤的發(fā)生機制以及腫瘤治療基因的功能機制。本研究通過克隆腫瘤抑制基因mda-7/IL-24,并構(gòu)建重組腺病毒載體包裝含目的基因的腺病毒以揭示其過表達對喉癌細胞的作用,并對其作用機制進行初步探討。結(jié)果顯示,成功構(gòu)建了含目的基因IL-24的腺病毒表達載體,并且病毒包裝成功。證明IL-24對喉癌細胞Hep2的增殖有較明顯的抑制作用,這種抑制作用有可能是通過改變凋亡基因與抗凋亡基因比例來實現(xiàn)的。這為進一步研究IL-24對喉癌的抑制作用機制以及喉癌的臨床基因治療提供了理論和技術(shù)依據(jù)。
[Abstract]:Background and objective: laryngeal carcinoma is a common malignant tumor in Otolaryngology, more than 90% are squamous cell carcinoma. The incidence is a rising trend in recent years, which improve the level of diagnosis and have a relationship, changes in lifestyle and air pollution. Statistics show that the incidence of cancer in the city than in rural areas that is higher than the light industry developed city in heavy industry city. Surgical treatment is the most commonly used method for the treatment of laryngeal carcinoma, with the development of head and neck surgery, laryngeal surgery continues to improve, with a 5 year survival rate and quality of life improved. But there are still 30%-40% patients after radical surgery and chemotherapy treatment after recurrence when the recurrence or metastasis. And / or distant metastasis patients on chemotherapy is not sensitive, 3 year survival rate is only 10%., so how to further improve the efficacy of laryngeal cancer. With the development of molecular biology and has become a pressing matter of the moment matrix Because of the rapid development of the project, the gene immunotherapy has been paid more and more attention, showing a good prospect of development.
Interleukin 24, also known as melanoma differentiation associated antigen 7 (melanoma differentiation associated antigen7, MDA-7), which is mainly composed of CD3~+T lymphocytes and monocytes expressed as a secretory protein, which belongs to the IL-10 family members. Research shows that IL-24 is currently found only a can inhibit tumor growth and neovascularization of cytokines it has immune stimulation. This effect is not dependent on p53, Rb and p16 tumor suppressor gene, almost no effect on normal cells. Tumor therapy has become a hot research.
The inhibitory effect of mda-7/IL-24 on tumor cells, but it is not clear whether it has a similar effect on HEp-2 cells. We cloned its cDNA, mediated by adenovirus in vitro overexpression effect on laryngeal carcinoma Hep2 cell proliferation were observed and discussed the mechanism. The specific work is as follows:
Method:
1., we use PHA to stimulate human peripheral blood lymphocytes to express IL-24 gene. RT-PCR method is used to get mda-7/IL-24 encoded cDNA. and construct T cloning vector PEasy-T1 Simple-IL-24..
2. from the T cloning vector PEasy-T1 Simple-IL-24 amplification mda-7/IL-24, digested fragment into pAdTrack-CMV shuttle plasmid, and pAdEasy plasmid for homologous recombination in E.coliBJ5183 adenovirus vector plasmid, and then transfected into 293A cell line package.TCID_ (50) virus titer test method, PCR and identification of recombinant Western-blot adenovirus Ad-mda-7/IL-24.3. infection of Ad-mda-7/IL-24 laryngeal carcinoma cells and related biological detection: Ad-mda-7/IL-24 and Adv were infected with human laryngeal carcinoma cell Hep2, MTT to observe the effect of Ad-mda-7/IL-24 on proliferation of human laryngeal carcinoma Hep2 cells, the expression of genes related to mRNA infection were detected by RT-PCR method, the expression of related protein was detected after Western-blot infection.
Result:
1., mda-7/ IL-24 encoding cDNA. was cloned from human peripheral blood lymphocytes stimulated by PHA from RT-PCR, and T clone vector PEasy-T1 Simple-IL-24 was successfully constructed. It was verified by enzyme digestion and sequencing to be the correct IL-24 cDNA..
2. by gene recombinant adenovirus expression vector of a series of related, CMV-mda-7/IL-24 Adenovirus Shuttle Plasmid pAdTrack-, plasmid pAd-mda-7/IL-24 and empty vector adenovirus plasmid containing pAd gene and verified by restriction enzyme digestion and sequencing. The successful construction of adenovirus expression vector. After series of linearized plasmid was transfected into 293A cells with liposomes after, can emit green fluorescence under fluorescent microscope. The RT-PCR expression was detected in IL-24 mRNA, Western Blot identified the expression of the target protein IL-24.
3.Ad-mda-7/IL-24 infected Hep2 cells strongly inhibited the proliferation activity of Hep2 cells, and the expression of BAX and Caspase-3 of apoptotic molecules increased significantly, while BCL-2 expression decreased.
Conclusion:
The main purpose of tumor therapy is to inhibit tumor growth and improve the prognosis of patients, for clarifying the mechanism of function in carcinogenesis and tumor gene therapy on the basis of the primary task. This study through cloning of tumor suppressor gene mda-7/IL-24, and construct the recombinant adenovirus vector containing the target gene of adenovirus packaging to reveal its over expression on laryngeal carcinoma cells, and to explore its mechanism. The results show that the successful construction of adenovirus expression vector containing IL-24 gene, and the virus packaged successfully. That the proliferation of IL-24 on laryngeal carcinoma cell Hep2 was inhibited obviously, this inhibition may be through the changes of apoptosis and anti apoptosis gene the proportion of genes to achieve. It provides theory and technology for clinical gene therapy further study the inhibition mechanism of IL-24 on laryngeal carcinoma and laryngeal carcinoma Basis.

【學(xué)位授予單位】：濟南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.65

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