Gene therapy for malignant brain tumor by infusion of antisense oligonucleotide

1.恶性胶质瘤的遗传学特征我们在31例人脑ﾟ胶质瘤中发现6例在10号染色体上存在微卫星不稳定性。在恶性胶质瘤中发现罕见的细胞凋亡。这些结果提示ﾟC基因异常与胶质瘤的恶性ﾟC转化密切相关。ﾟCorgans反义寡核苷酸定量检测方法的建立。取器官、脑、肝、肾等组织，用离心法和过滤法提取。DNA片段在丙烯酰胺凝胶上进行电泳，并转移到膜上。用RI标记的正义寡核苷酸对反义寡核苷酸进行定量检测。颈内动脉注射缓激肽选择性递送反义寡核苷酸我们从颈内动脉注射反义寡核苷酸给荷瘤大鼠。用上述方法测定递送的寡核苷酸。与对照研究相比，反义寡核苷酸选择性地进入脑瘤的能力提高了1.4倍。颈内动脉注射缓激肽对犬生命体征的影响我们用20只狗观察了颈内动脉注射缓激肽对生命体征的影响。在1和2.5伽马剂量下，颈内动脉注射缓激肽对生命体征无明显影响。另一方面，在5和10伽马剂量时，它改变了包括输液开始时的脑血流量在内的生命体征。

1. Genetic characterization of malignant gliomasWe recognized the microsatellite instability in the chromosome 10 in 6 out of 31 humanﾟCgliomas. And rare apoptosis was recognized in malignant gliomas. These findingsﾟCsuggested that the genetic abnormality strongly associated with the malignantﾟCtransformation of the gliomas.2. Establishment of quantitative measurement of antisenseoligonucleotide in theﾟCorgans.After obtaining the organs, brain, liver, and kidney, DNA fractions was extracted using centrifugation and filters. The DNA fractions were electrophorated on the acrylamide gel and trasbiotted to the membrane. A quantitative measurement of antisenseoligonucleotide was performed using RI-labeled senseoligonucleotide.3. Selective delivery of antisenseoligonucleotide by intracarotid bradykinin infusion We injected antisenseoligonucleotide from internal carotid artery in tumor-bearing rat. The delivered oligonucleotide was measured by the methods described above. The antisenseoligonucleotide was selectively delivered into the brain tumor by 1.4 fold, compared with the control study.3. Effect of intracarotid bradykinin infusion on vital signs in dogs.Using 20 dogs, we examined the effect of intracarotid bradykinin infusion on vital signs. At the dose of 1 and 2.5 gamma, intracarotid bradykinin infusion did not change the vital signs significantly. On the other hand, at the dose of 5 and 10 gamma, it changed the vital signs including cerebral blood flow at the beginning of the infusion.