本文關鍵詞：癌源性外泌體介導胰腺癌病程中骨骼肌細胞胰島素抵抗的產(chǎn)生及其機制探討　出處：《浙江大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 胰腺癌 骨骼肌 胰島素抵抗 外泌體 微小RNA 代謝重構 代謝交互

【摘要】：背景:臨床上常用的腫瘤學標記物等生化檢查手段及影像學檢查方法對胰腺癌早期診斷的準確性仍較差。因此,尋找早期診斷的標記物,理解胰腺癌細胞生物學行為,開發(fā)新的治療模式/靶點是目前胰腺癌研究領域的主要方向。胰腺癌有獨特的代謝重構現(xiàn)象,與外周組織間發(fā)生著基于胰島素抵抗的代謝交互作用。這不僅導致腫瘤自身代謝改變,還引起全身代謝紊亂,其中葡萄糖耐量異常是首先出現(xiàn)的代謝異常狀況,隨后發(fā)生的外周組織(骨骼肌、脂肪)胰島素抵抗則與癌瘤增殖、病情進展,癌癥惡液質密切相關。外泌體是細胞間物質、信息傳遞的重要載體,miRNAs則可在轉錄后水平調控基因表達。miRNAs憑借外泌體的保護避免體液中RNA酶的降解,外泌體則攜帶miRNAs特異性靶向受體細胞,參與調控基因表達、信號轉導,在多種疾病發(fā)生發(fā)展過程中發(fā)揮關鍵作用。大量研究表明,外泌體miRNAs可作為代謝疾病相關的分子標記物,并可作為治療靶點來糾正代謝紊亂。因此,本研究旨在通過探索胰腺癌源外泌體是否能導致骨骼肌細胞產(chǎn)生胰島素抵抗,并以外泌體中miRNAs為靶標,對其生物學功能進行初步檢測,從機制層面解釋病理現(xiàn)象,以期為開發(fā)用以臨床的糾正胰腺癌患者代謝紊亂、改進診療的潛在干預靶點提供實驗依據(jù)。方法:KrasLsL-G12D/+,Trp53LSL-R172H/+,Pdx1-Cre(KPC)小鼠是經(jīng)典的胰腺導管細胞癌動物模型,用其來源的胰腺癌細胞(KPC cells),傳代增殖,采用超速離心法從細胞培養(yǎng)上清中提取外泌體(KPC-exosomes),利用外泌體處理分化成熟的小鼠骨骼肌細胞(C2C12 cells),檢測骨骼肌細胞攝取葡萄糖的能力、細胞內(nèi)脂質沉積,及相關信號通路(InsulinPI3K/AktFoxO信號通路)上關鍵蛋白的表達情況,以驗證骨骼肌細胞是否出現(xiàn)胰島素抵抗。在此前提下,以小鼠胰腺導管上皮細胞(murine pancreatic ductal epithelial cell line,MPDC)所分泌的外泌體(MPDC-exosomes)為陰性對照,利用miRNA基因芯片技術,檢測KPC-exosomes中差異表達的miRNAs,并進行KEGG(Kyoto Encyclopedia of Genes and Genomes)信號通路富集分析,以驗證我們所關注信號通路被相關基因富集情況,其余信號通路信息亦可為后續(xù)研究提供思路。利用miRNAs模擬物(miRNA-mimics)及蛋白免疫印跡技術,對表達差異顯著的若干miRNAs進行初步驗證,觀測其對相關信號通路關鍵蛋白表達的影響,為后續(xù)利用人源細胞系、小鼠模型開展轉化研究,提供備選對象。結果:外泌體與分化成熟的骨骼肌細胞孵育24小時即可觀測到其進入肌管結構(外泌體濃度20μg/ml);胰腺癌源外泌體處理骨骼肌細胞24小時內(nèi)(外泌體濃度5μg/ml),并不引起后者大量死亡,但足以導致明顯的細胞內(nèi)脂質沉積(外泌體濃度0.2—0.8μg/ml)、葡萄糖攝取能力明顯下降(外泌體濃度1μg/ml)、IRS/PI3K/Akt信號軸上關鍵蛋白表達下調(外泌體濃度0.2—0.8μg/ml);作為Akt下游重要蛋白FoxO1和Glut4,前者在細胞核內(nèi)發(fā)揮轉錄活性,與骨骼肌胰島素抵抗、脂質沉積關系密切,后者與骨骼肌細胞攝取葡萄糖直接相關。我們利用小鼠尾靜脈注射胰腺癌源外泌體(20μg/只)的方式,借助免疫熒光技術,明確觀測到肌肉組織中Fox01核內(nèi)聚集增多,而Glut4蛋白細胞膜表達明顯減少,此結果在細胞實驗中亦得到證實;我們進一步對外泌體中的miRNAs進行分析,以MPDC-exosomes為對照,利用miRNA芯片技術,獲得KPC-exosomes中差異表達的miRNAs,后續(xù)KEGG通路富集分析印證了這批差異表達miRNAs的靶基因主要涉及通路包括Insulin信號通路,PI3K/Akt信號通路,FOXO通路,以及泛素蛋白酶降解途徑(ubiquitin proteasome system,UPS);我們對miRNA芯片結果行RT-qPCR驗證后,利用免疫印跡技術,檢測了 KPC-exosomes中前9個高表達miRNA對胰島素相關信號通路的影響,發(fā)現(xiàn)miR-450b-3p及miR-151-3p可明顯下調骨骼肌內(nèi)IRS1及P110α的表達。結論:本研究結果提示胰腺癌源外泌體,可以導致骨骼肌細胞胞內(nèi)脂質沉積增多、攝取葡萄糖能力下降,最終促使胰島素抵抗發(fā)生。此病理發(fā)展過程與PI3K/Akt/FoxO1信號通路密切相關,而外泌體中miRNAs可能發(fā)揮重要作用,有望成為改善胰腺癌診療的干預靶點。
[Abstract]:Background: imaging methods commonly used in clinical oncology and other biochemical markers and imaging examination methods on the accuracy of the early diagnosis of pancreatic cancer is still poor. Therefore, looking for a marker of early diagnosis of pancreatic cancer cells, understanding the biological behavior, treatment mode / develop new target is the main direction of the research field. The metabolism of pancreatic cancer the reconstruction of the unique phenomenon of pancreatic cancer, and peripheral tissues between the metabolic interaction based on insulin resistance. This not only leads to the change of tumor metabolism, also cause systemic metabolic disorders, including impaired glucose tolerance is metabolism appear first, followed by the peripheral tissues (muscle, fat) in insulin with the proliferation of tumor resistance, disease, cancer cachexia is closely related. Exosomes are intercellular substance, an important carrier of information transmission, miRNAs can control water level in the post transcriptional gene expression .miRNAs with protection exosomes to avoid degradation of RNA enzyme in the body fluid, exosomes carry miRNAs targeting receptor cells involved in the regulation of gene expression, signal transduction, play a key role in the process of the occurrence and development of many diseases. Many studies show that exosomes miRNAs molecular markers as related metabolic diseases, and can be used as a therapeutic target to correct metabolic disorder. Therefore, this study aims to explore whether pancreatic cancer exosomes derived from skeletal muscle cells can lead to insulin resistance, and miRNAs urinary body as the target, the initial detection of its biological function, explain the pathological phenomena from the level of mechanism, in order to with the development of the metabolism of patients with pancreatic