本文選題：垂體ACTH腺瘤 + 信號通路��； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：背景：垂體腺瘤是常見的顱內(nèi)良性腫瘤之一,發(fā)病率居顱內(nèi)腫瘤第二位,約占顱內(nèi)腫瘤的10%-15%,并呈現(xiàn)逐年增加的趨勢。隨著腫瘤不斷生長,可壓迫蝶鞍區(qū)周圍結(jié)構(gòu),如視神經(jīng)、海綿竇、腦底動脈、下丘腦等,甚至累及額葉、腦干,而導(dǎo)致嚴重的功能障礙。同時,腫瘤生長還可導(dǎo)致垂體激素分泌紊亂。按照外周血激素水平,垂體腺瘤分為無功能型垂體腺瘤(NFPA)和功能型垂體腺瘤,包括：泌乳素腺瘤(PRL)、促腎上腺皮質(zhì)激素腺瘤(Cushing)、生長激素腺瘤(GH)、促甲狀腺激素腺瘤(TSH)、促性腺激素腺瘤(PGA)以及混合激素分泌腺瘤等。其中垂體促腎上腺皮質(zhì)激素腺瘤(ACTH-PAs)臨床又稱為庫欣病(Cushing's Disease)是一種伴有促腎上腺皮質(zhì)激素(ACTH)分泌的功能型垂體腺瘤約占全部垂體腺瘤的14%,約占庫欣綜合征(Cushing's syndrome)的70%。該病在歐美國家的發(fā)病率為39／百萬,我國尚缺乏大規(guī)模流行病學(xué)數(shù)據(jù)。由于該病的診斷主要基于實驗室激素水平檢查、患者臨床癥狀體征、影像學(xué)檢查及病理學(xué)免疫組織化學(xué)檢測,因此對于庫欣病的早期診斷十分困難,容易誤診�；颊叱３Ｓ捎诟咂べ|(zhì)醇血癥而引起多種嚴重并發(fā)癥,如高血壓、糖尿病、高脂血癥、骨質(zhì)疏松及精神抑郁等就診。在治療上,對于絕大多數(shù)庫欣病人而言,經(jīng)鼻蝶竇入路垂體腺瘤切除術(shù)仍然是臨床首選治療方法,放射治療以及帕瑞肽、酮康唑等藥物治療常作為難治性庫欣病的術(shù)后輔助治療方式。雖然文獻報道外科手術(shù)成功率在65%-90%,但是由于腫瘤自身生物學(xué)行為的不同及術(shù)者操作水平的差異,腫瘤復(fù)發(fā)率為3%-47%,平均復(fù)發(fā)時間為16～49個月,復(fù)發(fā)患者預(yù)后較差,病死率高。近年來針對庫欣病的分子水平研究主要集中在腫瘤的發(fā)生、發(fā)展、腫瘤的侵襲性及激素分泌等方面,但目前對于庫欣病的發(fā)病機制尚不完全清楚,因此臨床上亟待尋找有助于庫欣病診斷、治療及判斷預(yù)后的潛在靶點。本研究通過新一代測序技術(shù)(Next generation sequencing, NGS),對垂體ACTH腺瘤全轉(zhuǎn)錄組進行高通量測序,在轉(zhuǎn)錄組水平篩選相關(guān)差異表達基因和microRNA;并與患者臨床表型進行關(guān)聯(lián)分析,探究垂體ACTH腺瘤所致臨床表型的分子機理,此外,考慮到血清游離miRNA具有作為腫瘤標(biāo)志物的潛力,篩選差異表達的血清miRNA,并對差異表達的血清miRNA進行了驗證,為深入研究庫欣病的發(fā)病機制,篩選診斷標(biāo)志物提供依據(jù)。以期最終實現(xiàn)垂體腺瘤患者個性化治療。第一部分：基于全轉(zhuǎn)錄組深度測序的垂體ACTH腺瘤mRNA差異表達分析目的：通過新一代高通量測序技術(shù)對垂體ACTH腺瘤行轉(zhuǎn)錄組測序,探究垂體ACTH腺瘤與瘤周正常垂體組織間的差異表達基因,以期揭示腫瘤發(fā)生發(fā)展過程中可能的存在分子機制,并為后續(xù)的microRNA組學(xué)研究提供完整的分子生物學(xué)基礎(chǔ)。方法：收集單一中心68例垂體ACTH患者的組織標(biāo)本(腫瘤及瘤周正常垂體),20例無功能垂體腺瘤,18例生長激素型垂體腺瘤和8例泌乳素型垂體腺瘤,同時采集患者相關(guān)臨床信息。運用新一代測序技術(shù)對9對配對樣本(腫瘤和瘤周正常垂體組織)進行轉(zhuǎn)錄組深度測序,通過生物信息學(xué)分析,獲取垂體ACTH腺瘤基因差異表達譜。進一步對篩選出的差異表達基因行GO term和PATHWAY分析,并挑選差異表達顯著基因進行驗證和功能機制研究,探究其在垂體ACTH腺瘤中可能存在的分子調(diào)節(jié)機制。結(jié)果：通過對垂體ACTH腺瘤mRNA-seq數(shù)據(jù)做生物信息學(xué)分析,我們篩選出166個上調(diào)的差異表達基因和288個下調(diào)的差異表達基因,發(fā)現(xiàn)了庫欣病腫瘤發(fā)生發(fā)展、激素分泌和腫瘤侵襲性等相關(guān)的基因和相關(guān)信號通路。并從中挑選未經(jīng)報道的TIMP3基因做進一步驗證,發(fā)現(xiàn)與瘤周正常垂體組織相比TIMP3在mRNA和蛋白水平的表達在各類型垂體腺瘤中顯著下調(diào),并與腫瘤的體積、侵襲性、Ki-67等顯著相關(guān)。結(jié)論：通過建立垂體ACTH腺瘤基因的差異表達譜,揭示庫欣病可能存在的發(fā)病機制及臨床表型相關(guān)機制,并為后續(xù)的庫欣病microRNA組學(xué)分子靶向研究提供理論基礎(chǔ)。第二部分：基于全轉(zhuǎn)錄組深度測序的垂體ACTH腺瘤microRNAs差異表達分析目的：運用新一代測序技術(shù),建立垂體ACTH腺瘤microRNA差異表達譜。鑒定相關(guān)microRNA在垂體ACTH腺瘤組織中及患者血清中的表達情況,以期作為診斷庫欣病和預(yù)后監(jiān)測的分子標(biāo)志物。方法：經(jīng)蝶竇入路手術(shù)治療庫欣病患者,術(shù)中收集垂體ACTH腺瘤患者腫瘤標(biāo)本及瘤周垂體組織共9對。運用Illumina Genome Analyzer Ⅱx行全轉(zhuǎn)錄組microRNA測序,結(jié)合生物信息學(xué)分析建立垂體ACTH腺瘤差異表達的microRNA譜。并在55例腫瘤組織和15例瘤周正常垂體組織中運用熒光定量PCR進行驗證,將篩選得到的microRNA在197例病人和120例健康人的血清中運用熒光定量PCR行驗證檢測,并進行靶基因的預(yù)測及與第一部分mRNA-seq結(jié)果做關(guān)聯(lián)分析,在組織水平作相關(guān)基因功能驗證,得到一組microRNA panel作為針對庫欣病診斷和預(yù)后觀察的分子標(biāo)志物。結(jié)果：所有9對樣品建庫成功,Illumina Genome Analyzer Ⅱx深度測序平均讀取10M/樣品。我們發(fā)現(xiàn),垂體ACTH腺瘤與瘤周垂體相比,有67個microRNA顯著差異表達。通過對測序數(shù)據(jù)分析發(fā)現(xiàn),垂體ACTH腺瘤相對瘤周正常垂體組織比較,根據(jù)Fold change2, Recurrence≥5,發(fā)現(xiàn)67個差異表達的microRNA,其中包括39個上調(diào)的microRNA和28個下調(diào)的microRNA,在腫瘤和瘤周正常垂體組織中驗證得到6個上調(diào)(miR-9-5p, miR-9-3p, miR-190b, miR-137, miR-885-5p, miR-592)和1個下調(diào)的mir-551b-3p,并且在患者和正常人的血清中進行檢測,也得到了1個下調(diào)的mir-551b-3p,其表達水平與組織中一致。受試者工作特征曲線分析顯示,mir-551b-3p對于庫欣病的診斷具有較高的敏感性和特異性。血清中mir-551b-3p的表達水平與患者的腫瘤體積,血皮質(zhì)醇和ACTH水平顯著相關(guān)。結(jié)論：研究結(jié)果表明microRNA的差異表達可能參與垂體ACTH腺瘤的發(fā)生發(fā)展。microRNA有可能成為庫欣病人特異性的,外周血無創(chuàng)診斷的分子標(biāo)志物。
