异体骨髓干细胞移植后假肥大型肌营养不良症模型鼠膈肌dystrophin表达及病理改变

Dystrophin expression and pathology of diaphragm muscles of mdx mice after xenogenic bone marrow stem cell transplantation

目的探讨骨髓干细胞移植对假肥大型肌营养不良症(DMD)模型鼠-mdx鼠膈肌的治疗效果。方法取雄性SD大鼠骨髓干细胞经尾静脉植入放疗处理后的8周龄雌性mdx鼠(n=18)。于移植后4、8、12周各取6只mdx鼠的膈肌行 HE染色、抗肌萎缩蛋白(dystrophin)免疫荧光检测以及dystrophin mRNA的RT-PCR分析,同时用正常C57鼠及放疗而未移植的mdx鼠作为对照,另进行PCR反应检测实验鼠膈肌内Sry(Y染色体的性别决定区)基因。结果移植后mdx 鼠膈肌间质内炎性细胞浸润较放疗而未移植mdx鼠有所减少;移植后核中心移位纤维比例[移植后4、8、12周分别为 (15．58±0．91)％、(12．50±1．87)％、(10．17％±1．17)％]较未移植mdx鼠(19．5±1．87)％显著减少。移植后dystrophin免疫荧光阳性细胞比例[移植后4、8、12周分别为(1．00±0．32)％、(6．00±1．05)％、(11．92±1．11％)]较未移植mdx鼠(O．17±0．41)％显著增加。RT-PCR结果显示C57鼠膈肌的dystrophin mRNA相对含量(0．63±0．04)最高;而未移植mdx鼠的膈肌中未检测到dystrophin mRNA;移植后的mdx鼠膈肌中mRNA的表达水平(移植后4、8、12周分别为0．19±0．05、0．26±0.06、0．36± 0．04)随时间推移逐渐增高;移植后各时间点mdx鼠膈肌Sry基因均为阳性。结论骨髓干细胞系统移植mdx鼠可以恢复部分膈肌的dystrophin表达,改善膈肌的病理,骨髓干细胞移植有希望成为全身治疗DMD的有效方法。

Objective To investigate the effect of bone marrow stem cell transplantation （BMT） on the diaphragm muscles of mdx mice, a mouse model of Duchenne muscular dystrophy （DMD）. Methods The bone marrow-derived stem cells form male SD rats was transplanted through the tail vein into 18 female 8-week-old mdx mice, which were sacrificed at 4, 8 and 12 weeks aRer BMT （6 at each time point）, respectively. The diaphragm muscles of the mice were subjected to HE staining, immunofluorescence detection of dystrophin, reverse transcription （RT）-PCR analysis of dystrophin mRNA transcripts and PCR analysis of Sty （sex-determining region on the Y chromosome） gene, with age-matched female C57 mice and untreated mdx mice as the controls. Results The proportion of centrally nucleated fibers （CNF） in the diaphragm muscle of the recipient mdx mice was （15.58±0.91）%, （12.50±1.87）% and （10.17±1.17）% at 4, 8 and 12 weeks aRer BMT, respectively, significantly smaller than that of untreated mdx mice [（19.5±1.87）%], and the fibers after BMT showed less inflammatory infiltration. Compared with the untreated mice, the recipient mdx mice showed greet,, fluorescence on significantly more diaphragm muscle cell membranes [with the proportion of dystrophin-positive fibers of（1.00±0.32）%, （6.00±1.05）% and （11.92±1.11）% at 4, 8, and 12 weeks after BMT]. RT-PCR of dystrophin mRNA also demonstrated significantly higher relative levels of dystrophin in the recipient mdx mice （0.19±0.05, 0.26±0.06 and 0.36±0.04 at 4, 8 and 12 weeks after BMT） than in untreated mdx mice, and Sry gene was present in the recipient mice. Conclusions BMT can partially restore dystrophin expression and ameliorate the pathology in the diaphragm muscles of mdx mice, and has great potential to produce general therapeutic effect in patients with DMD.