本文選題：非霍奇金淋巴瘤 + 彌漫大B細(xì)胞淋巴瘤　； 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：研究背景:隨著人類基因組計(jì)劃的完成及高通量基因測(cè)序和轉(zhuǎn)錄組學(xué)研究的不斷深入,研究人員發(fā)現(xiàn)基因的精準(zhǔn)表達(dá)調(diào)控對(duì)維持機(jī)體穩(wěn)態(tài)和正常的生理活動(dòng)至關(guān)重要。非編碼RNA (non-coding RNA,ncRNA)是不存在編碼蛋白能力RNA的統(tǒng)稱,可通過多種機(jī)制在RNA水平干預(yù)基因調(diào)控網(wǎng)絡(luò),并廣泛參與細(xì)胞代謝、分化、增殖等一系列生命進(jìn)程。微小RNA (microRNA, miRNAs)作為非編碼RNA重要的一員,主要通過與靶基因mRNA配對(duì),沉默靶基因表達(dá)發(fā)揮作用。通常認(rèn)為,一種miRNA可以通過識(shí)別靶基因的mRNA3'UTR區(qū)域的互補(bǔ)結(jié)合序列,調(diào)控多個(gè)靶基因活性、穩(wěn)定性,進(jìn)而影響相應(yīng)靶基因干擾細(xì)胞功能;多種miRNAs也可以識(shí)別干擾同一個(gè)靶基因?qū)ζ湎掠涡盘?hào)通路產(chǎn)生網(wǎng)絡(luò)交互調(diào)控影響;因此,miRNAs表達(dá)異�？山�(jīng)由復(fù)雜的基因交互網(wǎng)絡(luò)參與細(xì)胞表觀遺傳調(diào)控、細(xì)胞自噬調(diào)控、糖代謝調(diào)控以及腫瘤相關(guān)基因調(diào)控等發(fā)揮強(qiáng)大的生物學(xué)功能,是生命科學(xué)領(lǐng)域?qū)＜谊P(guān)注的熱點(diǎn)。由于50%以上的miRNAs基因位于腫瘤相關(guān)基因組區(qū)域/脆性位點(diǎn),因此在人類腫瘤發(fā)生過程中起到重要作用,而miRNAs在腫瘤中的重要意義促使“腫瘤相關(guān)miRNAs”這一概念的提出,即“Oncomirs”。為此,尋找疾病特異性miRNAs并明確其調(diào)控機(jī)制,對(duì)疾病診斷、治療、預(yù)后均有重要意義。彌漫大B細(xì)胞淋巴瘤(Diffuse large B cell lymphoma, DLBCL)是侵襲性非霍奇金淋巴瘤(non-Hodgkin lymphoma NHL)中最常見的亞型,我國(guó)彌漫大B細(xì)胞淋巴瘤的發(fā)病率約為非霍奇金淋巴瘤的30%-45%。近十余年,隨著以美羅華為主的免疫化療方案(R-CHOP)廣泛應(yīng)用于彌漫大B細(xì)胞淋巴瘤的治療,半數(shù)以上的患者獲得治愈,但仍有超過30%的患者面臨復(fù)發(fā)的風(fēng)險(xiǎn),復(fù)發(fā)難治病例通常對(duì)現(xiàn)有藥物敏感度差,亟需新型治療措施。miRNAs替代治療是目前生命科學(xué)領(lǐng)域的一個(gè)熱點(diǎn),大量研究從事疾病差異表達(dá)miRNAs的確定工作,并對(duì)其發(fā)揮作用的相應(yīng)靶基因進(jìn)行驗(yàn)證,為尋找腫瘤可藥性miRNAs提供理論基礎(chǔ)。miR-10a是一個(gè)重要的腫瘤相關(guān)性miRNA,已有研究證實(shí)miR-10a在腦膠質(zhì)瘤、卵巢癌、以及非霍奇金淋巴瘤中存在表達(dá)差異,并可通過靶基因調(diào)控、免疫調(diào)控等多種機(jī)制發(fā)揮抑癌作用,但miR-10a在彌漫大B細(xì)胞淋巴瘤中的研究未見報(bào)道。本課題組前期研究發(fā)現(xiàn)彌漫大B細(xì)胞淋巴瘤患者血漿中miR-10a表達(dá)較健康對(duì)照組顯著降低,因此我們猜測(cè)miR-10a在彌漫大B細(xì)胞淋巴瘤的惡性轉(zhuǎn)化過程中發(fā)揮作用,并擬研究其對(duì)彌漫大B細(xì)胞淋巴瘤生物學(xué)功能的影響。研究目的:本研究旨在探討miR-10a在彌漫大B細(xì)胞淋巴瘤中表達(dá)情況及其在彌漫大B細(xì)胞淋巴瘤發(fā)生發(fā)展過程中的生物學(xué)功能,尋找miR-10a下游作用靶基因,并進(jìn)一步探討這種靶向作用對(duì)于彌漫大B細(xì)胞淋巴瘤細(xì)胞生物學(xué)功能的影響的作用機(jī)制,以期為彌漫大B細(xì)胞淋巴瘤的靶向治療提供新的思路及途徑。研究方法:1.收集彌漫大B細(xì)胞淋巴瘤組織及作為對(duì)照的非腫瘤性淋巴組織,使用qRT-PCR評(píng)估m(xù)iR-10a在彌漫大B細(xì)胞淋巴瘤組織及對(duì)照組織中的表達(dá)水平。2.使用miR-10a過表達(dá)慢病毒質(zhì)粒構(gòu)建miR-10a差異過表達(dá)細(xì)胞系,通過移植瘤模型驗(yàn)證miR-10a對(duì)彌漫大B細(xì)胞淋巴瘤腫瘤生成的影響。使用細(xì)胞轉(zhuǎn)染技術(shù)干預(yù)細(xì)胞內(nèi)miR-10a表達(dá)水平,經(jīng)qRT-PCR檢驗(yàn)miR-10a轉(zhuǎn)染效率,通過流式細(xì)胞術(shù)、細(xì)胞周期實(shí)驗(yàn)、CCK8細(xì)胞增殖實(shí)驗(yàn)在細(xì)胞水平驗(yàn)證miR-10a對(duì)彌漫大B細(xì)胞淋巴瘤細(xì)胞生物學(xué)功能的影響。3.利用生物信息學(xué)軟件TargetScan、miRanda、PicTar并結(jié)合文獻(xiàn)檢索,預(yù)測(cè)miR-10a潛在靶基因,使用雙熒光素酶報(bào)告基因的方法進(jìn)行驗(yàn)證;通過細(xì)胞轉(zhuǎn)染技術(shù)干預(yù)細(xì)胞內(nèi)miR-10a及相應(yīng)靶基因表達(dá),使用Western blot技術(shù)驗(yàn)證miR-10a對(duì)其靶基因的表達(dá)水平的調(diào)控作用;借助流式細(xì)胞術(shù)、細(xì)胞周期實(shí)驗(yàn)、CCK8細(xì)胞增殖實(shí)驗(yàn)在細(xì)胞水平驗(yàn)證miR-10a確通過調(diào)控其下游靶基因的表達(dá)水平,影響彌漫大B細(xì)胞淋巴瘤細(xì)胞生物學(xué)功能。