[Abstract]:Background: pituitary adenoma is one of the most common benign intracranial tumors, with the incidence of second of intracranial tumors, accounting for 10%-15% of intracranial tumors and increasing trend year by year. As the tumor grows, it can oppress the surrounding structure of the sella region, such as the optic nerve, cavernous sinus, the cerebral artery, the hypothalamus, and even the frontal and brain stem. At the same time, tumor growth can also lead to a disorder of pituitary hormone secretion. According to peripheral blood hormone levels, pituitary adenomas are divided into nonfunctional pituitary adenomas (NFPA) and functional pituitary adenomas, including prolactin adenoma (PRL), adrenocorticotropin adenoma (Cushing), growth hormone adenoma (GH), thyroid stimulating hormone adenoma (TSH), and promoting pituitary adenomas (TSH). Gonadotropin adenoma (PGA) and mixed hormone secretory adenoma. The pituitary adrenocorticotropin adenoma (ACTH-PAs) is also known as Cushing's disease (Cushing's Disease), a functional pituitary adenoma secreted by adrenocorticotropin (ACTH), about 14% of all pituitary adenomas, accounting for Cushing's syndrome (Cushing's syndrome). The incidence of 70%. in European and American countries is 39 per million. There is still a lack of large-scale epidemiological data in our country. Because the diagnosis of this disease is mainly based on laboratory hormone level examination, clinical symptoms and signs, imaging examination and pathological examination of Pathology, it is very difficult for early diagnosis of Cushing's disease and easy to be misdiagnosed. Patients often cause a variety of severe complications due to hypercortisol, such as hypertension, diabetes, hyperlipidemia, osteoporosis, and depression. In the treatment of most Cushing patients, transsphenoidal pituitary adenoma resection is still the preferred treatment, radiotherapy, Parein, and ketkang. Azoles are often used as adjuvant therapy for refractory Cushing's disease. Although the literature reports that the success rate of surgery is 65%-90%, the recurrence rate of the tumor is 3%-47%, the recurrence time is 16~49 months due to the difference of the biological behavior of the tumor and the difference of the operation level. The prognosis of the recurrent patients is poor and the mortality is high. Recent studies on the molecular level of Cushing's disease are mainly focused on the occurrence, development, invasiveness and hormone secretion of the tumor, but the pathogenesis of Cushing's disease is not yet