Asselin I, Tremblay M, Vilquin J T, Guérette B, Roy R, Tremblay J P
Université Laval, Département d'Anatomie, PQ, Canada.
Muscle Nerve. 1995 Sep;18(9):980-6. doi: 10.1002/mus.880180909.
A mutagenesis RT-PCR method was used to detect normal dystrophin mRNA following the injection of normal myoblasts in mdx mice using two immunosuppressors. A specific sequence of the dystrophin mRNA (257 bp) was amplified by RT-PCR from the muscle total RNA. MaeIII digestion of the amplified products allowed us to distinguish the normal messenger of dystrophin from the dystrophic one and to establish the percentage of normal and of dystrophic (mdx) dystrophin mRNA. Normal dystrophin mRNA was detected using this technique in mdx muscles transplanted with histocompatible normal myoblasts. For this type of transplantation, no significant difference in the percentage of normal dystrophin mRNA was observed between immunosuppressed mice and those not immunosuppressed. No normal dystrophin mRNA was, however, observed in mdx mice following histoincompatible normal myoblast transplantation without immunosuppression. When such transplantations were done in mice immunosuppressed with cyclosporine or FK-506, normal dystrophin mRNA accounted for 31% and 36% of the total dystrophin mRNA, respectively. In fact, one animal immunosuppressed with FK-506 expressed as much as 57% of normal dystrophin mRNA. These results thus show that FK-506 makes it possible to restore dystrophin expression to a level comparable to that observed in DMD carriers that are usually asymptomatic.
采用诱变逆转录聚合酶链反应(RT-PCR)方法,在使用两种免疫抑制剂的情况下,于mdx小鼠体内注射正常成肌细胞后检测正常抗肌萎缩蛋白mRNA。通过RT-PCR从肌肉总RNA中扩增出抗肌萎缩蛋白mRNA的特定序列(257 bp)。对扩增产物进行MaeIII酶切,使我们能够区分正常抗肌萎缩蛋白信使与营养不良型抗肌萎缩蛋白信使,并确定正常和营养不良型(mdx)抗肌萎缩蛋白mRNA的百分比。使用该技术在移植了组织相容性正常成肌细胞的mdx肌肉中检测到了正常抗肌萎缩蛋白mRNA。对于这种类型的移植,免疫抑制小鼠与未免疫抑制小鼠之间在正常抗肌萎缩蛋白mRNA百分比方面未观察到显著差异。然而,在未进行免疫抑制的情况下,组织不相容的正常成肌细胞移植后的mdx小鼠中未观察到正常抗肌萎缩蛋白mRNA。当用环孢素或FK-506对小鼠进行免疫抑制后进行此类移植时,正常抗肌萎缩蛋白mRNA分别占总抗肌萎缩蛋白mRNA的31%和36%。事实上,一只用FK-506免疫抑制的动物表达的正常抗肌萎缩蛋白mRNA高达57%。因此,这些结果表明FK-506能够将抗肌萎缩蛋白表达恢复到与通常无症状的杜氏肌营养不良症(DMD)携带者中观察到的水平相当。