Chorioallantoic membrane assay:: Vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model
Chorioallantoic membrane assay:: Vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model
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DOI：
10.1016/s0022-5347(05)65820-x
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发表时间：
2001-10-01
影响因子：
6.6
通讯作者：
Gschwend, JE
中科院分区：
文献类型：
--
作者：
Kunzi-Rapp, K;Genze, F;Gschwend, JE
目的：多年来，绒毛尿囊膜一直被用作一种可靠的生物医学检测系统。在育种的早期阶段，鸡蛋介于体外和体内系统之间，但可能提供一个免疫缺陷的、血管化的测试环境。材料和方法：将人前列腺癌LNCaP、PC-3和Tsu-PRL单细胞悬液以及2株永生化的人前列腺上皮细胞株接种于受精鸡受精卵的绒毛尿囊膜上。立体显微镜下观察肿瘤生长情况和胚胎存活情况。第10天，取出膜，石蜡包埋。苏木精-伊红染色后观察细胞形态。免疫组织化学检测细胞角蛋白、前列腺特异性抗原和雄激素受体的表达及细胞凋亡诱导作用。结果：肿瘤细胞接种于胚外血管系统的绒毛尿囊膜3天后，细胞生长和三维肿瘤形成百分率达到100%。在已建立的肿瘤和侵入绒毛尿囊膜基质的肿瘤细胞旁边检测到强烈的新生血管生成。细胞角蛋白表达以及前列腺特异性抗原和雄激素受体在LNCaP细胞中的表达证实了人类前列腺癌的起源。静脉注射佛波酯12-0-十四酰佛波酯-13-醋酸酯后对LNCaP细胞的体内定量诱导凋亡的评估证实了该模型是一个通用的体内系统。结论：培育的鸡卵血管良好的绒毛尿囊膜是一种适合于早期体内癌症研究的系统。前列腺癌细胞系的可靠生长是可行的，并且可以评估血管内或局部应用抗癌药物后的增殖和凋亡诱导。利用这种检测方法可以大大减少或取代后续的动物实验。
Purpose: Chorioallantoic membranes have been used as a reliable biomedical assay system for many years. Chicken eggs in the early phase of breeding are between in vitro and in vivo systems but may provide an immunodeficient, vascularized test environment. We tested this model as an in vivo system for prostate cancer research.Materials and Methods: Single cell suspensions of LNCaP, PC-3 and Tsu-Prl human prostatic cancer cell lines as well as 2 immortalized normal human prostate epithelial cell lines were inoculated on the chorioallantoic membrane of fertilized chicken eggs on day 5 or 6 of breeding. Tumor growth and viability of the embryo was evaluated by stereo microscopy. At day 10 the membranes were removed and embedded in paraffin. Cell morphology was assessed after hematoxylin and eosin staining. Cellular expression of cytokeratin, prostate specific antigen and androgen receptor as well as apoptosis induction was confirmed by immunohistochemistry.Results: Three days after tumor cell inoculation on the extraembryonic vascular system of the chorioallantoic membrane cell growth and formation of 3-dimensional tumors became apparent in 100% of inoculated membranes. Strong neo-angiogenesis was detected next to the established tumors and tumor cells invading the stroma of the chorioallantoic membrane. Cytokeratin expression as well as prostate specific antigen and androgen receptor in LNCaP cells confirmed the human prostate tumor origin. Assessment of quantitative in vivo apoptosis induction in LNCaP cells after intravenous injection of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate confirmed the model as a versatile in vivo system.Conclusions: The well vascularized chorioallantoic membrane of bred chicken eggs is a suitable system for early in vivo cancer research. Reliable growth of prostate cancer cell lines is feasible and allows the evaluation of proliferation and apoptosis induction after intravascular or topic application of anticancer drugs. Exploitation of this assay enables a substantial reduction in or substitution for subsequent animal experiments.