發(fā)布時間：2018-03-21 16:39

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

【摘要】：目的:建立人前列腺癌原代細胞模型,得到足夠數量的腫瘤細胞。建立成功率高的人前列腺癌上皮細胞的培養(yǎng)方法。本實驗的細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)的轉變過程。原代細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管電泳/質譜聯用(CE/MS)技術,從代謝水平全新的角度研究雄激素阻斷治療(ADT)前后CD44陽性細胞系間的差異代謝組學,為進一步研究雄激素耐藥機制提供了新思路,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記物。本實驗為此研究大方向的第一步,是探索新方向的預實驗。方法:原代細胞培養(yǎng):前列腺癌病人行前列腺根治術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,適當消化過的肉湯樣溶液經離心收集及清洗得到上皮細胞團塊。細胞團塊高密度接種于鋪好Matrigel的T25培養(yǎng)瓶內用PrEGM完全培養(yǎng)基培養(yǎng),7-10天內生長成單層細胞。DHT處理:用Trypsin/EDTA溶液消化細胞以1:2傳代培養(yǎng),細胞生長到70%-80%融合時給予DHT藥物(ADPC組:10nM DHT;ADT 組:1nM DHT,1μM Casodex)處理 24小時。MACS CD44磁珠抗體分選細胞:24小時后根據MACS CD44磁珠抗體說明書分選CD44陽性與陰性細胞。分選完畢四組細胞分別計數以保證細胞數一致。胞內代謝物提取:分液漏斗中,向膜中央傾倒細胞消化液使細胞均勻的分布在濾膜上。溶液完全濾掉后,用1OmL去離子水清洗細胞三次。取下濾膜,依附有細胞的一面朝下,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)皿使細胞懸浮于甲醇中。將1.6mL甲醇細胞懸液轉移至一個新的離心管中,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相。水相分裝于6個超濾管中,蓋緊,4℃離心3-5小時直至全部溶液過濾完畢。經過濾的溶液開口低溫凍干,上機分析。結果:我們首先建立了成功率較高的原代細胞培養(yǎng)方法,幾乎做到每一例標本都成功,保證標本不被浪費。磁珠分選后發(fā)現ADT治療后的CD44陽性細胞普遍增高,提示CD44陽性細胞可在前列腺癌由ADPC轉為CRPC的過程中起重要的作用,在代謝水平研究CD44陽性細胞對CRPC的發(fā)生機制有意義。一共檢測到106種代謝物,最后,通過對比ADT前后CD44陽性細胞的代謝物,發(fā)現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。結論:這些結果提示我們這7種差異化合物有可能是CD44陽性細胞在對抗缺乏雄激素環(huán)境下生存的重要原因。但是由于原代細胞數的有限性,更多的代謝物沒有被檢測到,實驗方法將在后續(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，

本文編號：1644678