An appropriate drug delivery system of gene therapy for cerebrovascular disease

脑血管疾病基因治疗的合适药物递送系统

• 批准号: 12470282
• 负责人: OHKUMA Hiroki
• 金额: $ 3.14万
• 依托单位: Hirosaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470282/
• 关键词: Cerebrovascular disease; Gene therapy; Drug delivery system; ドラッグデリバリ-システム

Efficacy of liposome and hydrogel-polymer as vector for gene transfer to cerebral arteries by intrathecal administration was investigated.1. Liposome vectorCationic lipid, N,N,N',N^1-bis(2-hydroxyethyl)-2,3,-dioleoyloxy-1,4-butanediammonium iodide / L-dioleoyl phosphatidylethanolamine, was used.1) in vitro evaluationSafety of cationic lipid was evaluated using cultured rat smooth muscle cells (SMC). Cationic lipid was dissolved in cultured solution at a concentration of 10, 20, 40, 80, 160μM, and SMC was induced in the solution for 24 hours. Cytotoxicity was evaluated by microculture tetrazolium method. At a concentration of 80 and 160μM, cytotoxicity was seen, and a concentration of 40μM was used in the following experiment. β galactosidase control vector was mixed with cationic lipid and solved in the cultured solution at a concentration of 40μM, in which SMC was incubated for 48 hours. β galactosidase assay revealed expression of β galactosidase in the SMC incubated in the solution … More with β galactosidase mixed with cationic lipid was seen about eight times as those incubated in the solution with β galactosidase alone.2) in vivo evaluationβ galactosidase mixed with cationic lipid was injected into cisterna magna of SD rat, and expression of β galactosidase at the basilar artery was serially evaluated. The basilar artery was taken, homogenized, and lysed, and expression of β galactosidase was measured using β galactosidase assay. Until 8 days after the injection, expression of β galactosidase was seen, however, on 14 days after the injection, it was markedly decreased.The results indicated that cationic lipid was useful for gene transfer in the acute cerebrovascular disease such as cerebral vasospasm after subarachnoid hemorrhage.2. Hydrogel polymer1) Evaluation of safetyRabbit middle cerebral artery was exposed under operative microscope after craniotomy and it was coated with hydrogel polymer. Two weeks to 3 months after, the brain was taken and histopathological change was examined under light microscopy. No abnormal histological changes were seen.2) gene transferRabbit middle cerebral artery was coated with hydrogel polymer mixed with Luciferase gene in the same manner as described above, and the middle cerebral artery was serially taken 2 weeks to 3 months after the surgery. The middle cerebral artery was homogenized and lysed to examine Luciferase activity in the arterial wall using Luciferase assay system. Two weeks and 1 month after Luciferase activity was markedly seen, however, it decreased 2 months after, and it was not detected 3 months after.The results indicated that hydrogel polymer can offer a long and slow transfer of gene into cerebral artery. Less

研究了脂质体和水凝胶聚合物作为鞘内注射基因转移至脑动脉的载体的功效。 1．使用二油酰氧基-1,4-丁烷碘化二铵/L-二油酰磷脂酰乙醇胺。1)体外评价安全性使用培养的大鼠平滑肌细胞(SMC)评价阳离子脂质将阳离子脂质以10、20、40、80、160μM的浓度溶解在培养液中，并在溶液中诱导SMC 24小时来评价细胞毒性。微量培养四唑法，在80和160μM浓度下，可见细胞毒性，本实验中使用的浓度为40μM。接下来的实验将β半乳糖苷酶对照载体与阳离子脂质混合并以40μM的浓度溶解在培养液中，其中SMC孵育48小时，β半乳糖苷酶检测显示在溶液中孵育的SMC中β半乳糖苷酶的表达。 β半乳糖苷酶与阳离子脂质混合的情况是单独与β半乳糖苷酶在溶液中孵育的情况的约8倍。2)体内评价β半乳糖苷酶混合的情况将含有阳离子脂质的注射液注入SD大鼠小脑延髓池，连续评价基底动脉处β半乳糖苷酶的表达，取基底动脉，匀浆并裂解，并使用β半乳糖苷酶测定法测定β半乳糖苷酶的表达。注射后第1天，β半乳糖苷酶出现表达，但注射后第14天，β半乳糖苷酶表达明显下降。阳离子脂质可用于急性脑血管疾病如蛛网膜下腔出血后脑血管痉挛的基因转移。2.水凝胶聚合物1)安全性评价开颅手术后在手术显微镜下暴露兔子大脑中动脉，并在其上涂上水凝胶聚合物。几个月后取脑，光镜下观察组织病理学变化，未见异常组织学变化。2)基因转入兔中。以与上述相同的方式用混合有荧光素酶基因的水凝胶聚合物涂覆大脑动脉，并在手术后2周至3个月连续取大脑中动脉，将大脑中动脉匀浆并裂解以检查大脑中动脉中的荧光素酶活性。动脉壁荧光素酶检测系统在两周和1个月后明显可见荧光素酶活性，但2个月后荧光素酶活性下降，3个月后未检测到。结果表明，水凝胶聚合物可以提供长而缓慢的基因转移到脑动脉中。