Study of electro-gene therapy on diabetic neuropathy

1.以小鼠肌肉总RNA为模板，通过RT-PCR方法合成了含有单肽序列的表达载体。将该片段亚克隆到含有β-肌动蛋白启动子的pcaggs载体中，并在骨骼肌中高效表达。将该表达载体(pcag-Mv3)导入COS7细胞，Western印迹分析证实其表达。小鼠VEGF64基因转移至正常小鼠骨骼肌及对骨膜神经和器官的影响C57B1/6小鼠胫前肌注射50ug pCag-Mv3后进行电穿孔。术后3周和9周，取坐骨神经用戊二醛固定，甲苯胺蓝染色。肌肉、肝、脾、肾用4%多聚甲醛固定，苏木精-伊红染色。3周后，胫前肌切片可见明显的毛细血管增生和单核细胞侵入，提示局部合成和分泌VEGF64。在肝脏中，可以观察到血窦的伸展和内皮细胞的增殖，这可能反映了静脉在肌肉中产生的VEGF64的循环。没有反映静脉在肌肉中产生的VEGF64的循环。注射组小鼠的脾、肾组织形态与对照组无明显差异。术后3周和9周，巩膜神经恢复正常。虽然POE综合征患者血清中的血管内皮生长因子水平显著升高，并且推测血管内皮生长因子可能导致了这种疾病的神经病变，但我们的数据表明，我们的方法短期内升高的血中血管内皮生长因子水平并不损害周围神经。我们正在尝试将VEGF64基因转移到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