人類胎盤絨毛膜蛻膜間葉細胞促進小鼠模型血管再生緩解後肢缺血

Abstract

周邊動脈疾病(PAD)是指缺血性靜息痛、動脈功能不全、潰瘍、壞疽和下肢低血流。按照Fontaine分級(III-IV期)或Rutherford分級(4-6級)，重症肢體缺血(CLI)是本病PAD的晚期，通常伴隨著相當高的發病率和死亡率。到目前為止，標準的治療方案，如手術、血管內介入治療、侵入性手術對PAD的病人進行早期微創處理，但仍然是效果不佳。越來越多的研究表明，來自不同組織的間葉幹細胞（MSCs）具有促進缺血性肢體血管生成和改善機體血液供應的潛力。在此，我們提出從人類胎盤絨毛膜中提取的間葉幹細胞(pcMSCs)可以在小鼠動物模型中幫助組織再生和促進血管生成，表明pcMSCs是CLI/PAD治療應用的潛在候選者。在無血清培養體系和IV型膠原蛋白條件下，pcMSCs仍具有紡錘形，並具有分化為脂肪、硬骨和軟骨的能力，此外，它們還將顯示出間質基質細胞的標誌。在基因集富集分析(GSEA)中，pcMSCs的血管新生基因富集度相較BMMSCs有高度表現，有趣的是纖維母細胞生長因子9(FGF-9)在pcMSCs當中是獨有且大量表現的，在血管新生及組織再生中，這可能是pcMSCs優於BMMSCs的關鍵因子。細胞實驗也可以發現，pcMSCs在內皮細胞沒有生長因子的情況下，促進了內皮細胞的增殖、遷移和血管的形成，並保護內皮細胞及減少細胞凋亡。在活體外的實驗也發現，pcMSCs可以促進血管生成，加速動脈環的萌發。在活體內的實驗發現，pcMSCs輔助血液灌注、後肢缺血程度減弱，並且肌肉的壞死程度也較小。結合臨床經驗，為了加強細胞治療的效果，我們結合pcMSCs的免疫調節能力，利用常見的發炎因子調節pcMSCs的反應，希望將缺血性疾病的進程區分的更細以利於投予促進發炎抑或是抑制發炎的pcMSCs來加速組織的修復。

Peripheral artery disease (PAD) is defined as ischemic rest pain, arterial insufficiency ulcers, gangrene and hypoperfusion of the lower extremity. According to the Fontaine classification (stage III-IV) or the Rutherford classification (grades 4–6), critical limb ischemia (CLI) is an advanced stage of PAD, which is usually accompanied with a considerable high incidence and mortality. So far, standard treatment programs such as surgery, intravascular interventional therapy, and invasive surgery, have performed early minimally invasive treatment for patients with PAD; however, the effect is still not great. More and more studies have shown that mesenchymal stem cells (MSCs) from different tissues have the potential to promote ischemic limb angiogenesis and improve the body's blood supply. Here, we propose that mesenchymal stem cells (pcMSCs) extracted from human placental chorion can help tissue regeneration and promote angiogenesis in mouse animal models, indicating that pcMSCs are potential candidates for CLI/PAD therapeutic applications. Under the condition of a serum-free culture system and type IV collagen, pcMSCs still have a spindle shape and have the ability to differentiate into fat, hard bone, and cartilage. In addition, they will also show signs of mesenchymal stromal cells. In gene set enrichment analysis (GSEA), pcMSCs has a higher degree of enrichment of angiogenesis genes than BMMSCs. What is interesting is that fibroblast growth factor 9 (FGF-9) is unique and abundant in pcMSCs. In angiogenesis and tissue regeneration, this may be a key factor for pcMSCs to be superior to BMMSCs. Cell experiments can also find that pcMSCs promote the proliferation and migration of endothelial cells and the formation of blood vessels in the absence of growth factors in endothelial cells, protects endothelial cells, and reduces apoptosis. In vitro experiments have also found that pcMSCs can promote angiogenesis and accelerate the germination of arterial rings. In vivo experiments found that pcMSCs assisted blood perfusion, reduced hind limb ischemia, and reduced muscle necrosis. In addition to clinical experience, we combined the immunomodulatory ability of pcMSCs and used common inflammatory factors to regulate the response of pcMSCs and strengthen the effect of cell therapy. We hope to distinguish the process of ischemic disease more finely to facilitate the administration to promote inflammation or inhibit inflamed pcMSCs from accelerating tissue repair.