外泌體microRNA定量PCR 實(shí)驗(yàn) 外泌體(Exosome)是由細(xì)胞分泌而來(lái)的微小囊泡,直徑約為30-100 nm,具有杯狀形態(tài)、雙層膜結(jié)構(gòu),天然存在于血液、尿液、唾液、母乳和細(xì)胞培養(yǎng)基等生物體液中。包括腫瘤細(xì)胞在內(nèi)幾乎所有類型的細(xì)胞(免疫細(xì)胞、神經(jīng)細(xì)胞、干細(xì)胞),都可以產(chǎn)生并釋放exosome。Exosome可通過(guò)細(xì)胞膜受體直接激活受體細(xì)胞,也可運(yùn)輸?shù)鞍踪|(zhì)、mRNA、miRNA、lncRNA、circRNA,甚至細(xì)胞器進(jìn)入受體細(xì)胞,參與細(xì)胞間通訊。Exosome在免疫應(yīng)答、炎癥反應(yīng)、血管生成、凋亡、凝血和廢物處理等生理過(guò)程發(fā)揮關(guān)鍵作用,可作為多種疾病的早期診斷標(biāo)記物,也能作為靶向藥物的載體進(jìn)行疾病治療。 miRNA是一類22nt左右大小的非編碼分子, 它可以通過(guò)外泌體從一個(gè)地方轉(zhuǎn)運(yùn)到另外一個(gè)地方行使基因沉默功能。通過(guò)檢測(cè)外泌體中miRNA表達(dá)變化,可以發(fā)現(xiàn)外泌體中miRNA特異性功能。 圖1外泌體產(chǎn)生過(guò)程的示意圖 圖2外泌體miRN PCR擴(kuò)增曲線 圖3外泌體miRN PCR擴(kuò)增曲線 實(shí)驗(yàn)流程: 樣品要求 血清樣品:2-4ml 外泌體樣品:1ml RNA樣品:500ng 參考文獻(xiàn) Tkach M, et al. Communication by Extracellular Vesicles: Where We Are and Where We Need to Go. Cell. 2016 Mar 10;164(6):1226-32 Enderle D, Spiel A, Coticchia CM, Berghoff E, Mueller R, Schlumpberger M, et al.Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method. PLoS ONE (2015)10(8): e0136133. doi:10.1371/journal.pone.0136133 Zhou W, Fong M Y, Min Y, et al. Cancer-Secreted miR-105 Destroys Vascular Endothelial Barriers to Promote Metastasis[J]. Cancer cell. 2014, 25(4): 501-515. Takahashi KJ, Yan IK, Wood J, et al. Involvement of Extracellular Vesicle Long Noncoding RNA (linc-VLDLR) in Tumor Cell Responses to Chemotherapy. Mol Cancer Res.2014 ,12(10): 1377–87. Li Y, Zheng QP, Bao CY, et al. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis. Cell Res. 2015, 25(8):981-4. Melo SA, Sugimoto H, O'Connell JT, et al. Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis. Cancer Cell, 2014. |