研究結(jié)果:1.相較于對(duì)照的非腫瘤性淋巴瘤組織,miR-10a在彌漫大B細(xì)胞淋巴瘤腫瘤組織中表達(dá)水平明顯降低。2.動(dòng)物移植瘤模型示miR-10a可在動(dòng)物水平抑制彌漫大B細(xì)胞淋巴瘤生成;細(xì)胞層面實(shí)驗(yàn)顯示:過表達(dá)miR-10a可以促進(jìn)彌漫大B細(xì)胞淋巴瘤細(xì)胞凋亡、抑制細(xì)胞增殖;而彌漫大B細(xì)胞淋巴瘤miR-10a表達(dá)水平受到抑制時(shí),細(xì)胞凋亡率降低、細(xì)胞增殖活性增強(qiáng)。3.生物信息學(xué)預(yù)測(cè)提示Bcl-6為miR-10a潛在調(diào)控靶點(diǎn);雙熒光素酶實(shí)驗(yàn)驗(yàn)證Bcl-6和miR-10a存在靶向調(diào)控關(guān)系。Bcl-6在彌漫大B細(xì)胞淋巴瘤組織中的表達(dá)水平較對(duì)照正常組織明顯升高。過表達(dá)彌漫大B細(xì)胞淋巴瘤細(xì)胞內(nèi)miR-10a水平,Bcl-6的表達(dá)受抑制;抑制細(xì)胞內(nèi)miR-10a表達(dá)水平,Bcl-6的表達(dá)水平升高。Bcl-6能夠抑制細(xì)胞凋亡、促進(jìn)細(xì)胞增殖,且miR-10a和Bcl-6對(duì)彌漫大B細(xì)胞淋巴瘤的生物學(xué)功能成負(fù)調(diào)控模式。研究結(jié)論:1.彌漫大B細(xì)胞淋巴瘤組織中miR-10a表達(dá)水平較反應(yīng)增生性組織明顯降低。2. miR-10a促進(jìn)彌漫大B細(xì)胞淋巴瘤細(xì)胞凋亡,并抑制其增殖。3.在彌漫大B細(xì)胞淋巴瘤細(xì)胞中,miR-10a通過靶向調(diào)控Bcl-6促進(jìn)細(xì)胞凋亡、抑制細(xì)胞增殖。
[Abstract]:Background: with the completion of the human genome project and the continuous deepening of high throughput gene sequencing and transcriptional studies, researchers have found that the precise regulation of gene expression is essential to maintain the homeostasis and normal physiological activities of the body. Non coded RNA (non-coding RNA, ncRNA) is a general name for the absence of the ability to code protein, RNA. Many mechanisms interfere with the gene regulation network at RNA level, and participate in a series of life processes, such as cell metabolism, differentiation and proliferation. As an important member of non coded RNA, micro RNA (microRNA, miRNAs) is mainly matched with the target gene mRNA to silence the target gene expression and volatiles. It is generally believed that a miRNA can be identified by the target group. The complementary binding sequence of the mRNA3'UTR region regulates the activity and stability of multiple target genes, and then affects the function of the target genes to interfere with the cell function, and a variety of miRNAs can also identify the interference of the same target gene to the downstream signaling pathway of the target gene. Therefore, the abnormal expression of miRNAs can be mediated by a complex gene interaction network. Cell epigenetic regulation, regulation of autophagy, regulation of cell autophagy, regulation of glucose metabolism, and regulation of tumor related genes play a very important role in the field of life science. More than 50% of miRNAs gene is located in the region of the tumor related genome / fragile site, so it plays an important role in the process of human cancer. The significance of miRNAs in the tumor promotes the concept of "tumor related