cancer clinical disorders correction, provide experimental basis for potential intervention targets improved diagnosis and treatment. Methods: KrasLsL-G12D/+, Trp53LSL-R172H/+, Pdx1-Cre (KPC) is a classic mouse pancreatic duct The animal model of cancer cells, with pancreatic cancer cell origin (KPC cells), the proliferation, extraction of exosomes from the cell culture supernatant by ultracentrifugation (KPC-exosomes), using exosomes processing the differentiation and maturation of mouse skeletal muscle cells (C2C12 cells), the ability to detect skeletal muscle glucose uptake. Intracellular lipid deposition, and related signaling pathway (InsulinPI3K/AktFoxO pathway) expression of key proteins, to verify whether the skeletal muscle insulin resistance. Under this premise, the mouse pancreatic ductal epithelial cells (murine pancreatic ductal epithelial cell line, MPDC) exosomes secreted (MPDC-exosomes) as negative control miRNA, using gene chip technology to detect the expression of KPC-exosomes in different miRNAs, and KEGG (Kyoto Encyclopedia of Genes and Genomes) signal pathway enrichment analysis, to verify our Pay attention to the signaling is related gene enrichment, the signaling information can also provide ideas for further research. Simulation using miRNAs (miRNA-mimics) and Western blot analysis, preliminary verification to some significant differences in the expression of miRNAs, observing its influence on the key signal pathway related protein expression, for the subsequent use of human derived cells Study on transformation, mouse models provide alternatives. Results: exosomes and skeletal muscle cells were differentiated 24 hours incubation can be observed in myotubes (exosomes concentration 20 g/ml); pancreatic cancer exosomes derived from treatment of skeletal muscle cells within 24 hours (exosome concentration of 5 g/ml), not the latter is caused by the large number of deaths, but enough to cause significant intracellular lipid deposition (exosomes concentration of 0.2 - 0.8 g/ml), glucose uptake was significantly decreased (exosome concentration 1 g/ml, IRS/PI3K/Akt) No. shaft key expression (exosome concentration of 0.2 - 0.8 g/ml); as an important downstream of Akt protein FoxO1 and Glut4, the former play transcriptional activity in the nucleus, and skeletal muscle insulin resistance, lipid deposition is closely related to the latter is directly related to glucose transport in skeletal muscle cells. We use the mouse tail vein injection of pancreas exosomes derived from cancer (20 g/) way, by immunofluorescence technique, clearly observed increased aggregation of nuclear Fox01 in muscle tissue, and cell membrane Glut4 protein expression was significantly reduced, the results of in vitro experiments also confirmed; we further foreign body in the urinary miRNAs was analyzed by MPDC-exosomes. As control, using miRNA chip technology, obtained KPC-exosomes was differentially expressed in miRNAs, subsequent KEGG pathway enrichment analysis confirms these differentially expressed miRNAs target genes mainly involved include Insulin signal path Road, PI3K/Akt pathway, FOXO pathway, and the ubiquitin proteasome degradation pathway (ubiquitin proteasome, system, UPS); miRNA RT-qPCR on our microarray results after validation by Western blot, detect the effect of KPC-exosomes in the first 9 high expression of miRNA on insulin signaling pathway, miR-450b-3p and miR-151-3p could downregulate the expression of skeletal intramuscular IRS1 and P110 alpha. Conclusion: the results of this study suggest that pancreatic cancer derived exosomes, can cause lipid deposition in skeletal muscle cells increased, decreased glucose uptake, ultimately promote insulin resistance. The pathological process and PI3K/Akt/FoxO1 signaling pathway is closely related to the outer body of urinary miRNAs may play an important role. The intervention is expected to become a target for improving the treatment of pancreatic cancer.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.9

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本文編號：1368538