生物钟基因Per2抑制人骨肉瘤细胞生长的实验研究

发布时间：2018-07-24 10:17

【摘要】：研究背景骨肉瘤是一种起源于成骨性间叶组织的最常见的原发性恶性肿瘤,好发于儿童和青少年,约占所有骨肿瘤的20%。目前由于早期诊断和化疗手段迅速的发展使得患者肺转移率得到明显控制,5年生存率提高了50%以上,但约有30%患者化疗效果不佳,仍有半数以上的患者死于骨肉瘤的转移和复发,骨肉瘤的治疗处于一个平台期。近年来尤其是进入二十一世纪以来,由于分子生物学领域理论和技术水平的不断提高,使得医学科技工作者对骨肉瘤的发生机制等基础研究方面的认识在分子水平达到了前所未有的程度,期待在骨肉瘤发病机制及基因治疗研究方面有突破性的进展。各种生物体的生理、生化及行为过程都表现出一定的昼夜节律变化。昼夜节律生物钟可看作是机体内的定时系统,因其存在使机体内多种生理活动、行为模式得以表现出近似24h的规律性节奏,从而对其所生存的具有周期性的环境做出适应性反应。生物节律是生命活动的基本特征之一,不仅整个机体之中,而且器官、组织、包括游离的单个细胞之中也广泛存在。机体内维持这些节律的基本结构即是生物钟(circadian clock),而产生和维持生物节律运转的分子生物学基础即是生物钟基因(circadian clock genes)。至目前为止,在哺乳动物体内至少已发现10多种生物钟基因。昼夜节律发生的物质基础被称作生物振荡器(oscillator),它是由一系列呈节律表达的生物钟基因及其相应蛋白产物作为特异的核心元件所构成,而由这些特异的核心元件构成的转录-翻译反馈环路生物节律的即是其基本分子机制。生物钟基因是生命活动的时序控制器,从机体微观方面影响、控制着机体的生长、发育及疾病、衰亡等,通过调控细胞增殖周期、细胞凋亡、神经内分泌和免疫功能等方面与还与恶性肿瘤的发生、发展、治疗和预后等关系密切,国内外大量报道证实肿瘤的发生与生物节律紊乱密切相关。生物钟基因Period家族中的Per2基因是重要的生物钟基因之一,在机体中枢系统、外周组织以及肿瘤细胞中都有存在,除了本身的生物周期节律调节功能,还具有非节律功能即在恶性肿瘤的发生、发展中起重要作用。现已发现在肺癌、乳腺癌、前列腺癌、胃癌等恶性肿瘤中均可检测出Per2的异常表达,但在人骨肉瘤中的作用尚未见报道,因此进一步研究Per2将为明确骨肉瘤的发病机制,将有助于探索一条针对骨肿瘤的诊断与治疗、判断预后及转归等的新方法。目的构建并鉴定pEGFP-N1-hPer2真核表达载体,观察其在骨肉瘤细胞MG63中的表达,并通过细胞水平的体外实验探讨hPer2对人骨肉瘤细胞MG63作用及其机制。方法 (1)应用RT-PCR技术从MG63细胞中扩增hPer2,并将PCR产物经双酶切后定向克隆入pEGFP-N1的多克隆位点,hPer2重组质粒经双酶切及测序鉴定,从而成功构建pEGFP-N1-hPer2真核表达载体。(2)采用脂质体介导转染骨肉瘤细胞MG63,qRT-PCR和Western blot分别检测hPer2的表达情况。(3)利用脂质体法将hPer2真核表达质粒pEGFP-N1-hPer2和空质粒pEGFP-N1分别转染入MG63,分为pEGFP-N1-hPer2转染组、pEGFP-N1空质粒转染组和不做任何处理的空白组3组。应用RT-PCR、Western blot检测Per2基因和蛋白的表达,CCK-8法、流式细胞仪和Transwell小室法分别检测细胞增殖、凋亡、周期分布和侵袭能力的变化。结果 (1)重组质粒pEGFP-N1-hPer2经PstⅠ、KpnⅠ双酶切和测序与hPer2基因序列一致。(2)利用脂质体介导将pEGFP-N1-hPer2重组质粒成功转染到骨肉瘤细胞MG63中并获得了hPer2基因的过表达。(3)与对照组和空白组比较,实验组通过hPer2过表达使肿瘤细胞增殖能力下降、凋亡率增加及穿膜细胞数减少。结论成功构建pEGFP-N1-hPer2真核表达载体,并能够在骨肉瘤细胞MG63中过表达。hPer2在人骨肉瘤细胞系MG63中过表达对其具有生长抑制作用,因此利用生物钟基因hPer2可以作为人骨肉瘤基因治疗的可能方法之一。
[Abstract]:Background osteosarcoma is the most common primary malignant tumor originating in the osteogenic mesoleaf tissue. It is found in children and adolescents. The 20%., which accounts for all bone tumors, is currently significantly controlled by early diagnosis and rapid development of chemotherapy. The 5 year survival rate has increased by more than 50%, but it is about 30%. More than half of the patients died of metastasis and recurrence of osteosarcoma, and the treatment of osteosarcoma was in a platform period. In recent years, especially since twenty-first Century, the basic research on the mechanism of osteosarcoma caused by medical scientists and technicians has been improved by the continuous improvement of the theory and technology level in the field of molecular biology. The understanding has reached an unprecedented level at the molecular level, looking forward to a breakthrough in the pathogenesis of osteosarcoma and the research of gene therapy. The physiological, biochemical and behavioral processes of various organisms show certain circadian rhythms. The circadian clock can be considered as a timing system in the body because of its existence. A variety of physiological activities in the body, the behavior pattern can show the regular rhythm of the approximate 24h, and thus make adaptive response to the periodic environment of its existence. Biological rhythm is one of the basic characteristics of life activities, not only in the whole body, but also in the organs, tissues, and free individual cells. The basic structure that maintains these rhythms is the circadian clock, and the molecular biological basis for the production and maintenance of biological rhythms is the biological clock gene (circadian clock genes). Up to now, at least 10 kinds of biological clock genes have