Analysis of the effect with the, expanded CTG repeat for cell toxicity, especially tauopathy in the central nervous system in patietnt of myotonic dystrophy type 1 (DM 1)

携带mRNA的扩增的CUG三联重复序列似乎是1型强直性肌营养不良（DM 1）的原因。为了研究DM 1的发病机制，特别是其在CNS中的tau蛋白病，我们使用PC 12神经元细胞系构建了DM 1细胞培养模型，并筛选了改善这种mRNA获得功能的黄酮类化合物。将扩增的250个CTG重复序列亚克隆到荧光素酶基因的3 ′-非翻译区中，产生稳定的PC 12双链。用荧光素酶评价扩增重复序列对该细胞的顺式效应。为了找到改变扩增的CTG重复序列的毒性作用的试剂，筛选了235种生物素。当用神经生长因子诱导分化时，发现该细胞的顺式效应和细胞毒性增加。此外，在该DM 1模型细胞中也证实了tau基因的可变剪接模式的改变。用抗caspase-3抗体进行的Western blotting表明，细胞死亡是由凋亡引起的。筛选分析证实，黄酮类化合物、黄酮类化合物、黄烷酮和DHEA-S防止了扩增的CTG重复序列的细胞毒性和顺式效应，并且黄烷酮、两种黄酮类化合物和黄嘌呤苷强烈抑制了CTG重复序列的顺式效应。总之，我们发现，这种神经元细胞系，表达CUG重复轴承mRNA，通过报告基因和神经元死亡后，在体外细胞分化显示顺式效应。此外，一些黄酮类化合物和DHEA-S抑制顺式效应和细胞毒性，表明它们的化学结构可以改善这些毒性作用。该系统使得评估扩展的CTG重复序列的毒性作用变得容易，因此对于筛选其他DM 1治疗的功效应该是有用的。

Expanded CUG triplet repeats carrying mRNA seem to be responsible for myotonic dystrophy type 1 (DM1). To study the pathogenesis of DM 1, especially of its tauopathy in CNS, we constructed a DM 1 cell culture model using a PC 12 neuronal cell line and screened flavonoids that ameliorate this mRNA gain of function. The expanded 250 CTG repeat was subcloned into the 3'-untranslated region of the luciferase gene yielding a stable transformant of PC 12. The cis-effect of expanded repeat for this cell was evaluated with luciferase. To find agents that alter the toxic effect of expanded CTG repeat, 235 bioflavonoids were screened. An increased cis-effect and cytotoxicity were found when this cell was treated with nerve growth factor to induce differentiation. Furthermore, modification of alternative splicing pattern of tau gene was also confirmed in this DM1 model cell. Western blotting with anti-caspase-3 antibody suggested that cell death was caused by apoptosis. Screening analysis confirmed that a flavone, an isoflavone, a flavanone and DHEA-S prevent both the cytotoxicity and cis-effect of expanded CTG repeat and that a flavanone, two isoflavones, and xanthylatin strongly inhibit the cis-effect of CTG repeats. In conclusion, we found that this neuronal cell line, which expresses the CUG repeat-bearing mRNA, showed cis-effects through the reporter gene and neuronal death after cell differentiation in vitro. Moreover, some flavonoids and DHEA-S inhibit both the cis-effect and cytotoxicity, indicate that their chemical structures work to ameliorate both these toxic effects. This system makes it easy to evaluate the toxic effects of expanded.CTG repeats and therefore should be useful for screening other DM1 treatments for their efficacies.