碘-125标记PSA/hTERT双调控溶瘤腺病毒对激素非依赖性前列腺癌靶向治疗及瘤微环境影响的实验研究

Advanced prostate cancer is a clinical problem, radionuclide 125-I is a commonly used treatment method, but the lack of targeted.The Oncolytic adenovirus is concerned because of tumor specificity. The research group construct the prostate specific antigen ( PSA ) and telomerase ( hTERT ) promoter regulated oncolytic adenovirus,which could targeted therapy efficiently for prostate cancer cell. We labled radionuclide125-I to protein molecules of oncolytic adenovirus surface, and combine the two. Labeled radionuclide125-I with the virus reaches the prostate cancer site, separated radionuclide uptake in tumor cells after viral replication,to overcome the lack of radionuclide tumor targeting shortcomings.Because the virus can increase the sensitivity of radiotherapy on tumor tissue, and radionuclide 125-I makes viral oncolytic effect enhancement, thus kill all tumor cells completely, by the synergistic effect of tumor growth microenvironment. On the basis of detecting targeting and killing capacity of the markers for prostate cancer cell lines in vitro,established mouse prostate cancer model, by the methods of direct and intravenous injection, detecting the transfection efficiency, antitumor effect, radionuclide concentration distribution,tumor microvasculature,prostate cancer stem cells, tumor microenvironment changes and so on. Analysis the Anticancer therapeutic mechanism, Provide a new method of targeted therapy for prostate cancer.

晚期前列腺癌是临床治疗难题，核素碘-125是常用治疗方法但缺乏靶向性。溶瘤腺病毒因为具有肿瘤特异性而受到重视。本课题构建前列腺特异性抗原（PSA）和端粒酶（hTERT）启动子双调控的溶瘤腺病毒，使其对前列腺癌细胞有高效的靶向性。我们设想将碘-125标记到溶瘤腺病毒表面的蛋白质分子上而将二者结合起来，标记的碘-125可随病毒到达前列腺癌细胞，病毒复制后核素脱离而浓聚在肿瘤细胞内，克服核素缺乏肿瘤靶向性的缺点。因病毒能够增加瘤细胞对放疗的敏感性，而碘-125辐射使得病毒溶瘤作用增强，产生协同作用进而杀伤所有肿瘤细胞并影响肿瘤生长的微环境。在检测标记物对体外培养前列腺癌细胞株靶向性、杀伤能力基础之上，建立小鼠前列腺癌模型，通过直接注射和静脉内给药的方法，检测标记物体内转染效率、抗瘤效果、核素的浓聚分布、肿瘤内微血管和前列腺癌干细胞等瘤微环境的变化，分析抗癌治疗机制，为前列腺癌靶向治疗提供新方法。

[摘要] 目的 核素125I标记hTERT/PSA双调控增殖型溶瘤腺病毒，构建125I－RSOAds－hTERT/PSA核素-溶瘤病毒标记物，并考察不同反应条件下病毒标记物标记率的影响。方法 125I标记采用N-溴代琥珀酰亚胺(NBS)作为氧化剂进行标记，分别设定不同浓度的溶瘤病毒和NBS进行作用，确定最佳的标记条件。分别考察125I的用量、反应时间、pH 值、反应体积对125I－RSOAds－hTERT/PSA核素-溶瘤病毒标记物标记率的影响。采用凝胶柱层析法分离纯化核素-溶瘤腺病毒标记物；纸层析法测定125I－RSOAds－hTERT/PSA标记物不同时间的放射性纯度。结果 125I－RSOAds－hTERT/PSA放化纯度达95 %以上，4 ℃冰箱放置7 d 其放化纯度基本稳定，保持在93 %～94 %。125 I 用量为0.5ul （约0.2mCi，7 .4 MBq），NBS用量为25ug， 8×109 VP/ml 125 I－RSOAds－hTERT/PSA病毒液用量为100ul，反应时间为3min，pH 7.5，加入PBS体积120uL为最优条件。结论 核素125I标记hTERT/PSA双调控增殖型溶瘤腺病毒确切可行，标记物在一定条件下放化纯度稳定。