论文部分内容阅读
目的建立Duchenne型肌营养不良(DMD)模型dko小鼠的鉴定方法,评估干细胞移植后dystrophin的再生水平。方法采用SSP-PCR方法鉴定杂合子鼠交配产生的子代鼠的基因型。生化分析仪测定dko小鼠血浆肌酸激酶含量,HE染色观察肌肉组织学变化。扩增人脐带间充质干细胞并注射到dko小鼠后肢肌肉,2个月后免疫荧光染色法检测dystrophin的表达。结果杂合子鼠交配可以产生三个基因型的子代鼠,21.2%的子代鼠可以鉴定为dko小鼠的基因型(285 bp)。dko小鼠显示了肌营养不良的症状,血浆肌酸激酶含量高达(16,988.52±617.48)IU/L,典型的病理变化包括肌纤维大小不一,多见核中移细胞,结缔组织增生或炎性细胞浸润。将人脐带间充质干细胞注射到dko小鼠后肢肌肉,2个月后可检测到人dystrophin的表达。结论采用SSP-PCR可用于鉴定dko小鼠基因型,dko小鼠是研究干细胞治疗DMD的理想动物模型。
Objective To establish a method to identify Duchenne muscular dystrophy (DMD) model dko mice and evaluate the level of dystrophin regeneration after stem cell transplantation. Methods SSP-PCR was used to identify the genotypes of offspring mice produced by cross mating. The biochemical analyzer was used to determine the plasma creatine kinase level in dko mice. The histological changes were observed by HE staining. Human umbilical cord mesenchymal stem cells were expanded and injected into dko mouse hindlimb muscle. The expression of dystrophin was detected by immunofluorescence staining two months later. Results Hybrid mice produced three genotypes of offspring, while 21.2% of offspring could be identified as dko mice (285 bp). dko mice showed symptoms of muscular dystrophy with plasma creatine kinase levels as high as (16,988.52 ± 617.48) IU / L, with typical pathologic changes including varying sizes of myofibrils, more common in the nucleus of metastatic cells, connective tissue hyperplasia or inflammatory cells infiltration. Human umbilical cord mesenchymal stem cells were injected into dko mouse hindlimb muscles and the expression of human dystrophin was detected after 2 months. Conclusion SSP-PCR can be used to identify dko mouse genotype. Dko mouse is an ideal animal model for stem cell therapy of DMD.