VEGF/ANG-1基因纳米缓释微球靶向SVF细胞对游离移植脂肪血管化影响的研究

The efficacy of autologous fat transplantation is reduced by fat absorption and fibrosis due to fat necrosis. Enhanced transplant neovascularization early after transplantation may reduce these outcomes. Clinicans are looking for a kind of effective method, which is Suitable for clinical application, to improve the survival rate of fat transplantation. Stromal vascular fraction (SVF) is freshly isolated from adipose tissue. It contain multipotent mesenchymal stem cells that possess the ability of differentiating, in vitro, into cells of mesenchymal lineage, including adipocytes, osteoblasts, chondrocytes, and myoblasts. Some cell factors ,such as vascular endothelial growth factor (VEGF) can promote this effect. However, the half-life (about 50 minutes) of VEGF is so short that it can not catch a effical concentration. In contrast, sustained local concentration of growth factors is necessary for the development of mature blood vessels. In this study, We try to use VEGF\ANG-1 nano-sustained release microspheres and ask whether VEGF/ANG-1 nano-sustained release microspheres plus SVF could improve fat transplant neovascularization and survival. We harvest SVF after collagenase digestion and Construct VEGF/ANG-1 sustained release microspheres though the method of ultrasonic emulsification in vitro. Human fat tissues are then mixed with the SVF cells(Group B) , SVF plus VEGF/ANG-1 nano-sustained release microspheres(Group A) and Dulbecco’s Modified Eagle Medium(Group C) as control. 18 nude mice were injected subcutaneously with all three preparations, with each of the three designated spots receiving one of these three mixtures in a random fashion. Two months later, transplanted tissue wet weight and histology are evaluated and neovascularization is quantified by counting the capillaries. Hematoxylin and eosin stains, anti-VEGF and anti-ANG-1 stains are used to visualize cell infiltration.

自体脂肪是最理想的的软组织填充材料，但是高达40％－80％吸收率限制了其临床应用。如何促进移植物尽快建立充分的血供以实现再血管化是保障其成活的关键。先期的研究发现，脂肪组织来源血管基质片段（SVF）结合VEGF 细胞因子能显著改善游离移植脂肪的存活率，大大降低存活移植物的纤维化率，并显著促进新生血管的形成。但由于VEGF较短的半衰期，限制了VEGF 促新生血管形成的作用。本研究拟采用细胞生物学技术、蛋白生化技术、体外三维血管构建技术、病理检测技术等，重点研究VEGF、ANG-1 双细胞因子缓释系统的构建和其对血管生成的协同作用及其靶向SVF细胞对游离移植脂肪颗粒血管化影响及机制。探讨SVF 细胞多向分化及在VEGF\ANG-1 缓释系统作用下参与新生血管的修剪和重塑的可能机制，为多细胞因子靶向SVF 细胞疗法在游离脂肪移植领域的广泛临床应用奠定坚实的理论及实验依据。

背景：自体脂肪移植在临床上备受关注，是整形和修复重建手术中理想的填充材料。但是自体脂肪移植后不确定的存活率极大地限制了它在临床应用中的进一步普及。为此，学者们对如何提高移植后脂肪的存活率进行了广泛的研究和探索。SVFs可以自我复制，具有多向分化的潜能，并能够分泌多种细胞因子，因此将SVFs和游离脂肪颗粒混合移植可以显著提高脂肪组织移植的存活率，降低液化、变形、纤维化、囊肿、坏死等不良反应。另一方面有学者也证实了移植后的脂肪受多种血管生成因子的调节，如VEGF，Ang-1等。这些血管生成因子可以促进移植物中新生血管的形成，实现再血管化。所以理论上联合SVFs和生长因子，可以进一步促进移植后的脂肪组织血管化，改善存活率。然而，由于这些生长因子的半衰期短，不能在体内充分发挥其生物学效应。PLGA缓释微球技术的引入为这一难题的解决提供了很好的解决思路。研究内容：本实验通过构建裸鼠体内脂肪移植模型，将生长因子缓释微球VEGF/Ang-1-PLGA联合SVFs应用于游离脂肪移植，利用两者的协同作用，加速移植物内新生血管的形成，研究其对自体脂肪移植存活率的影响。结果：8周后移植物测量结果显示，实验组的平均体积（1.08±0.069ml）与其他两组（0.62±0.036ml、0.83±0.059ml）相比显著性增大。进一步的组织学分析表明，同时添加了SVFs和VEGF/ANG-1-PLGA缓释微球组的移植物与其它两组相比，纤维化程度更低，脂肪细胞更饱满，并且所含新生血管的数量更多（A(5.08±1.22)个/HP，B(8.32±1.40)个/HP，C(12.88±2.17)个/HP）。结论：该实验结果证实VEGF/Ang-1-PLGA的双重缓释系统联合SVFs可以显著地促进移植后的脂肪颗粒新生血管的形成，从而提高脂肪存活率。我们所开发的这一双重缓释系统在裸鼠身上很好地展现了与SVFs的协同效应，为今后的实验研究提供了新思路，为临床脂肪移植辅助治疗提供了方向和基础。