GFP标记的人胎盘间充质干细胞移植治疗急性肝衰竭小型猪的作用机制研究

Acute 1iver failure (ALF) is one of the most intractable problems in medicine. Orthotopic liver transplantation is the most effective treatment that improves the survival rate in patients with ALF. However, the death rate of ALF remains high due to the unavailability of healthy liver donor. Recently, studies in rodent animal models have demonstrated the therapeutic potential of mesenchymal stem cells (MSC) in the treatment of liver injury. MSC transplantation is expected to be the alternative treatment for hepatic failure in near future. To more closely evaluate the efficacy and safety of MSC transplantation for ALF, our group established the ALF model in Chinese experimental miniature pigs whose livers mimic the anatomic and physiological properties of human liver. Our preliminary study shows that transplantation of mesenchymal stem cell derived from human placental (hPMSC) into ALF pigs significantly increases its survival rate and improves liver function as well as promotes the regeneration and repair of damaged liver. However, the underlying mechanisms remains unknown. We hypothesize that the transplanted hPMSC may take effect by (1) differentiating into normal hepatocytes or (2) fusing with hepatocytes and initiating proliferation or (3) supporting and activating liver progenitor cells, which further initiate endogenous cell differentiation and proliferation or (4) secreting multiple cytokines and growth factors to inhibit inflammation and accelerate liver restoration. In the present project, we aim to elucidate the above proposed mechanisms in single cell level as well as systematic level by transplanting enhanced green fluorescent protein (EGFP) labeled hPMSC into ALF pigs. We will employ immunofluorescence, flow cytometry, proteomics and metabonomics etc to study the underlying mechanisms of the curative effect of hPMSC on ALF. Our research can provide a theoretical basis for the clinical application of stem cell therapy.

急性肝功能哀竭(ALF)病死率很高。近年来一系列动物实验证实了间充质干细胞（MSC）在ALF治疗中的潜力，然而其机制存在分歧，尚无确切定论。这些实验多采用啮齿类动物模型，难以对MSC治疗ALF的临床疗效和安全性进行评估。申请人在国际上较早以肝脏解剖结构和生理指标更接近人的大型动物中国小型猪为实验模型，采用人胎盘间充质干细胞（hPMSC）为种子细胞，建立了完整的移植方法，显著提高了ALF模型猪的存活率，促进了受损肝脏的再生和修复。然而其治疗机制还有待进一步研究，推测可能是移植细胞定植后支持与激活受体肝干细胞，从而促进肝干细胞分化或者移植细胞分泌多种细胞因子发挥免疫调节作用。本项目拟采用课题组已成功构建的表达绿色荧光蛋白的hPMSC移植到ALF模型猪中，在单细胞水平和系统水平深入研究细胞的归巢和移植治疗的具体机制，为干细胞疗法的临床应用提供理论基础和实验依据。

急性肝功能哀竭 (ALF)是一类严重的肝功能障碍性疾病，病死率高达70~80%，肝移植是唯一确切有效的治疗方法。间充质干细胞（MSC）移植作为一种可能的替代疗法近年来得到广泛关注。MSC移植治疗是否在肝脏定植，确切机制如何，这些关键问题的探讨对MSC移植临床应用至关重要。MSC来源广泛，人胎盘来源的间充质肝细胞（hPMSCs）具有来源广泛、无创取材、无伦理学限制等方面的优势，成为有临床应用前景的干细胞源。本项目在前期工作基础上，以hPMSCs为细胞源，通过ALF中国小型猪、鼠等实验模型，评价hPMSCs移植治疗ALF的安全性和有效性，并阐明其机制。取得了以下成果：1）制备了表达绿色荧光蛋白（GFP）的hPMSCs（GFP+ hPMSCs），通过检测转染前后hPMSCs表型谱、活力以及成脂、成骨和成肝诱导分化潜能；体外评估定向诱导分化的GFP+肝细胞样细胞的代谢、合成和分泌功能，证实了转染不影响细胞的生物学特性，使用慢病毒载体转染GFP基因是可靠的MSC标记和跟踪方法；2）hPMSCs移植可显著提高ALF中国小型猪的生存率，7天存活率从0%提高到66.7%，生化在移植1周后逐渐恢复到正常水平，病理结果显示hPMSCs移植治疗3周后，存活动物的肝脏结构接近正常。抗体芯片结果表明MSC分泌大量的细胞因子，流式质谱技术绘制了肝脏全免疫细胞图谱，揭示了MSC移植治疗ALF过程关键的免疫细胞亚群。这些结果都证实了MSC通过分泌相关细胞因子和激活其它免疫细胞从而发挥免疫调节功能,亦可抑制受体肝细胞凋亡并刺激内源性组织修复。3）利用多组学技术和生物信息学分析，深入研究hPMSC移植治疗急性肝损伤的关键蛋白、功能基因和通路。包括线粒体及能量代谢相关蛋白共29种、糖酵解及糖原分解通路、凝血通路及一些脂类及氨基酸代谢通路；首次研究了干细胞移植对肠道微生态的影响，发现MSC移植后小鼠肠道微生态发生显著改变，这些改变通过促进肠粘膜结构完整、维持肠内环境稳态从而促进肝脏修复。. 上述成果在成果发表SCI收录论文12篇；申请国家发明专利2项（1项已授权）；作为主要完成人获得浙江省教学成果奖一等奖1项。