Development of New Technique to Concentrate Intra-vascular Injected Gene Vector or Anti-cancer Drugs into Tumor Tissue

本研究的目的是开发一种新的技术，使抗癌药物和基因载体能够在体内积聚到肿瘤细胞中，当这些药物被推注到血管中时。我们利用体内电穿孔技术进行了多学科治疗的动物实验，讨论了临床实验的可能性和局限性。结果1.我们研制了一套动物实验系统，可以利用荧光体视显微镜对肿瘤的血流动力学进行延时观察。我们观察了静脉注射右旋糖酐，俄勒冈绿对SIDD和C3H小鼠肿瘤荧光的时程变化。我们在注射开始后的20秒内测量并比较每个累积的荧光。相对累积荧光在注射开始后20-40秒达到最大。在俄勒冈州绿葡聚糖中加入造影剂并不影响荧光的时间推移积累。结果2.…实验选用8周龄C3H型小鼠。我们将小鼠纤维肉瘤FSA-II细胞移植到C3H小鼠的大腿上。当肿瘤长到65mm3时，用250 mg/kg的环磷酰胺(CY)和体内电穿孔(EP)治疗。将小鼠分为5组：对照组、单纯环磷酰胺(CY)组、单纯环磷酰胺(EP)组、环磷酰胺冲击组(EP-CY)、环磷酰胺冲击后EP间隔20-40秒组(CY-EP)。每组至少使用16只小鼠，总共使用114只小鼠。治疗结束后，每周测量肿瘤体积3次，共120天。用肿瘤生长(Tg)-50法评价肿瘤生长情况。TG-50(天)是肿瘤数量的一半从65mm3增加到800mm3的时间。TG-50(天)：对照组13天，单纯EP组15天，CY组24天，EP-CY组26天，CY-EP组31天。这些结果表明，电穿孔联合CY静脉推注可显著提高CY的治疗效果。结果3.实验选用8周龄C3H型和SID型小鼠。我们将小鼠纤维肉瘤FSA-II细胞移植到小鼠的大腿上。当肿瘤达到65mm3时，在体外表达增强型绿色荧光蛋白(EGFP)的pEGFP-N1质粒被团注。间隔20-40秒后，进行体内电穿孔治疗，用荧光体视显微镜观察每24小时的荧光，连续3天。在使用的条件下，没有观察到肿瘤上的荧光。较少

The aim of this study is to develop a new technology which enables anti-cancer drugs and gene-vector to be accumulated into tumor cells in vivo, when these agents are given bolus into vessels. We underwent animal experiments of multi-disciplinary treatments using chemotherapy and gene-therapy using in vivo electroporation, to discuss the possibility and limits for clinical trials.Result 1.We developed an animal experiment system which enables time-lapse observation of tumor hemodynamics using a fluorescence stereomicroscope. We observed the time-lapse change of fluorescence from tumors in skid and C3H mice using Dextran, Oregon Green which was bolus injected intravenously. We measured and compared each accumulated fluorescence for 20 sec after the start of injection. Relative accumulated fluorescence reached maximum during 20-40 sec after the start of injection. The addition of contrast media into Dextran, Oregon Green did not affect time-lapse accumulation of fluorescence.Result 2.The … More C3H mice were used for experiments at age of 8 weeks. We transplanted murine fibrosarcoma, FSa-II cells onto rt.thigh of C3H mice. When tumors reached 65 mm3, tumors were treated with cyclophosphamide(CY) of 250mg/kg, and in vivo electroporation(EP). We divided mice into 5 groups ; control, CY alone, EP alone, EP following by CY bolus injection (EP-CY), CY bolus injection following by EP with interval 20-40 sec (CY-EP). At least 16 mice were used for each group, and total mice used were 114. After treatments the volume of tumors were measured 3 times a week for 120 days. Tumor growth were evaluated with tumor growth (TG) -50 methods. The TG-50 (days) is the time for half number of tumors to reach 800mm3 from 65 mm3. The TG-50s (days) were 13 for control, 15 for EP alone, 24 for CY alone, 26 for EP-CY, and 31 for CY-EP. These indicates that electroporation combined with bolus intravenous injection of CY significantly enhances the effect of CY, when the timing between two treatments was adequate.Result 3.The C3H and skid mice were used for experiments at age of 8 weeks. We transplanted murine fibrosarcoma, FSa-II cells onto rt.thigh of mice. When tumors reached 65 mm3, the pEGFP-N1 plasmid, which has expressed enhanced green fluorescent protein (EGFP) in vitro, was bolus injected. After the interval of 20-40 sec., tumors were treated with in vivo electroporation The fluorescence were observed at each 24 hours for 3 days using a fluorescence stereomicroscope. No fluorescence was observed on tumors under the used condition. Less