Development of apoptosis-inducing gene therapy using prostate specific membrane antigen (PSMA) antibody

裸鼠制备前列腺癌模型前列腺癌细胞系采用PSMA抗原存在的LNCaP细胞、C4-2细胞及其亚系、PSMA阴性的PC-3细胞。1×10^6分别皮下注射LNCaP和C4-2细胞。C4-2细胞比LNCaP细胞（70%）更容易形成肿瘤团块（100%），适合作为模型。PSMA抗体偶联Caspase 8质粒的体外研究首先制备hTERT启动子驱动的Caspase 8质粒。首先，将克隆的hTERT启动子（384bp）整合到pGL3基本载体中。接下来，将pGL3载体的荧光素酶基因替换为Caspase 8基因。然后，将PSMA抗体与上述制备的载体或pGL3对照载体（sv40o启动子驱动荧光素酶基因）进行生物分离。同时对PSMA抗体进行生物鉴定。将PSMA抗体偶联的荧光素酶质粒（PSMA抗体和pGL3对照载体）和PSMA抗体C…More aspase B DNA质粒（PSMA抗体和hTERT启动子驱动的Caspase 8质粒）分别与亲和素偶联制备。将PSMA抗体结合的荧光素酶质粒引入LNCaP、C4-2和PC-3细胞进行荧光素酶检测。当PC-3细胞的荧光素酶活性为1.0时，LNCaP和C4-2细胞的荧光素酶活性较高，分别为10.3和28.4。此外，在通道染色后，通过流式细胞仪证实了PSMA抗体Caspase 8 DNA质粒在各细胞系中的凋亡情况。结果pc3为1.2%，LNCaP为3.0%，C4-2为4.2%，差异无统计学意义。PSMA抗体偶联Caspase 8质粒1、10和100 μg PSMA抗体偶联荧光素酶质粒分别给药于同源前列腺癌模型（PC-3和C4-2）。给药3天后取肿瘤，用抗荧光素酶抗体进行免疫染色。C4-2肿瘤在10和100 μg剂量下部分染色，PC-3肿瘤完全不染色。随后，在同源前列腺癌模型（PC-3和C4-2）肿瘤生长至直径10mm时，每天给药100 μg PSMA抗体Caspase 8 DNA质粒，观察其抗增殖作用。与对照组（PSMA抗体偶联荧光素酶质粒给药组）相比，虽有一定的抗增殖作用，但差异无统计学意义。少

Preparation of prostate cancer model with nude miceFor prostate cancer cell lines, LNCaP cells in which existence of the PSMA antigen has been confirmed, C4-2 cells, their subline, and PSMA-negative PC-3 cells were used. 1×10^6 LNCaP and C4-2 cells were subcutaneously injected, respectively. The C4-2 cells formed a tumor mass more easily (100%) than LNCaP cells (70%) showing appropriate as a model.In vitro study using PSMA antibody conjugated Caspase 8 plasmidAt the beginning, the hTERT promoter driven Capase 8 plasmid was prepared. First, the cloned hTERT promoter (384bp) was integrated into the pGL3 basic vector. Next, the luciferase gene of the pGL3 vector was replaced by the Caspase 8 gene. Then, the PSMA antibody and the vector prepared above or pGL3 control vector (SV4O promoter driven luciferase gene) were biotynilated. At the same time, the PSMA antibody was biotynilated. The PSMA antibody conjugated luciferase plasmid (PSMA antibody and pGL3 control vector) and PSMA antibody C … More aspase B DNA plasmid (PSMA antibody and hTERT promoter driven Caspase 8 plasmid) were prepared by conjugating the biotynilated vector and antibody with avidin, respectively. The luciferase assay was conducted by introducing the PSMA antibody conjugated luciferase plasmid into the LNCaP, C4-2 and PC-3 cells. The luciferase activities of the LNCaP and C4-2 cells were high, showing 10.3 and 28.4, respectively, when letting those of the PC-3 cells to be 1.0. In addition, the status of apoptosis in each cell line in administration of the PSMA antibody Caspase 8 DNA plasmid was confirmed by the FACS following the tunel staining. The results were 1.2% inPC3, 3.0% in LNCaP and 4.2% in C4-2, showing no significant difference.In vivo study using PSMA antibody conjugated Caspase 8 plasmid1, 10 and 100 μg of the PSMA antibody conjugated luciferase plasmid were administered to the homologous prostate cancer models(PC-3 and C4-2),respectively. The tumor was taken three days after administration and immunostained with the anti-luciferase antibody. The C4-2 tumor was partially stained in 10 and 100 μg administration, whereas the PC-3 tumor was not stained at all. Next, 100 μg of the PSMA antibody Caspase 8 DNA plasmid was administered daily when the tumor grew to the size of 10mm in diameter in the homologous prostate cancer models(PC-3 and C4-2),and its antiproliferative effects were examined. There was no significant difference though some antiproliferative effects were found, compared with the control group (the PSMA antibody conjugated luciferase plasmid administration group). Less