DMD模型鼠基因型鉴定及其肌组织病理学与超微结构研究

Genotype evaluation, muscle histopathology and ultrastructure in a Duchenne muscular dystrophy animal model

目的:探讨DMD动物模型dko小鼠的基因型鉴定方法及其肌组织的病理学与超微结构情况。方法:运用序列特异性引物PCR-SSP技术鉴定杂合子种鼠的子代基因型,并对子代dko小鼠的骨骼肌组织冰冻切片进行HE染色和dystrophin/utrophin的SABC-Cy3荧光免疫组织化学检测以及肌组织的透射电镜观察。结果:112只子代鼠中,mdx小鼠28只,dko小鼠26只,杂合子鼠58只,按百分比计算,分别占25.0％、23.2％、51.8％,基因型鉴定结果符合孟德尔遗传规律;dko小鼠肌组织HE染色显示细胞大小形态不均,轮廓变圆,核中移,肌间隙变宽,有炎症细胞浸润与结缔组织增生现象;免疫荧光检测肌膜未见有dystrophin/utrophin表达;电镜观察有肌膜断裂不完整、膜与膜下组织分离并水肿、肌原纤维结构松散、结缔组织增生及炎症细胞浸润等现象。结论:PCR-SSP技术鉴定子代鼠基因型快捷准确;dko小鼠的病理生理学表现与DMD极为相像,是DMD临床治疗研究的理想疾病动物模型。

AIM: To evaluate the genotype, muscle histopathology and ultrastructure in dko mice. METHODS: Dystrophin/Utrophin-deficient double knockouts (dko) mice were obtained from university of Oxford, UK. Genotype of filial generation of heterozygote was evaluated by PCR-SSP. HE staining and fluorescent immunohistochemistry by SABC-Cy3 were used to detect striated muscle of dko mouse, and the muscle ultrastructure was observed by transmission electron microscope(TEM). RESULTS: In 112 filial generation mice, there were 28 mdx (25.0%), 26 dko (23.2%) and 58 heterozygote (51.8%), which coincided with the law of Mendelian inheritance. HE staining showed that the myocytes were not very uniform, there were phenomenon of round outline, centrally nucleated fibers, widening interspace, inflammatory cell infiltration and connective tissue proliferation in dko mice. There were no any immunofluorescent expression of dystrophin and utrophin in sarcolemma in dko mice. TEM showed sarcolemma breakage, separation and edema, and loose myofibril texture, inflammatory cell infiltration and connective tissue proliferation in dko mice. CONCLUSION: PCR-SSP is a very quick and accurate way for genotype evaluation of filial generation. The pathophysiology of dko mouse was very similar to Duchenne muscular dystrophy (DMD), and dko mouse is an ideal animal model for study of DMD clinical therapy.