本文選題：鼻咽癌　切入點：腫瘤干細胞　出處：《醫(yī)學研究生學報》2014年11期

【摘要】：目的目前分離和鑒定鼻咽癌干細胞的方法仍不成熟。探索鼻咽癌細胞系干細胞微球體培養(yǎng)方法,并對CNE-2細胞微球體是否具有腫瘤干細胞生物特性(干性)進行鑒定。方法人鼻咽癌細胞CNE2、C666-1細胞在含生長因子的無血清培養(yǎng)基(serum-free medium,SFM)中懸浮培養(yǎng)。流式細胞術(shù)、Transwell小室實驗、裸鼠成瘤實驗、分別檢測CNE2貼壁細胞(CNE2 monolayer,CNE2-MN)及CNE2微球體細胞(CNE2 sphere cells,CNE2-SC)CD133+標記細胞比例,體外侵襲能力、體內(nèi)成瘤能力;CNE2-SC在含血清培養(yǎng)基中貼壁培養(yǎng)觀察其分化能力;實時熒光定量PCR分析CNE2-MN及CNE2-SC相關(guān)干性基因Bmi-1、Oct4、Twist1RNA的表達。結(jié)果 2種細胞系均能在特殊配制的SFM中形成可以穩(wěn)定傳代的微球體,SFM新鮮配制、用細胞分離劑Accutase替代胰酶傳代以及保持細胞的懸浮狀態(tài)均有利于微球體的形成和增殖;在含血清培養(yǎng)基中培養(yǎng)能貼壁分化成CNE2-MN而無明顯差異;CNE2-SC中CD133+細胞比例(98.79%)顯著高于CNE2-MN(0.98%),差異有統(tǒng)計學意義(P0.01);與CNE2-MN相比,CNE2-SC高表達干細胞相關(guān)基因Bmi-1、Oct4、Twist1及EMT標志物N-cadherin、Vimentin。Transwell小室實驗示CNE2-SC與CNE2-MN的穿膜細胞數(shù)分別為(122±6)個/視野及(36±7)個/視野(P0.05);裸鼠成瘤實驗示1×104個CNE2-SC 3周內(nèi)即能成瘤,成瘤率33.3%,而CNE2-MN不能成瘤,1×105個CNE2-SC與CNE2-MN成瘤體積比為(1.750±0.613)cm3vs(0.457±0.291)cm3(P0.05),而1×106個以上2種細胞成瘤體積比為(2.332±0.549)cm3vs(0.669±0.278)cm3(P0.01)。結(jié)論利用特制SFM懸浮培養(yǎng)法可得到鼻咽癌CNE2-SC,這種微球體富集了腫瘤干細胞,將為后續(xù)鼻咽癌干細胞研究打下基礎(chǔ)。
[Abstract]:Objective the method of isolation and identification of nasopharyngeal carcinoma stem cells is still immature.To explore the method of cell microsphere culture of nasopharyngeal carcinoma (NPC) cell line and to identify whether the microsphere of CNE-2 cell has the biological characteristics of tumor stem cells (dryness).Methods Human nasopharyngeal carcinoma cell line CNE2C666-1 was cultured in serum-free medium containing growth factor (SFM).CNE2 adherent cells CNE2 monolayerus CNE2-MNs and CNE2 microspheres somatic cells CNE2 sphere cells CNE2-SCE2-CD133 were detected by flow cytometry and tumorigenesis in nude mice, respectively, and the invasiveness of CNE2 cells in vitro was detected.The differentiation ability of CNE2-SC was observed by adherent culture in serum-containing medium in vivo, and the expression of CNE2-MN and CNE2-SC related dry gene Bmi-1Oct4 Twist1 mRNA was analyzed by real-time fluorescence quantitative PCR.Results two kinds of cell lines could form stable microspheres in specially prepared SFM. It was beneficial for the formation and proliferation of microspheres to replace trypsin passage with Accutase and to maintain the suspension state of the cells.鍦ㄥ惈琛，

本文編號：1711930