淋巴管畸形（LM）是常见先天性脉管畸形。研究表明，PIK3CA突变并过度磷酸化与LM的发生密切相关，但具体机制不明。本研究提出假说：①PIK3CA突变并过度磷酸化，使PI3K/AKT信号高度激活，VEGF-C表达上调。②VEGF-C与VEGFR-3结合，抑制AKT磷酸化，使其介导淋巴管内皮细胞（LECs）及淋巴管平滑肌细胞（LSMCs）迁移、增殖障碍，从而导致LM发生。③设立不同浓度的雷帕霉素（RAPA），注射于斑马鱼模型，通过抑制PI3K/AKT/mTOR信号通路，促使LM消退。为验证此假说，建立ECs特异性表达PIK3CA突变（E542K）转基因斑马鱼模型，荧光显微镜观察斑马鱼发育各阶段淋巴管系统的发育及LSMCs迁移及增殖情况。应用免疫组化及Whole-Mount原位杂交、RT-PCR、免疫印迹等技术，检测RAPA作用前后PIK3CA、VEGF-C及AKT的表达及磷酸化水平的变化。

Lymphatic Malformation（LM) are common congenital symptomatic vascular anomalies. The mechanistic of PIK3CA mutations related to LM and hypo-phosphorylation remain elusive. To gain insight into the cause of LM， we hypothesized: 1.The expression of PIK3CA mutations lead to activating PI3K/AKT path and up-regulation of VEGF-C expression.2. VEGF-C combined with VEGFR-3， induce AKT dephosphorylation, and decrease its ability to recruit LECs and LSMCs which lead to the formation of LM. 3. Concentration gradient of RAPA administer in the zebrafish model. RAPA promotes LM to degrade through inhibit PI3K/AKT/mTOR signal path. To test above hypothesis in vivo and ex vivo, this research is designed. We construct transgenic zebrafish model and cell lines which express PIK3CA-E542K targeted to ECs, and observe the development of Lymphatic system and migration of LSMCs, as well as analyzed the expression level of VEGF-C and AKT through fluorescence microscope、Western blotting、immunoprecipitation、RT-PCR and Whole-mount hybridization in situ after RAPA administation. This research is significant to explore the pathogenesis of LM, and can provide molecular target for treatment of LM.

根据研究提出假说，进行了相关的实验，首先，建立内皮细胞（ECs）特异性表达PIK3CA突变（E542K）转基因动物模型，荧光显微镜观察动物发育各阶段淋巴管系统的发育及淋巴管平滑肌细胞（LSMCs）迁移及增殖情况。我们发现：①PIK3CA突变并过度磷酸化，使PI3K/AKT信号高度激活，VEGF-C表达上调。②VEGF-C与VEGFR-3结合，抑制AKT磷酸化，使其介导LECs及LSMCs迁移、增殖障碍，从而导致淋巴管畸形（LM）发生。接下来，设立不同浓度的雷帕霉素（RAPA），注射于斑马鱼模型，我们发现随药物浓度逐渐升高，抑制PI3K/AKT/mTOR信号通路的作用越强，促使LM消退。应用免疫组化及RT-PCR、免疫印迹等技术，检测RAPA作用前后PIK3CA、VEGF-C及AKT的表达。通过上述研究，我们发现构建的LM动物模型能够基本上模拟人LM的形态特征，为药物治疗研究提供了平台，为进一步阐明LM的发病机制打下了基础。