癌源性外泌体介导胰腺癌病程中骨骼肌细胞胰岛素抵抗的产生及其机制探讨

发布时间：2018-01-02 09:01

本文关键词：癌源性外泌体介导胰腺癌病程中骨骼肌细胞胰岛素抵抗的产生及其机制探讨　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:临床上常用的肿瘤学标记物等生化检查手段及影像学检查方法对胰腺癌早期诊断的准确性仍较差。因此,寻找早期诊断的标记物,理解胰腺癌细胞生物学行为,开发新的治疗模式/靶点是目前胰腺癌研究领域的主要方向。胰腺癌有独特的代谢重构现象,与外周组织间发生着基于胰岛素抵抗的代谢交互作用。这不仅导致肿瘤自身代谢改变,还引起全身代谢紊乱,其中葡萄糖耐量异常是首先出现的代谢异常状况,随后发生的外周组织(骨骼肌、脂肪)胰岛素抵抗则与癌瘤增殖、病情进展,癌症恶液质密切相关。外泌体是细胞间物质、信息传递的重要载体,miRNAs则可在转录后水平调控基因表达。miRNAs凭借外泌体的保护避免体液中RNA酶的降解,外泌体则携带miRNAs特异性靶向受体细胞,参与调控基因表达、信号转导,在多种疾病发生发展过程中发挥关键作用。大量研究表明,外泌体miRNAs可作为代谢疾病相关的分子标记物,并可作为治疗靶点来纠正代谢紊乱。因此,本研究旨在通过探索胰腺癌源外泌体是否能导致骨骼肌细胞产生胰岛素抵抗,并以外泌体中miRNAs为靶标,对其生物学功能进行初步检测,从机制层面解释病理现象,以期为开发用以临床的纠正胰腺癌患者代谢紊乱、改进诊疗的潜在干预靶点提供实验依据。方法:KrasLsL-G12D/+,Trp53LSL-R172H/+,Pdx1-Cre(KPC)小鼠是经典的胰腺导管细胞癌动物模型,用其来源的胰腺癌细胞(KPC cells),传代增殖,采用超速离心法从细胞培养上清中提取外泌体(KPC-exosomes),利用外泌体处理分化成熟的小鼠骨骼肌细胞(C2C12 cells),检测骨骼肌细胞摄取葡萄糖的能力、细胞内脂质沉积,及相关信号通路(InsulinPI3K/AktFoxO信号通路)上关键蛋白的表达情况,以验证骨骼肌细胞是否出现胰岛素抵抗。在此前提下,以小鼠胰腺导管上皮细胞(murine pancreatic ductal epithelial cell line,MPDC)所分泌的外泌体(MPDC-exosomes)为阴性对照,利用miRNA基因芯片技术,检测KPC-exosomes中差异表达的miRNAs,并进行KEGG(Kyoto Encyclopedia of Genes and Genomes)信号通路富集分析,以验证我们所关注信号通路被相关基因富集情况,其余信号通路信息亦可为后续研究提供思路。利用miRNAs模拟物(miRNA-mimics)及蛋白免疫印迹技术,对表达差异显著的若干miRNAs进行初步验证,观测其对相关信号通路关键蛋白表达的影响,为后续利用人源细胞系、小鼠模型开展转化研究,提供备选对象。结果:外泌体与分化成熟的骨骼肌细胞孵育24小时即可观测到其进入肌管结构(外泌体浓度20μg/ml);胰腺癌源外泌体处理骨骼肌细胞24小时内(外泌体浓度5μg/ml),并不引起后者大量死亡,但足以导致明显的细胞内脂质沉积(外泌体浓度0.2—0.8μg/ml)、葡萄糖摄取能力明显下降(外泌体浓度1μg/ml)、IRS/PI3K/Akt信号轴上关键蛋白表达下调(外泌体浓度0.2—0.8μg/ml);作为Akt下游重要蛋白FoxO1和Glut4,前者在细胞核内发挥转录活性,与骨骼肌胰岛素抵抗、脂质沉积关系密切,后者与骨骼肌细胞摄取葡萄糖直接相关。我们利用小鼠尾静脉注射胰腺癌源外泌体(20μg/只)的方式,借助免疫荧光技术,明确观测到肌肉组织中Fox01核内聚集增多,而Glut4蛋白细胞膜表达明显减少,此结果在细胞实验中亦得到证实;我们进一步对外泌体中的miRNAs进行分析,以MPDC-exosomes为对照,利用miRNA芯片技术,获得KPC-exosomes中差异表达的miRNAs,后续KEGG通路富集分析印证了这批差异表达miRNAs的靶基因主要涉及通路包括Insulin信号通路,PI3K/Akt信号通路,FOXO通路,以及泛素蛋白酶降解途径(ubiquitin proteasome system,UPS);我们对miRNA芯片结果行RT-qPCR验证后,利用免疫印迹技术,检测了 KPC-exosomes中前9个高表达miRNA对胰岛素相关信号通路的影响,发现miR-450b-3p及miR-151-3p可明显下调骨骼肌内IRS1及P110α的表达。结论:本研究结果提示胰腺癌源外泌体,可以导致骨骼肌细胞胞内脂质沉积增多、摄取葡萄糖能力下降,最终促使胰岛素抵抗发生。此病理发展过程与PI3K/Akt/FoxO1信号通路密切相关,而外泌体中miRNAs可能发挥重要作用,有望成为改善胰腺癌诊疗的干预靶点。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.9

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