Study for Proliferative Vascular Disease Treatment by Bone Morphogenetic

本研究构建了在CAG启动子AV-CAG-EGFP控制下表达增强型绿色荧光蛋白（EGEP）的重组腺相关病毒（rAAV）载体。当大鼠颈总静脉暴露于AV-CAG-EGFP时，静脉标本中未检测到显著的EGFP表达。相反，我们发现存在于胎盘绒毛中的人胎盘源性间充质细胞（hPDMCs）通过AV-CAG-EGFP转导能高效表达EGFP。在腺病毒介导AV-CAG-EGFP后，hPDMCs的egfp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，表达egfp的hPDMCs呈离散阳性，约占AV-CAG-EGFP感染细胞的15-20%。因此，hPDMCs中的一些细胞群可能对raav介导的基因转导高度敏感。此外，在感染AV-CAG-EGFP后4周，约1%的hPDMCs中观察到稳定的EGEP表达。总的来说，hPDMCs具有有利于raav介导的基因表达的特性（微生物学）。免疫学杂志，2003,47:109-116)。同时，我们发现hPDMCs能大量产生VEGF等血管生长因子。然后，我们研究了hPDMCs在小鼠后肢缺血模型中的移植效果，该模型可能是人类周围血管疾病动脉硬化闭塞症的动物模型。激光多普勒血管功能分析和组织学分析显示，移植hPDMCs在该模型中血管再生方面有良好的结果。因此，raav介导的治疗基因转导可能使hPDMCs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.