been found in mammals. The substance basis of the circadian rhythm is called the biological basis. A biological oscillator (oscillator) is made up of a series of rhythmic clock genes and their corresponding protein products as a specific core component. The biological rhythm of the transcriptional translation feedback loop, composed of these specific core components, is the basic molecular mechanism. The clock gene is a timing controller for life activities. From the microcosmic aspect of the body, the growth, development, disease and decay of the body are controlled, and the relationship between the cell proliferation cycle, cell apoptosis, neuroendocrine and immune function is closely related to the occurrence, development, treatment and prognosis of malignant tumor. The Per2 gene in the Period family of biological clock gene is one of the important biological clock genes. It exists in the central system of the body, peripheral tissue and tumor cells. Besides its biological cycle rhythm regulation function, it also has the non rhythmic function that plays an important role in the development of malignant tumor. The abnormal expression of Per2 can be detected in breast, prostate and gastric cancer, but the role of Per2 in human osteosarcoma has not yet been reported. Therefore, further study on the pathogenesis of osteosarcoma will be helpful to explore a new method for diagnosis and treatment of bone tumor, and to determine the prognosis and prognosis. The expression of pEGFP-N1-hPer2 eukaryotic expression vector was identified and its expression in osteosarcoma cell MG63 was observed. The effect of hPer2 on human osteosarcoma cell MG63 and its mechanism were investigated by the cell level in vitro. Method (1) hPer2 was amplified from MG63 cells by RT-PCR technology, and the product of PCR was cloned into pEGFP-N1 by double enzyme digestion. The recombinant plasmid of hPer2 was identified by double enzyme digestion and sequencing, and the eukaryotic expression vector of pEGFP-N1-hPer2 was successfully constructed. (2) the expression of hPer2 was detected by liposome mediated transfection of osteosarcoma cells MG63, qRT-PCR and Western blot respectively. (3) the eukaryotic expression plasmid pEGFP-N1-hPer2 and empty plasmid pEGFP-N1 were transferred by liposome method respectively. MG63 was injected into MG63, divided into pEGFP-N1-hPer2 transfection group, pEGFP-N1 empty plasmid transfection group and blank group without any treatment. RT-PCR and Western blot were used to detect the expression of Per2 gene and protein, CCK-8, flow cytometry and Transwell cell method were used to detect cell proliferation, apoptosis, periodic distribution and invasion ability. Results (1) recombinant plasmid The particle pEGFP-N1-hPer2 was cut and sequenced by Pst I, Kpn I and sequencing with the hPer2 gene sequence. (2) the pEGFP-N1-hPer2 recombinant plasmid was successfully transfected into the osteosarcoma cell MG63 by liposome mediated and the overexpression of the hPer2 gene was obtained. (3) compared with the control group and the blank group, the test group was able to proliferate the tumor cells through the over expression of hPer2. Conclusion the pEGFP-N1-hPer2 eukaryotic expression vector can be successfully constructed and the overexpression of.HPer2 in osteosarcoma cell MG63 can inhibit its growth in human osteosarcoma cell line MG63. Therefore, the biological clock gene hPer2 can be used as a possible method of gene therapy for human osteosarcoma. One of.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738.1

【相似文献】