光动力纳米载体联合si-P3H4治疗膀胱癌的初步探索  

Photodynamic nanocarriers combined with siP3H4 for precise targeting therapy in the treatment of bladder cancer: a pilot study

作　　者：顾志波 郝林[2] 陆明[1] 陈建刚[1] Zhibo Gu;lin Hao;Ming Lu;Jiangang Chen(Department of Urology,Nantong First People's Hospital(the Second Affiliated Hospital of Nantong University),Jiangsu 226200,China;Department of Urology,Xuzhou Central Hospital(Xuzhou Clinical College of Xuzhou Medical University),Jiangsu 221009,China)

机构地区：[1]南通市第一人民医院(南通大学第二附属医院)泌尿外科,江苏226000 [2]徐州市中心医院(徐州医科大学徐州临床学院)泌尿外科,江苏221009

出　　处：《中华腔镜泌尿外科杂志（电子版）》2023年第6期633-641,共9页Chinese Journal of Endourology(Electronic Edition)

基　　金：2020年南通市科技局面上项目(MS12020025);江苏省自然科学基金面上项目(BK20221220)。

摘　　要：目的探讨光动力纳米载体联合小干扰-脯氨基3-羟化酶家族成员(si-P3H4)精准治疗膀胱肿瘤的疗效。方法RT-qPCR和Western Blot分别检测转染siRNA后膀胱癌EJ和T24细胞株中的P3H4mRNA和蛋白表达量。CCK8、划痕实验和Transwell小室检测敲低P3H4后对EJ和T24膀胱癌细胞增殖、迁移、侵袭的影响。以氨基树脂为底物合成高分子纳米载体CH3-R9-cRGD,将纳米载体药物包裹si-P3H4、光敏剂Ce6转染至膀胱癌细胞(HCV29细胞),检测不同pH及激光照射条件下药物体外释放情况。同时,探索纳米药物与膀胱癌细胞靶向结合内吞机制,检测细胞内活性氧(ROS)水平。将不同分组纳米复合物转染至膀胱癌细胞,CCK8法检测各组细胞活力,体内试验进一步探索不同分组纳米复合物肿瘤抑制能力。结果RT-qPCR显示EJ组和T24组P3H4mRNA表达量分别降低至对照组的68.4%和57.1%。Western Blot显示EJ组和T24组P3H4蛋白表达较阴性对照组分别下降至20.3%和36.5%。CCK8实验吸光度A值为EJ组相对于对照组96 h:(0.785±0.084) vs (1.358±0.064),t=12.06,P<0.01;T24组相对于对照组96 h:(0.833±0.065) vs (1.346±0.545),t=13.415,P<0.01。划痕实验细胞愈合率:EJ组相对于对照组为(47.8±4.32)% vs (68.60±4.39)%,t=7.545,P<0.01;T24组相对于对照组为(50.40±3.64)% vs (70.61±3.85)%,t=8.521,P<0.01。Transwell细胞穿膜数:EJ组相对于对照组为[(302.71±7.56) vs (130.42±3.95)]个,t=53.40,P<0.01;T24组[(99.56±4.50) vs (35.22±6.28)]个,t=24.974,P<0.01。成像结果显示多肽纳米载体上具有良好的渗透性,可以同时将si-P3H4和Ce6靶向运输至膀胱癌细胞中。CCK8法检测结果显示当纳米复合物CH3-R9-cRGD&Ce6的浓度为50 μg/ml时,激光照射组的细胞活力均低于无激光照射组,且纳米探针+激光+siP3H4组细胞活力最低(F=299.57,P<0.05)。体内试验显示纳米复合物/si-P3H4/激光抑制肿瘤细胞增强效果最为显著。结论纳米载体可以同时将si-P3H4和Ce6靶向运输�Objective To explore the efficacy of photodynamic nanocarriers combined with si-P3H4 in the precise treatment of bladder tumors.Methods RT-qPCR and Western Blot were used to detect the expression of P3H4 mRNA and protein in EJ and T24 cell lines of bladder cancer after siRNA transfection.CCK8,scratch test and Transwell chamber were used to detect the effect of P3H4 knockdown on proliferation,migration and invasion of EJ and T24 bladder cancer cells.The polymer nanocarrier CH3-R9-cRGD was synthesized with amino resin as substrate,and the drug encapsulated si-P3H4 and photosensitizer Ce6 were transfected into bladder cancer cells(HCV29 cells)to detect the drug release in vitro under different pH and laser irradiation conditions.At the same time,the mechanism of targeted binding endocytosis of nano drugs and bladder cancer cells was explored,and the level of intracellular reactive oxygen species(ROS)was detected.Different groups of nanocomposites were transfected into bladder cancer cells,and the cell viability of each group was detected by CCK8 method.In vivo experiments were conducted to further explore of the tumor inhibitory ability of different groups of nanocomposites.Results The P3H4mRNA expression levels in the EJ group and T24 group decreased to 68.4%and 57.1%of the control group,respectively,according to RT qPCR.Western blot showed that the expression of P3H4 protein in the EJ and T24 groups decreased to 20.3%and 36.5%,respectively,compared to the negative control group.The absorbance A value of CCK8 experiment was(0.785±0.084)vs(1.358±0.064)in the EJ group compared to the control group at 96 hours,t=12.06,P<0.01;Compared to the control group at 96 hours,the T24 group showed(0.833±0.065)vs(1.346±0.545),t=13.415,P<0.01.The cell healing rate in the scratch experiment was(47.8±4.32)%in the EJ group compared to the control group,(68.60±4.39)%,t=7.545,P<0.01;The T24 group compared to the control group was(50.40±3.64)%vs(70.61±3.85)%,t=8.521,P<0.01.Transwell cell penetration count:The EJ group had[(302.7

关 键 词：膀胱癌 光动力 纳米载体 小干扰RNA(siRNA) P3H4 

分 类 号：R737.14[医药卫生—肿瘤]