miRNAs", namely, "Oncomirs". For this purpose, it is important to find the disease specific miRNAs and clarify its regulatory mechanism for the diagnosis, treatment, and prognosis of the disease. Diffuse large B cell lymphoma (Diffuse large B cell lymphoma, DLBCL) is an invasive non Hodgkin. The most common subtype of non-Hodgkin lymphoma NHL, the incidence of diffuse large B cell lymphoma in our country is about ten years in non Hodgkin's lymphoma, and with the immunotherapy (R-CHOP) based on the most widely used for the treatment of diffuse large B cell lymphoma, more than half of the patients have been cured. More than 30% of the patients still face the risk of relapse. The relapsed and refractory cases are usually poor in sensitivity to existing drugs. It is urgent for the new treatment measures to replace.MiRNAs as a hot spot in the field of life science. MiRNAs provides a theoretical basis for finding tumor drug-resistant miRNAs..miR-10a is an important tumor associated miRNA. There has been a study on the expression of miR-10a in glioma, ovarian cancer, and non Hodgkin's lymphoma, and can play a role in inhibiting cancer by targeting gene regulation, immunoregulation and so on, but miR-10a is diffuse to large B cells. There is no report in the study of Ba tumor. Our previous study found that the expression of miR-10a in the plasma of diffuse large B cell lymphoma was significantly lower than that of the healthy control group. Therefore, we speculate that miR-10a plays a role in the malignant transformation of diffuse large B cell lymphoma and is intended to study the biological function of diffuse large B cell lymphoma. The purpose of this study is to investigate the expression of miR-10a in diffuse large B cell lymphoma and its biological functions in the development of diffuse large B cell lymphoma, to find the target gene for the downstream effect of miR-10a, and to further explore the effect of this target on the biological function of diffuse large B cell lymphoma cells. The mechanism of action is expected to provide new ideas and ways for the targeting therapy of diffuse large B cell lymphoma. Research methods: 1. collect diffuse large B cell lymphoma and non tumor lymphoid tissue as control, and use qRT-PCR to evaluate the expression level of miR-10a in diffuse large B cell lymphoma tissue and control tissues using miR-10a over miR-10a The expression of lentivirus plasmid was expressed by miR-10a differentially overexpressed cell lines, and the effect of miR-10a on the formation of diffuse large B cell lymphoma was verified by transplantation tumor model. Cell transfection technique was used to intervene the level of miR-10a expression in cell, qRT-PCR test of