completely clear, so it is urgent to find the potential targets for the diagnosis, treatment and prognosis of Cushing's disease. Next generation sequencing (NGS), high flux sequencing of pituitary ACTH adenoma complete transcriptional group, screening related differentially expressed genes and microRNA at the level of the transcriptional group, and associated with the clinical phenotype of the patients, to explore the molecular mechanism of the clinical phenotype of pituitary ACTH adenoma, and to consider the free miRNA in serum. The differential expression of serum miRNA was screened as the potential of tumor markers, and the differential expression of serum miRNA was verified to provide a basis for the in-depth study of the pathogenesis of Cushing's disease and screening diagnostic markers. The first part: the pituitary ACTH gland based on the full transcriptional sequence based on the full transcriptional sequence. Differential expression analysis of tumor mRNA Objective: To explore the differentially expressed genes between pituitary ACTH adenomas and normal pituitary tissues by a new generation of high-throughput sequencing technology to explore the differentially expressed genes between pituitary ACTH adenomas and normal pituitary tissues, in order to reveal the possible molecular mechanisms in the process of tumor development and to provide a complete study for subsequent microRNA studies. Molecular biology basis. Methods: a single center 68 case of pituitary ACTH patients (tumor and tumor Zhou Zhengchang pituitary), 20 non functional pituitary adenomas, 18 growth hormone pituitary adenomas and 8 prolactin pituitary adenomas were collected, and the patient's clinical information was collected. 9 pairs of paired samples (tumors and) were used in the new generation sequencing technique. The differential expression profiles of pituitary ACTH adenomas were obtained by bioinformatics analysis. The differentially expressed genes were further analyzed by GO term and PATHWAY analysis, and the differentially expressed genes were selected to verify and function the mechanism of the pituitary adenoma, and to explore the possibility of it in pituitary ACTH adenomas. Results: through bioinformatics analysis of the mRNA-seq data of pituitary ACTH adenoma, we screened 166 up-regulated differentially expressed genes and 288 down regulated differentially expressed genes, and found genes and related signaling pathways related to the development of Cushing's disease tumor, hormone secretion and tumor invasion. The unreported TIMP3 gene was selected to further verify that the expression of TIMP3 at mRNA and protein levels was significantly downregulated in all types of pituitary adenomas compared with normal pituitary tissue, and was significantly related to the volume, invasiveness and Ki-67 of the tumor. Conclusion: the differential expression profile of the gene of the ACTH adenoma of the body was established to reveal Cushing's disease. Possible pathogenesis