Duchenne型肌营养不良小鼠模型鉴定及干细胞移植后dystrophin的变化

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.

Identification of the method of establishment of a DKO mouse model of Duchenne muscular dystrophy and regeneration of dystrophin expression in vivo after stem cell transplantation

Objective To establish a method of identification of DKO mouse model of Duchenne muscular dystrophy, and to assess the dystrophin regeneration after stem cell transplantation. Methods Heterozygous mice were mated and the resulting offspring were used to identify their genotype by SSP-PCR. The plasma creatine kinase level was measured by biochemical analyzer and histological changes in the DKO mice were analyzed using HE staining. Human umbilical cord mesenchymal stem cells were prepared and injected into the DKO mice hindlimb muscle, and dystrophin expression was detected by immunofluorescence staining at 2 months after injection. Results Mating of heterozygous mice generated three kinds of genotype offsprings, and 21.2% of the offsprings were identified as DKO genotype (285 bp). DKO mice showed dystrophic symptoms, their plasma creatine kinase level was as high as 16988.52±617.48 IU/L, and significant histological changes including diverse myocyte sizes, numerous centrally nucleated cells and connective tissue proliferation or inflammatory cells infiltration. Human dystrophin expression was detected in the DKO mouse hindlimb muscle at two months after injection of human umbilical cord mesenchymal stem cells. Conclusion DKO mouse genotype can be identified by SSP-PCR, and DKO mouse is an ideal animal model for studies of stem cell therapy for Duchenne muscular dystrophy.