Preliminary Study of in vivo electroporation of VEGF-C Gene Transfer for Lymphedema Model

我们研究了血管内皮生长因子(VEGE-家族)与癌症性质(Mori，Saitou，Kuboi等)之间的关系。另一方面，接受腋窝清扫和乳腺癌术后放射治疗的患者会在受累的手臂产生难以治愈的淋巴水肿(Kitajima et al.)。我们进行了淋巴管生长因子-C基因转移的实验研究，以确定其对淋巴水肿动物模型的治疗效果和安全性。方法：利用pCAGGS-VEGF-C表达载体，在体内评价电穿孔基因转移到肌肉中的效率。将血管内皮生长因子C基因插入含有CAG启动子的pCAGGS表达载体中，构建成表达载体。用CUY21EDIT基因转移设备进行体内电穿孔。用Kawashima方法在大鼠一侧小腿造成外科淋巴水肿。每只大鼠注射50μg的闭合环状DNA(pCAGGS-VEGFC，对照pCAGGS)。进行周长测量，计算差值百分比和周长缩小率。处死大鼠胫骨前肌标本，10%福尔马林固定于PBS中24小时。连续切片行苏木精-伊红(H-E)染色和免疫组织化学染色，评价淋巴管的组织学表现(血管密度和血管面积比)。结果：血管内皮细胞生长因子治疗组与对照组比较，差异有统计学意义(P<0.05)。但治疗组与对照组比较，差异无统计学意义。治疗组淋巴管密度和血管面积比也有所增加，但两组间无显着性差异。结论：体内电穿孔基因转移血管内皮细胞生长因子C基因对淋巴水肿有较好的治疗作用。

We have examined the relationship between the vascular endothelial growth factors (VEGE-family) and the character of cancer (Mori, Saitou, Kuboi, et al.). On the other hand, the patients who received axillary dissection and postoperative irradiation for the breast cancer produce the lymphedema of the suffered arm that are difficult to cure (Kitajima et al.). We conducted the experimental study of the gene transfer of VEGF-C (lymphatic duct growth factor) to determine the effect and safety of this therapy for lymphatic edema animal model.Method : The pCAGGS-VEGF-C expression plasmid was used to assess the efficiency of gene transfer into muscle by electroporation in vivo. This vector was prepared by inserting a VEGF-C cDNA into the pCAGGS expression vector containing the CAG promoter. In vivo electroporation method was performed using the gene transfer equipment CUY21EDIT. The surgical lymphedema by the method of Kawashima was created in the leg of one side of the rat. The rat were injected with 50 μg each of closed circular plasmid DNA (pCAGGS-VEGF-C, control pCAGGS). Circumference measurement was performed to calculated percent difference and circumference reduction rate. The tibialis anterior muscles samples for sacrificed rat were fixed 10% formalin in PBS for 24hours. Serial cut sections were sliced for hematoxylin-eosin (H-E) and immunohistochemical staining to evaluate lymphatic vessel findings (vessel density and vessel area rate) histologically. Results : The percent difference and the circumference reduction rate respectively showed between VEGF group and control. However, a significant difference was not found between the treatment group and control group. Lymphatic vessels density and vessel area rate were also increased in treatment group however ; there was no significant difference between the two groups. Conclusion : In vivo electroporation gene transfer of VEGF-C cDNA was demonstrated likelihood of effective to the lymphedema.