ADT治疗前后人前列腺癌CD44阳性细胞的代谢组学初步研究

发布时间：2018-03-21 16:39

  本文选题：前列腺癌　切入点：原代细胞培养　出处：《天津医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的:建立人前列腺癌原代细胞模型,得到足够数量的肿瘤细胞。建立成功率高的人前列腺癌上皮细胞的培养方法。本实验的细胞模型很好的模拟了前列腺癌从雄激素依赖性(ADPC)向非依赖性(CRPC)的转变过程。原代细胞模型可排除很多人为环境干扰因素。在方法上利用高精密度高灵度的毛细管电泳/质谱联用(CE/MS)技术,从代谢水平全新的角度研究雄激素阻断治疗(ADT)前后CD44阳性细胞系间的差异代谢组学,为进一步研究雄激素耐药机制提供了新思路,试图寻找比前列腺特异性抗原(PSA)更加特异的前列腺癌肿瘤标记物。本实验为此研究大方向的第一步,是探索新方向的预实验。方法:原代细胞培养:前列腺癌病人行前列腺根治术取下的肿瘤组织用无血清培养基漂洗表面血污,切成1mm3的小组织块,再次清洗至溶液清亮,加入胶原酶溶液37℃消化过夜,适当消化过的肉汤样溶液经离心收集及清洗得到上皮细胞团块。细胞团块高密度接种于铺好Matrigel的T25培养瓶内用PrEGM完全培养基培养,7-10天内生长成单层细胞。DHT处理:用Trypsin/EDTA溶液消化细胞以1:2传代培养,细胞生长到70%-80%融合时给予DHT药物(ADPC组:10nM DHT;ADT 组:1nM DHT,1μM Casodex)处理 24小时。MACS CD44磁珠抗体分选细胞:24小时后根据MACS CD44磁珠抗体说明书分选CD44阳性与阴性细胞。分选完毕四组细胞分别计数以保证细胞数一致。胞内代谢物提取:分液漏斗中,向膜中央倾倒细胞消化液使细胞均匀的分布在滤膜上。溶液完全滤掉后,用1OmL去离子水清洗细胞三次。取下滤膜,依附有细胞的一面朝下,放入装有2mL甲醇的培养皿中,用封口膜封闭培养皿,超声波水浴震荡培养皿使细胞悬浮于甲醇中。将1.6mL甲醇细胞悬液转移至一个新的离心管中,加入1.6mL氯仿和640μL去离子水,震荡混匀,4℃离心5min。溶液被分为两层:糖代谢物溶于上层水相;脂代谢物溶于下层有机相。水相分装于6个超滤管中,盖紧,4℃离心3-5小时直至全部溶液过滤完毕。经过滤的溶液开口低温冻干,上机分析。结果:我们首先建立了成功率较高的原代细胞培养方法,几乎做到每一例标本都成功,保证标本不被浪费。磁珠分选后发现ADT治疗后的CD44阳性细胞普遍增高,提示CD44阳性细胞可在前列腺癌由ADPC转为CRPC的过程中起重要的作用,在代谢水平研究CD44阳性细胞对CRPC的发生机制有意义。一共检测到106种代谢物,最后,通过对比ADT前后CD44阳性细胞的代谢物,发现7种差异代谢物在ADT治疗后升高,分别为甲基苯胺,m/o/p-氨基酚,苯乙醇胺和酪胺。结论:这些结果提示我们这7种差异化合物有可能是CD44阳性细胞在对抗缺乏雄激素环境下生存的重要原因。但是由于原代细胞数的有限性,更多的代谢物没有被检测到,实验方法将在后续的研究过程中继续改良优化。
[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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