Study for Proliferative Vascular Disease Treatment by Bone Morphogenetic

本研究构建了CAG启动子调控下的增强型绿色荧光蛋白（EGEP）重组腺相关病毒载体AV-CAG-EGFP。当大鼠颈总静脉暴露于AV-CAG-EGFP时，在静脉标本中未检测到显著的EGFP表达。与此相反，我们发现，人类胎盘来源的间充质细胞（hPDMCs），这驻留在胎盘绒毛，通过与AV-CAG-EGFP转导显示出有效的EGFP表达。腺病毒介导AV-CAG-EGFP后，hPDMCs的EGEP表达水平明显高于人脐静脉内皮细胞和大鼠主动脉平滑肌细胞。此外，流式细胞术分析显示了表达EGFP的hPDMC的离散阳性分数，其为用AV-CAG-EGFP感染的细胞的约15-20%。因此，hPDMCs中的某些细胞群体可能对rAAV介导的基因转导高度敏感。此外，在感染后4周，在约1%的用AV-CAG-EGFP感染的hPDMC中观察到稳定的EGEP表达。总的来说，hPDMC具有有利于rAAV介导的基因表达的特征（Microbiol. Immunol.2003，47：109-116）。同时，我们发现hPDMCs分泌大量的血管生长因子如VEGF。然后，我们在小鼠后肢缺血模型中检查了hPDMCs移植的效果，该模型被认为是人外周血管疾病闭塞性动脉硬化症的动物模型。使用激光多普勒和组织学分析的血管收缩分析显示，在该模型中，就血管再生而言，hPDMCs移植具有有利的结果。因此，rAAV介导的治疗性基因转导可能使hPDMCs的移植更有益。

In this study, we constructed recombinant adeno-associated virus (rAAV) vector expressing enhanced green fluorescent protein (EGEP) under the control of CAG promoter, AV-CAG-EGFP. When the rat common carotid veins were exposed to AV-CAG-EGFP, significant EGFP expression was not detected in the venous specimen. In contrast, we found that human placenta-derived mesenchymal cells (hPDMCs), which reside in placental villi, showed efficient EGFP expression by transduction with AV-CAG-EGFP. After the transduction of AV-CAG-EGFP in the adenoviruses, hPDMCs showed much higher level of EGEP expression than human umbilical vein endothelial cells or rat aortic smooth muscle cells. Moreover, flow cytometric analysis showed discrete positive fraction of EGFP-expressing hPDMCs, which is about 15-20% of the cells infected with AV-CAG-EGFP. Therefore, some cell population in hPDMCs might be highly susceptible to rAAV-mediated gene transduction. In addition, stable EGEP expressions were observed in about 1 % of hPDMCs infected with AV-CAG-EGFP at 4 weeks post-infection. Collectively, hPDMCs have characters favorable for rAAV-mediated gene expression (Microbiol. Immunol. 2003, 47: 109-116). Meanwhile, we found that hPDMCs produce vascular growth factors such as VEGF vigorously. Then, we examined the effect of transplantation of hPDMCs in mouse hindlimb ischemic model, which is supposed to be an animal model of human peripheral vascular disease, arteriosclerosis obliterans. The analysis of vascular ftmction using Laser Doppler and histological analysis showed a favorable outcome by transplantation of hPDMCs in terms of vascular regeneration in this model. Thus, it is possible that rAAV-mediated transduction of therapeutic gene might make transplantation of hPDMCs more beneficial.