發(fā)布時(shí)間：2018-07-24 10:17

【摘要】：研究背景骨肉瘤是一種起源于成骨性間葉組織的最常見的原發(fā)性惡性腫瘤,好發(fā)于兒童和青少年,約占所有骨腫瘤的20%。目前由于早期診斷和化療手段迅速的發(fā)展使得患者肺轉(zhuǎn)移率得到明顯控制,5年生存率提高了50%以上,但約有30%患者化療效果不佳,仍有半數(shù)以上的患者死于骨肉瘤的轉(zhuǎn)移和復(fù)發(fā),骨肉瘤的治療處于一個(gè)平臺(tái)期。近年來尤其是進(jìn)入二十一世紀(jì)以來,由于分子生物學(xué)領(lǐng)域理論和技術(shù)水平的不斷提高,使得醫(yī)學(xué)科技工作者對(duì)骨肉瘤的發(fā)生機(jī)制等基礎(chǔ)研究方面的認(rèn)識(shí)在分子水平達(dá)到了前所未有的程度,期待在骨肉瘤發(fā)病機(jī)制及基因治療研究方面有突破性的進(jìn)展。各種生物體的生理、生化及行為過程都表現(xiàn)出一定的晝夜節(jié)律變化。晝夜節(jié)律生物鐘可看作是機(jī)體內(nèi)的定時(shí)系統(tǒng),因其存在使機(jī)體內(nèi)多種生理活動(dòng)、行為模式得以表現(xiàn)出近似24h的規(guī)律性節(jié)奏,從而對(duì)其所生存的具有周期性的環(huán)境做出適應(yīng)性反應(yīng)。生物節(jié)律是生命活動(dòng)的基本特征之一,不僅整個(gè)機(jī)體之中,而且器官、組織、包括游離的單個(gè)細(xì)胞之中也廣泛存在。機(jī)體內(nèi)維持這些節(jié)律的基本結(jié)構(gòu)即是生物鐘(circadian clock),而產(chǎn)生和維持生物節(jié)律運(yùn)轉(zhuǎn)的分子生物學(xué)基礎(chǔ)即是生物鐘基因(circadian clock genes)。至目前為止,在哺乳動(dòng)物體內(nèi)至少已發(fā)現(xiàn)10多種生物鐘基因。晝夜節(jié)律發(fā)生的物質(zhì)基礎(chǔ)被稱作生物振蕩器(oscillator),它是由一系列呈節(jié)律表達(dá)的生物鐘基因及其相應(yīng)蛋白產(chǎn)物作為特異的核心元件所構(gòu)成,而由這些特異的核心元件構(gòu)成的轉(zhuǎn)錄-翻譯反饋環(huán)路生物節(jié)律的即是其基本分子機(jī)制。生物鐘基因是生命活動(dòng)的時(shí)序控制器,從機(jī)體微觀方面影響、控制著機(jī)體的生長、發(fā)育及疾病、衰亡等,通過調(diào)控細(xì)胞增殖周期、細(xì)胞凋亡、神經(jīng)內(nèi)分泌和免疫功能等方面與還與惡性腫瘤的發(fā)生、發(fā)展、治療和預(yù)后等關(guān)系密切,國內(nèi)外大量報(bào)道證實(shí)腫瘤的發(fā)生與生物節(jié)律紊亂密切相關(guān)。生物鐘基因Period家族中的Per2基因是重要的生物鐘基因之一,在機(jī)體中樞系統(tǒng)、外周組織以及腫瘤細(xì)胞中都有存在,除了本身的生物周期節(jié)律調(diào)節(jié)功能,還具有非節(jié)律功能即在惡性腫瘤的發(fā)生、發(fā)展中起重要作用�，F(xiàn)已發(fā)現(xiàn)在肺癌、乳腺癌、前列腺癌、胃癌等惡性腫瘤中均可檢測(cè)出Per2的異常表達(dá),但在人骨肉瘤中的作用尚未見報(bào)道,因此進(jìn)一步研究Per2將為明確骨肉瘤的發(fā)病機(jī)制,將有助于探索一條針對(duì)骨腫瘤的診斷與治療、判斷預(yù)后及轉(zhuǎn)歸等的新方法。目的構(gòu)建并鑒定pEGFP-N1-hPer2真核表達(dá)載體,觀察其在骨肉瘤細(xì)胞MG63中的表達(dá),并通過細(xì)胞水平的體外實(shí)驗(yàn)探討hPer2對(duì)人骨肉瘤細(xì)胞MG63作用及其機(jī)制。方法 (1)應(yīng)用RT-PCR技術(shù)從MG63細(xì)胞中擴(kuò)增hPer2,并將PCR產(chǎn)物經(jīng)雙酶切后定向克隆入pEGFP-N1的多克隆位點(diǎn),hPer2重組質(zhì)粒經(jīng)雙酶切及測(cè)序鑒定,從而成功構(gòu)建pEGFP-N1-hPer2真核表達(dá)載體。(2)采用脂質(zhì)體介導(dǎo)轉(zhuǎn)染骨肉瘤細(xì)胞MG63,qRT-PCR和Western blot分別檢測(cè)hPer2的表達(dá)情況。(3)利用脂質(zhì)體法將hPer2真核表達(dá)質(zhì)粒pEGFP-N1-hPer2和空質(zhì)粒pEGFP-N1分別轉(zhuǎn)染入MG63,分為pEGFP-N1-hPer2轉(zhuǎn)染組、pEGFP-N1空質(zhì)粒轉(zhuǎn)染組和不做任何處理的空白組3組。應(yīng)用RT-PCR、Western blot檢測(cè)Per2基因和蛋白的表達(dá),CCK-8法、流式細(xì)胞儀和Transwell小室法分別檢測(cè)細(xì)胞增殖、凋亡、周期分布和侵襲能力的變化。結(jié)果 (1)重組質(zhì)粒pEGFP-N1-hPer2經(jīng)PstⅠ、KpnⅠ雙酶切和測(cè)序與hPer2基因序列一致。(2)利用脂質(zhì)體介導(dǎo)將pEGFP-N1-hPer2重組質(zhì)粒成功轉(zhuǎn)染到骨肉瘤細(xì)胞MG63中并獲得了hPer2基因的過表達(dá)。(3)與對(duì)照組和空白組比較,實(shí)驗(yàn)組通過hPer2過表達(dá)使腫瘤細(xì)胞增殖能力下降、凋亡率增加及穿膜細(xì)胞數(shù)減少。結(jié)論成功構(gòu)建pEGFP-N1-hPer2真核表達(dá)載體,并能夠在骨肉瘤細(xì)胞MG63中過表達(dá)。hPer2在人骨肉瘤細(xì)胞系MG63中過表達(dá)對(duì)其具有生長抑制作用,因此利用生物鐘基因hPer2可以作為人骨肉瘤基因治療的可能方法之一。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R738.1

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