【作者】 郭宁；

【导师】 巩平；

【作者基本信息】 石河子大学 ， 免疫学， 2010， 硕士

【摘要】 目的：用自制鼠尾胶原建立胶滴肿瘤药敏检测技术(collagen gel droplet culture-drug sensitivity test, CD-DST),探讨该技术在肿瘤药物敏感性检测中的应用价值,从而为临床化疗方案的筛选提供一条有效途径。方法：首先,构建体外细胞培养的三维立体模型,即将Hela细胞加至由自制鼠尾胶原、10×F12培养基以及0.5 mol/L的NaOH溶液混合形成的胶原混合液中并使其凝固,形成利于细胞生长的鼠尾胶原凝胶滴,并观察培养在此模型内细胞的生长形态与二维培养形态学上的不同；进一步,建立胶滴肿瘤药敏检测技术(CD-DST),该技术主要包括Hela细胞在胶滴内培养、化疗药物处理、无药培养和图像分析四部分内容,同时与MTT检测法进行相关性比较；最后,将该技术初步应用于12例活检宫颈癌标本的体外药敏检测。结果：1、细胞-鼠尾胶原混合液置于37℃,CO2体积分数为5%的饱和湿度培养箱中15 min即可凝固,形成鼠尾胶原凝胶滴(即三维立体模型),培养在其内的Hela细胞生长良好,呈克隆样增殖,细胞以团块状分散在胶滴内；二维培养的细胞呈不规则多边形,以单细胞形式平铺分散样生长。2、用鼠尾胶原成功建立了胶滴肿瘤药敏检测技术(CD-DST),两种不同的方法对Hela细胞进行药敏检测时发现CD-DST法所测的药物抑制率明显高于MTT法(P<0.05),但两者的体外药敏结果有较好的一致性。3、12例宫颈癌标本通过CD-DST法进行药敏检测,其中6例标本培养成功并用于药敏检测,标本可评价率为50%(6/12)；不同标本对化疗药物的敏感性不同,体现了肿瘤患者之间存在个体差异。结论：1、本研究建立了体外细胞培养的三维立体模型,该模型的建立对于观察细胞在模拟类似人体环境中的生长状态和增殖方式提供了一个直观、可靠的体外培养模型,同时为以后研究癌细胞在体内的生长、转移、耐药及抗癌药物敏感性检测等方面奠定了基础。2、用自制鼠尾胶原建立的胶滴肿瘤药敏检测技术(CD-DST)可用于体外敏感性药物的筛选,并可反映出个体对肿瘤药敏的异质性；检测时细胞用量少(3×103个细胞/孔),扩大了临床应用范围,在体外药敏检测中具有可行性。更多还原

【Abstract】 Objective:To establish the collagen gel droplet embedded-culture drug-sensitivity test (CD-DST) with rat tail collagen and discuss the applied value of the technique above, and then to provide a available route for the scheme of clinical chemotherapy.Methods:The first, to construct three-diamensional formwork of cell culture in vitro, Id est, Hela cell suspension was added to a collagen solution which was a mixture of self-made rat-tail collagen, ten fold concentrated F-12 medium and 0.5 mol/L NaOH solution, and made collagen-cell mixture coagulate. Hela cells were cultured in the formwork, compared morphology with two-dimensional cultivation; then, to set up collagen gel droplet drug sensitivity test, this method consists of collagen gel droplet embedded culture step, exposure anticancer drugs, a drug-free culture step, and evaluation of anticancer effect by image analysis. drug sensitivity is detected by MTT and CD-DST, and evaluate the feasibility of this method; the final, applying this method to chemosensitivities test of twelve biopsy specimens in vitro.Results:1. Cell-collagen mixture could coagulate and form rat-tail collagen gel drop at 37℃in a CO2 incubator for 15 minutes. Hela cells cultured in three-diamensional formwork grew better, clonally proliferated, cells dispersed in it, appearance of cells cultured two-dimensionally was polygon、dispersal and individual.2. We have established the collagen gel droplet embedded-culture drug-sensitivity test (CD-DST) with rat tail-collagen. Comparing CD-DST with MTT, inhibition ratio with CD-DST was higher than that with MTT on identical drug concentration, but two results of the drug sensitivity had better consistency.3.12 cases of uterine cervix cancer had been detected by the CD-DST,6 cases could be separated tumor cells and be used for drug sensitive test, sample appreciation rate was 50% (6/12); drug sensitive of the 6 cases to 4 chemotherapeutics were different.Conclusions:1. This study establishes three-diamensional formwork of cell culture in vitro, It provides a visual and reliable in vitro model of observing the cells growth status and proliferation in the simulation of human environment similar, and lay a foundation in the future for the growth of cancer research in vivo, transfer, resistance and anticancer drug sensitivity test,etc.2. The collagen gel droplet embedded-culture drug-sensitivity test (CD-DST) with rat-tail collagen could be used for screening of drug-sensitivity test in vitro and reflect the heterogeneity of individuals to drug-sensitivity of tumors. Further more, the amount of cells for detecting is few (3×10 cells/hole) and enlarging the extension of clinical application. It is feasible for drug-sensitivity test in vitro.更多还原