and clinical phenotype related mechanisms, and provide a theoretical basis for subsequent study of Cushing's disease microRNA molecular targeting. The second part: analysis of differential expression of microRNAs in pituitary ACTH adenomas based on full transcriptional sequence analysis: using a new generation sequencing technology to establish a microRNA differential table for pituitary ACTH adenomas To identify the expression of microRNA in the pituitary adenoma and the sera of the pituitary ACTH, as a molecular marker for the diagnosis of Cushing's disease and prognosis. Methods: transsphenoidal approach to the patients with Cushing's disease, 9 pairs of pituitary ACTH adenomas and pituitary tissue were collected during the operation. Illumina Genome was used. Analyzer II X line full transcriptional group microRNA sequencing, combined with bioinformatics analysis to establish the microRNA spectrum of differential expression of pituitary ACTH adenoma, and using fluorescence quantitative PCR in 55 cases of tumor tissue and 15 cases of Zhou Zhengchang pituitary tissue, the selected microRNA was used in the serum of 197 patients and 120 healthy people. PCR was tested and the target gene was predicted and associated with the first part of the mRNA-seq results. A group of microRNA panel as a molecular marker for the diagnosis and prognosis of Cushing's disease was obtained at the tissue level. Results: all 9 pairs of samples were built successfully and Illumina Genome Analyzer II X was deep. 10M/ samples were read by degree sequencing. We found that there were 67 significant differences in the expression of the pituitary ACTH adenoma compared with the peritumoral pituitary. Through analysis of the sequencing data, the pituitary ACTH adenoma was compared to the Zhou Zhengchang pituitary tissue, and 67 differentially expressed microRNA were present according to Fold change2, Recurrence more than 5, including 39 up-regulated adenomas. MicroRNA and 28 down regulated microRNA were tested for 6 up-regulated (miR-9-5p, miR-9-3p, miR-190b, miR-137, miR-885-5p, miR-592) and 1 down-regulated mir-551b-3p in the tumor and normal pituitary tissue, and were detected in the sera of the patients and normal people, and 1 down-regulated mir-551b-3p were also obtained, the expression level and the tissue of the tissues. The results showed that mir-551b-3p had high sensitivity and specificity for the diagnosis of Cushing's disease. The expression level of mir-551b-3p in serum was significantly related to the tumor volume, plasma cortisol and ACTH levels in the patients. Conclusion: the results showed that the differential expression of microRNA may be involved in pituitary ACTH adenomas. The occurrence and development of.MicroRNA is likely to be a specific marker of Cushing's disease and a molecular marker for noninvasive diagnosis of peripheral blood.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R736.4

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