miR-10a transfection efficiency by flow cytometry, cell cycle experiment, and CCK8 cell proliferation. The effect of miR-10a on the biological function of diffuse large B cell lymphoma (.3.) was tested at the cell level. The bioinformatics software TargetScan, miRanda, PicTar and literature retrieval were used to predict the potential target genes of miR-10a, and the method of using the double luciferase reporter gene was tested, and the cell transfection technique was used to interfere in the intracellular miR-10a and the cell transfection technique. The expression of target gene and Western blot technique were used to verify the regulation of miR-10a on the expression level of the target gene. By flow cytometry, cell cycle experiment, and CCK8 cell proliferation test, the expression level of the target gene was regulated by miR-10a, and the biological function of diffuse large B cell lymphoma cells was influenced by miR-10a. Results: 1. compared with control non tumor lymphoma tissue, the expression level of miR-10a in diffuse large B cell lymphoma tumor tissue decreased significantly in.2. animal transplantation tumor model and miR-10a could inhibit the formation of diffuse large B cell lymphoma in animal level; the cell level experiment showed that overexpression of miR-10a could promote diffuse large B cell lymphomas Apoptosis and inhibition of cell proliferation, while the miR-10a expression level of the diffuse large B cell lymphoma was inhibited, the apoptosis rate was reduced, the proliferation activity enhanced by.3. bioinformatics prediction suggested that Bcl-6 was a potential target for miR-10a, and the dual luciferase experiment proved that Bcl-6 and miR-10a had a targeting regulation relationship with.Bcl-6 in the diffuse large B thin The expression level of cell lymphoma was significantly higher than that of normal control. Overexpression of miR-10a level in large B cell lymphoma cells was inhibited, the expression of Bcl-6 was inhibited, the expression level of miR-10a in cells was inhibited, and.Bcl-6 could inhibit apoptosis and promote cell proliferation by the expression level of Bcl-6, and miR-10a and Bcl-6 were diffuse large B cells. The biological function of lymphoma is negatively regulated. Conclusion: 1. the expression level of miR-10a in 1. diffuse large B cell lymphoma significantly reduces the apoptosis of diffuse large B cell lymphoma cells and inhibits the proliferation of.3. in diffuse large B cell lymphoid cells, and miR-10a promotes Bcl-6 in the diffuse large B cell lymphomas. Apoptosis and inhibition of cell proliferation.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.1

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