本文選題：前列腺癌　切入點(diǎn)：原代細(xì)胞培養(yǎng)　出處：《天津醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:建立人前列腺癌原代細(xì)胞模型,得到足夠數(shù)量的腫瘤細(xì)胞。建立成功率高的人前列腺癌上皮細(xì)胞的培養(yǎng)方法。本實(shí)驗(yàn)的細(xì)胞模型很好的模擬了前列腺癌從雄激素依賴性(ADPC)向非依賴性(CRPC)的轉(zhuǎn)變過程。原代細(xì)胞模型可排除很多人為環(huán)境干擾因素。在方法上利用高精密度高靈度的毛細(xì)管電泳/質(zhì)譜聯(lián)用(CE/MS)技術(shù),從代謝水平全新的角度研究雄激素阻斷治療(ADT)前后CD44陽性細(xì)胞系間的差異代謝組學(xué),為進(jìn)一步研究雄激素耐藥機(jī)制提供了新思路,試圖尋找比前列腺特異性抗原(PSA)更加特異的前列腺癌腫瘤標(biāo)記物。本實(shí)驗(yàn)為此研究大方向的第一步,是探索新方向的預(yù)實(shí)驗(yàn)。方法:原代細(xì)胞培養(yǎng):前列腺癌病人行前列腺根治術(shù)取下的腫瘤組織用無血清培養(yǎng)基漂洗表面血污,切成1mm3的小組織塊,再次清洗至溶液清亮,加入膠原酶溶液37℃消化過夜,適當(dāng)消化過的肉湯樣溶液經(jīng)離心收集及清洗得到上皮細(xì)胞團(tuán)塊。細(xì)胞團(tuán)塊高密度接種于鋪好Matrigel的T25培養(yǎng)瓶?jī)?nèi)用PrEGM完全培養(yǎng)基培養(yǎng),7-10天內(nèi)生長(zhǎng)成單層細(xì)胞。DHT處理:用Trypsin/EDTA溶液消化細(xì)胞以1:2傳代培養(yǎng),細(xì)胞生長(zhǎng)到70%-80%融合時(shí)給予DHT藥物(ADPC組:10nM DHT;ADT 組:1nM DHT,1μM Casodex)處理 24小時(shí)。MACS CD44磁珠抗體分選細(xì)胞:24小時(shí)后根據(jù)MACS CD44磁珠抗體說明書分選CD44陽性與陰性細(xì)胞。分選完畢四組細(xì)胞分別計(jì)數(shù)以保證細(xì)胞數(shù)一致。胞內(nèi)代謝物提取:分液漏斗中,向膜中央傾倒細(xì)胞消化液使細(xì)胞均勻的分布在濾膜上。溶液完全濾掉后,用1OmL去離子水清洗細(xì)胞三次。取下濾膜,依附有細(xì)胞的一面朝下,放入裝有2mL甲醇的培養(yǎng)皿中,用封口膜封閉培養(yǎng)皿,超聲波水浴震蕩培養(yǎng)皿使細(xì)胞懸浮于甲醇中。將1.6mL甲醇細(xì)胞懸液轉(zhuǎn)移至一個(gè)新的離心管中,加入1.6mL氯仿和640μL去離子水,震蕩混勻,4℃離心5min。溶液被分為兩層:糖代謝物溶于上層水相;脂代謝物溶于下層有機(jī)相。水相分裝于6個(gè)超濾管中,蓋緊,4℃離心3-5小時(shí)直至全部溶液過濾完畢。經(jīng)過濾的溶液開口低溫凍干,上機(jī)分析。結(jié)果:我們首先建立了成功率較高的原代細(xì)胞培養(yǎng)方法,幾乎做到每一例標(biāo)本都成功,保證標(biāo)本不被浪費(fèi)。磁珠分選后發(fā)現(xiàn)ADT治療后的CD44陽性細(xì)胞普遍增高,提示CD44陽性細(xì)胞可在前列腺癌由ADPC轉(zhuǎn)為CRPC的過程中起重要的作用,在代謝水平研究CD44陽性細(xì)胞對(duì)CRPC的發(fā)生機(jī)制有意義。一共檢測(cè)到106種代謝物,最后,通過對(duì)比ADT前后CD44陽性細(xì)胞的代謝物,發(fā)現(xiàn)7種差異代謝物在ADT治療后升高,分別為甲基苯胺,m/o/p-氨基酚,苯乙醇胺和酪胺。結(jié)論:這些結(jié)果提示我們這7種差異化合物有可能是CD44陽性細(xì)胞在對(duì)抗缺乏雄激素環(huán)境下生存的重要原因。但是由于原代細(xì)胞數(shù)的有限性,更多的代謝物沒有被檢測(cè)到,實(shí)驗(yàn)方法將在后續(xù)的研究過程中繼續(xù)改良優(yōu)化。
[Abstract]:Objective: to establish a primary cell model of human prostate cancer. A sufficient number of tumor cells were obtained. The culture method of human prostate cancer epithelial cells with high success rate was established. The cell model of this experiment is a good simulation of the transition of prostate cancer from androgen dependent ADPCs to independent CRPCs. The primary cell model can eliminate many man-made environmental interference factors. The method uses high precision and high precision capillary electrophoresis / mass spectrometry combined with CEP / MS, The differential metabolomics between CD44 positive cell lines before and after androgen blockade therapy was studied from a completely new point of view of metabolic level, which provided a new idea for further study of androgen resistance mechanism. We are trying to find more specific tumor markers for prostate cancer than prostate specific antigen (PSAs). Methods: primary cell culture: the tumor tissues taken from prostate cancer patients by radical prostatectomy were rinsed with serum-free medium to bleach the surface blood stain, cut into small tissue blocks of 1mm3, then washed again to clear the solution. Add collagenase solution to digest overnight at 37 鈩，

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