Adjuvant gene therapy for pancreatic cancer using hybrid type liposome vector

大肠埃希菌尿嘧啶磷酸核糖转移酶(UPRT)催化5-FU合成FUMP，增加FdUMP的生成，抑制胸腺嘧啶合成功能，从而产生细胞毒作用。我们构建了表达UPRT的突变腺病毒载体(AxElAdB-UPRT)，该载体可在无P53功能的肿瘤细胞中优先复制。用腺病毒载体(Ax-UPRT和Ax-LacZ)将UPRT或LacZ基因导入P53功能缺失的胰腺癌细胞，在含有不同浓度5-FU的培养液中培养。采用腹膜腔播散性ASPC-1肿瘤模型评价AxE1AdB-UPRT/5-FU治疗的优势。小鼠在接种肿瘤后10天开始腹腔注射载体，然后给予5-FU(10 mg/kg)，连续7天。4周后处死小鼠，测量肿瘤重量。5种胰腺癌细胞对5-FU的敏感性显著增加(7.1…-72倍以上)。Ax-Uprt/5-FU组肿瘤重量(0.24g)明显低于Ax-LacZ/5-FU组(0.56g)(P&lt；0.001)。此外，AxElAdB-UPrT/5-FU组与Ax-UPrT/5-FU组相比，其抑制率显著降低(P=0.042)。此外，由于减少了5-FU的剂量，FdUMP对消化道的不良影响也减少了。肿瘤的生长和转移能力依赖于血管生成。血管生成是肿瘤从一小群无害的突变细胞过渡到大量恶性生长的关键步骤之一，能够扩散到全身的其他器官。我们分别用腺病毒载体Adsflt和AdlacZ分别将可溶性血管内皮生长因子受体(Flt1)和LacZ基因导入PANC-1细胞。从植入后第8天至第12天，每天直接向肿瘤内注射Adsflt或AdlacZ(3.5×10~(-7)pfu)或100μL。此外，我们还将野生型PANC-1及其转基因载体植入背侧皮皱室，并利用体内显微镜系统监测肿瘤血管生成。所有细胞系条件培养液中的VEGF水平(平均为1969pg/ml；PANC-1,980pg/ml)均高于HUVEC培养上清液中的水平(8pg/ml)。RT-PCR和Northern印迹分析检测到ALL细胞中有血管内皮生长因子mRNA的表达，并经DNA测序证实。Adsflt感染细胞的肿瘤生长率低于AdlacZ组和未治疗组，差异有统计学意义。皮褶小室观察到野生型PANC-1和LacZ转染体能促进肿瘤血管生成，而可溶性Flt-1转染体不能起到这种作用。较少

E. coli uracil phosphoribosyltransferase (UPRT) catalyzes the synthesis of FUMP from 5-FU, resulting in increased production of FdUMP, which induces cytotoxic effect by inhibiting the function of thymidylate synthesis. We constructeded UPRT expressing mutant adenovirus vector (AxElAdB-UPRT) that replicates preferentially in tumor cells without p53 function. Pancreatic cancer cells lacking p53 function were transfected with either UPRT or lacZ gene using adenovirus vectors (Ax-UPRT and Ax-lacZ) and cultured in medium containing various concentration of 5-FU. Therapeutic advantage of AxE1AdB-UPRT/5-FU treatment was evaluated using intraperitoneal disseminated ASPC-1 tumor mice model. The mice were treated with intraperitoneal injection of vectors 10 days after tumor inoculation, followed by the administration of 5-FU(10 mg/Kg) for 7days. Mice were sacrificed and tumor weight was measured 4 weeks later. The sensitivity against 5-FU of 5 pancreatic cancer cells increased significantly (7.1 … More -72 fold) after the transfection of UPRT gene. Tumor weight in Ax-UPRT/5-FU group (0.24 g) was significantly reduced as compared to Ax-lacZ/5-FU group (0.56 g) (P<0.001). Furthermore, that of AxElAdB-UPRT/5-FU group was significantly decreased (0.15 g).(P=0.042 vs Ax-UPRT/5-FU group). Furthermore, the adverse effect of FdUMP on alimentary tract was diminished because of the decreasd dosage of 5-FU.The growth of a tumor and its ability to metastasize is angiogenesis-dependent. Angiogenesis is one of the crucial steps in tumor's transition from a small, harmless cluster of mutated cells to a large, malignant growth, capable of spreading to other organs throughout the body soluble. We transfected Panc-1 cells with soluble VEGF receptor (flt1) and as control with the lacZ gene using the adenovirus vectors Adsflt and AdlacZ, respectively. Adsflt or AdlacZ (3.5X10^7 PFU) or 100 μl of PBS were injected directly into tumors daily from day 8 through day 12 after the implantation. Furthermore, we implanted in dorsal skinfold chamber the wild-type Panc-1 and its transfectants and monitored the tumor angipgenesis using in vivo microscope system. VEGF levels in conditioned media were higher in all cell lines (mean, 1969 pg/ml ; Panc-1, 980 pg/ml), compared to the level in media of HUVEC (8 pg/ml). Expression of VEGF mRNA was detected in the all cell lines by RT-PCR and Northern blot analysis and it was confirmed by DNA sequencing. Tumor growth rate of Adsflt-infected cells was lower than those of AdlacZ group and no treatment group, and the difference was statistically significant. Wild type Panc-1 and the LacZ transfectant implantation prompted strong tumor angiogenesis as observed in skinfold chamber, whereas soluble flt-1 transfectants failed to exert such an effect. Less