Study of electro-gene therapy on diabetic neuropathy

1. 以小鼠肌肉总RNA为原料，采用RT-PCR方法合成含有信号肽序列的小鼠vegfl64 Cdna。该DNA片段被亚克隆到具有β-肌动蛋白启动子并在骨骼肌中强烈表达的Pcaggs载体中。该质粒（Pcag-Mv3）转染COS7细胞，并通过western blot分析证实其表达。小鼠vegfl64在正常小鼠骨骼肌中的基因转移及对周围神经和器官的影响将Pcag-Mv3注射50 ug至C57B1/6小鼠胫骨前肌后电穿孔。3周和9周后，将坐骨神经用谷二醛固定，甲苯胺蓝染色。肌肉、肝、脾、肾用4%多聚甲醛固定，苏木精和伊红染色。3周后，胫前肌切片毛细血管增生，单核细胞浸润，提示vegfl64的局部合成和分泌。在肝脏中，观察到窦的扩张和内皮细胞的增殖，可能反映了血管在肌肉中产生的vegfl64的循环。没有反映血管在肌肉中产生的vegfl64的循环。注射组与对照组脾、肾形态无明显差异。3周和9周后巩膜神经恢复正常。虽然有报道称POEM综合征患者血清中VEGF水平显著升高，并推测VEGF可能导致该疾病的神经病变，但我们的数据表明，通过我们的方法短期升高血液中VEGF水平并不会累及周围神经。我们正在尝试将vegfl64基因转移到患有糖尿病神经病变的糖尿病小鼠的骨骼肌中

1. Construction of vector which expresses mouse vegfl64 Mouse vegfl64 Cdna which contains singal peptide sequence was synthesized by RT-PCR from mouse muscle total RNA. This DNA fragment was subcloned into Pcaggs vector which has β-actin promoter and strongly expresses in the skeletal muscles. This plasmid (Pcag-Mv3) was transfected in COS7 cells and its expression was confirmed by western blot analysis2. Gene transfer of mouse vegfl64 into skeletal muscles of normal mice and the effect of vegfl64 on perioheral nerves and organs Fifty ug of Pcag-Mv3 was injected into the anterior tibial muscles of C57B1/6 mice followed by electroporation. After 3 and 9 weeks the sciatic nerves were fixed in glutalaldehyde and stained with toluidine blue. The muscles, liver, spleen and kidney were fixed in 4% paraformaldehyde and stained by hematoxylin and eosin. After 3 weeks the sections of the anterior tiblal muscles showed the marked proliferation of capillary and invasion of monocytes suggesting the local synthesis and secretion of vegfl64. In the liver the extension of sinusoids and the proliferation of endothelial cells were observed probably reflecting the circulation of vegfl64 producted in the muscles by veins. There was no reflecting the circulation of vegfl64 produced in the muscles by veins. There was no morphological differences of spleen and kidney between injected and control mice. After 3 and 9 weeks the sclatic nerves were normal. Though the marked elevation of serum VEGF level was reported in patients with POEM syndrome and it is postulated that VEGF may cause neuropathy in this disease, our data suggest that the short-term elevation of blood VEGF level by our method dose not invoive the peripheral nerves.We are trying vegfl64 gene transfer into skeletal muscles of diabetic mice with diabetic neuropathy