重组人IL-24腺病毒表达载体的构建及对喉肿瘤细胞的生物学效应

发布时间：2018-03-16 22:23

本文选题：喉癌　切入点：IL-24　出处：《济南大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景与目的喉癌是耳鼻咽喉科常见的恶性肿瘤,约90%以上是鳞状细胞癌。喉癌的发病率在近几年有上升的趋势,这与诊断水平的提高、生活习惯改变以及空气污染都有关系。统计数据显示:喉癌的发病率在城市高于农村,在重工业发达的城市高于轻工业发达城市。手术治疗是喉癌最常用的治疗手段,随着头颈外科的发展,喉外科手术不断完善,患者的5年生存率及生存质量明显提高。但仍有30%-40%的患者,在经过根治手术和放化疗治疗后复发或转移。当出现复发和/或远处转移时患者对放化疗多不敏感,3年生存率仅为10%。因此如何进一步提高喉癌的疗效成为当务之急。随着分子生物学和基因工程的飞速发展,基因免疫治疗日益受到重视,显示出良好的发展前景。白细胞介素24,又名黑色素瘤分化相关抗原7(melanoma differentiation associated antigen7,MDA-7),它主要由CD3~+T淋巴细胞和单核细胞表达,为分泌型蛋白,属于IL-10家族成员。研究显示IL-24是目前发现唯一一种既具有抑制肿瘤细胞生长和血管形成又具有免疫刺激作用的细胞因子。这种抑制作用并不依赖p53、Rb和p16等抑癌基因,对正常细胞几乎无影响。现已成为肿瘤治疗研究热点。 mda-7/IL-24对多种肿瘤细胞有抑制作用,但尚不清楚它对喉癌细胞是否具有相似的作用。为此我们对其进行了克隆,通过腺病毒介导进行体外过量表达,观察对喉癌细胞Hep2增殖的影响并对作用机制进行初步探讨。具体工作如下: 方法: 1.利用PHA刺激培养人外周血淋巴细胞使其表达IL-24基因,采用RT-PCR方法得到mda-7/IL-24编码的cDNA。构建T克隆载体PEasy-T1 Simple-IL-24。 2.从T克隆载体PEasy-T1 Simple-IL-24中大量扩增mda-7/IL-24,酶切回收目的片段插入到pAdTrack-CMV穿梭质粒中,与pAdEasy骨架质粒在E.coliBJ5183中进行同源重组为腺病毒载体质粒,线形化后转染293A细胞进行包装。TCID_(50)方法测得病毒效价、PCR和Western-blot鉴定重组腺病毒Ad-mda-7/IL-24。3. Ad-mda-7/IL-24感染喉癌细胞及相关生物学检测:Ad-mda-7/IL-24和Adv分别感染人喉癌细胞Hep2,MTT观察Ad-mda-7/IL-24对人喉癌细胞Hep2的增殖影响,RT-PCR法检测感染后相关基因mRNA表达情况,Western-blot检测感染后相关蛋白表达。结果: 1.通过RT-PCR从PHA刺激培养的人外周血淋巴细胞中克隆出mda-7/ IL-24编码cDNA。构建T克隆载体PEasy-T1 Simple-IL-24成功。经酶切和测序验证为正确的IL-24 cDNA。 2.经基因重组技术获得腺病毒表达相关的一系列载体,穿梭质粒pAdTrack- CMV-mda-7/IL-24、含目的基因的腺病毒质粒pAd-mda-7/IL-24和空载体腺病毒质粒pAd,经酶切和测序验证正确。成功构建了腺病毒表达系列载体。质粒线性化后经脂质体法转染293A细胞后,能够在荧光显微镜下发出绿色荧光,RT-PCR检测出IL-24 mRNA的表达,Western Blot鉴定有目的蛋白IL-24的表达。 3.Ad-mda-7/IL-24感染Hep2细胞后对Hep2细胞的增殖活性有较强抑制作用;凋亡相关分子BAX、Caspase-3表达有明显增强,BCL-2表达则下降。结论: 肿瘤治疗的主要目的就是抑制肿瘤的生长和改善患者的预后,基础研究的首要任务则为阐明肿瘤的发生机制以及肿瘤治疗基因的功能机制。本研究通过克隆肿瘤抑制基因mda-7/IL-24,并构建重组腺病毒载体包装含目的基因的腺病毒以揭示其过表达对喉癌细胞的作用,并对其作用机制进行初步探讨。结果显示,成功构建了含目的基因IL-24的腺病毒表达载体,并且病毒包装成功。证明IL-24对喉癌细胞Hep2的增殖有较明显的抑制作用,这种抑制作用有可能是通过改变凋亡基因与抗凋亡基因比例来实现的。这为进一步研究IL-24对喉癌的抑制作用机制以及喉癌的临床基因治疗提供了理论和技术依据。
[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.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.65

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