依赖PSCAE和CD46的组织特异性溶瘤腺病毒对膀胱癌干细胞的作用及机制研究

The recurrence, metastasis and resistance to radiation and chemotherapy of bladder cancer are related to the cancer stem cells. To improve the targeting of oncolytic adenovirus for cancer stem cells and explore the regulatory mechanism of androgen agonists by PSCAE, we constructed new oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1A and Ad5/F11p-PSCAE-UPII-E1A-AR, which precise control UPⅡ by stem cell affinity PSCAE and entrance the bladder cancer by CD46 after engineering cilia transformation. We will establish subcutaneous, orthotopic and heterotopic transplantation tumor model in nude mice applying the bladder cancer stem cells with ALDH positive selected by Flow Cytometry (FCM). The DNA damage, apoptosis, the expression of nucleic acid and protein related autophagy were observed by utilizing RT-PCR and Western Blot. Rotary Disc Type Living Cell Work Station, Clone Formation Assay, Transwell Invasion Assay, Detection of Circulating Tumor Cells in CellSearch System, Autophagy Analysis and other technologies are also introduced in vivo and in vitro experiments. We adopt the above experiment methods to investigate the oncolytic adenovirus related to invasion and dissolution cancer cells,dynamic process and the ability of virus infection, mechanism of the killing cancer cells, and the mechanism of new oncolytic adenovirus in combination with radiation and chemotherapy. We will also study the role of oncolytic adenovirus in the prevention of bladder cancer metastasis and invasion by observing the influence of oncolytic adenovirus on circulating cancer cells. The research project will definitely provide new evidence and viable option for tissue specific oncolytic adenovirus gene therapy to bladder cancer.

膀胱癌复发转移及放化疗抵抗与肿瘤干细胞有关。我们构建了通过亲干细胞性的PSCAE精确调控UPII和纤毛改造依赖CD46进入癌细胞的溶瘤病毒Ad5/F11p-PSCAE-UPII-E1A和Ad5/F11p-PSCAE-UPII-E1A-AR，通过PSCAE、AR和雄激素提高对肿瘤干细胞的靶向性。用流式细胞仪分选ALDH+膀胱癌干细胞，建立裸鼠皮下、原位及异位移植瘤模型，用RT-PCR、Western Blot观察DNA损伤、凋亡、细胞自噬相关核酸及蛋白表达。采用转盘式活细胞检测、克隆形成实验、Transwell侵袭实验、CellSearch循环癌细胞检测、细胞自噬分析等技术进行体内、外实验。观察溶瘤动态过程和感染能力，探讨杀瘤及放化疗协同作用和机理。明确溶瘤病毒对循环癌细胞的影响和在预防膀胱癌转移和侵袭中的作用。为组织特异性溶瘤病毒基因治疗膀胱癌提供新的依据。

膀胱癌复发转移及放化疗抵抗与肿瘤干细胞有关。我们构建了通过亲干细胞性的PSCAE精确调控UPII和纤毛改造依赖CD46进入癌细胞的溶瘤病毒Ad5/F11p-PSCAE-UPII-E1A和Ad5/F11pPSCAE-UPII-E1A-AR，通过PSCAE、AR和雄激素⚳㖬对肿瘤干细胞的靶向性。用流式细胞仪分选ALDH+膀胱癌干细胞，建立裸鼠皮下、原位及异位移植瘤模型，用RT-PCR、Western Blot观察DNA损伤、凋亡、细胞自噬相关核酸及蛋白表达。采用转盘式活细胞检测、克隆形成实验、Transwell侵袭实验、CellSearch循环癌细胞检测、细胞自噬分析等技术进行体内、外实验。观察溶瘤动态过程和感染能力，探讨杀瘤及放化疗协同作用和机理。明确溶瘤病毒对循环癌细胞的影响和在预防膀胱癌转移和侵袭中的作用。为组织特异性溶瘤病毒基因治疗膀胱癌提供新的依据。