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Pathogenesis and Treatment of beta-Galactosidase-Deficient Knockout Mice

β-半乳糖苷酶缺陷型基因敲除小鼠的发病机制和治疗

基本信息

批准号：
08457058
负责人：
SUZUKI Yoshiyuki
金额：
$ 4.93万
依托单位：
The Tokyo Metropolitan Institute of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

We succeeded in producing a mouse model of human G_<M1>-gangliosidosis by disruption of the murin beta-galactosidase gene, in order to analyze its pathogenesis and to try therapeutic approaches. Clinically the mutant mouse developed a progressive neurological disease 4 months after birth, manifesting itself as spastic diplegia. They died of severe nervous system dysfunction and extreme emaciation at 7-11 months of age. Neuronal cytoplasmic swelling due to storage of undigested substrates was observed in every area of the central nervous system, and the storage material appeared as membranous cytoplasmic bodies electron microscopically. This morphological change progerssed rapidly between 4 and 8 weeks of age. beta-Galactosidase activity was almost compeltely deficient in all tissues and body fluids examined.Biochemical analysis revealed a marked storage of ganglioside G_<M1> and its asialo derivative G_<A1>D in the central nervous system and some solid tissues, such as liver and spleen. G_<A1> storage was more remarkable as compared to that in human patients. These results indicated that this model animal is an authentic murine counterpart of human G_<M1>-gangliosidosis. However, there was no bone dysplasia or keratan sulfaturia in these disease mice. Urinary oligosaccharides showed an abnormal pattern on thin-layr chromatography which was similar to that in infantile G_<M1>-gangliosidosis. As an experimental trial, an adenovirus-mediated intravenous injection of beta-galactosidase cDNA was preformed into the mutant newborn mouse. The beta-galactosidase activity was expressed in the central nervous system 2 weeks after injection at the 10% normal lavel. At this stage, storage of G_<M1> and G_<A1> was significantly reduced as compared to animals without treatment. We concluded that the gene introduced in the vascular system has reached the central nervous system through the undeveloped blood-brain barrier in the neonatal period.

我们成功地通过阻断鼠β-半乳糖苷酶基因建立了人神经节苷脂沉积症的小鼠模型，以分析其发病机制，并尝试治疗方法。临床上，突变小鼠在出生4个月后发展为进行性神经系统疾病，表现为痉挛双瘫。他们在7-11个月大时死于严重的神经系统功能障碍和极度消瘦。中枢神经系统各区均可见因未消化底物储存引起的神经元胞浆肿胀，电镜下可见储存物质呈膜性胞浆小体。这种形态变化在4-8周龄时迅速发展。生化分析表明，神经节苷脂G&lt；M1&gt；及其去唾液衍生物G&lt；A1&gt；D显著储存在中枢神经系统和一些固体组织中，如肝和脾。与人类患者相比，G&lt；A1&gt；存储更显著。这些结果表明，该模型动物是真正的人类神经节苷脂沉积症的小鼠对应物。然而，在这些疾病小鼠中，没有出现骨发育不良或角质蛋白硫尿症。尿寡糖在薄层层析上表现为异常图谱，与婴儿神经节苷脂沉积症相似。作为一项实验，将腺病毒介导的β-半乳糖苷酶基因静脉注射到突变的新生小鼠体内。注射后2周，在10%正常水平的中枢神经系统可见β-半乳糖苷酶活性的表达。在这个阶段，与未处理的动物相比，G&lt；M1&gt；和G&lt；A1&gt；的贮藏量显著减少。我们的结论是，在新生儿期，引入血管系统的基因已经通过未发育的血脑屏障到达中枢神经系统。