人羊膜上皮细胞与胞外基质成分透明质酸联合干预1型糖尿病的协同效应机制

Human amniotic epithelial cells (hAECs) are recognized as a promising and noncontroversial source of stem cells for cell transplantation and regenerative medicine. Regretfully, previous studies showed that only a few hAECs could be observed in pancreas, and did not differentiate into insulin-producing cells (IPCs) in following hAECs transplantation into type 1 diabetes mellitus (T1DM) model. So no desirable outcomes were obtained in the past years. Based on our results and the related research progress on stem cell, we think carefully that these authors possibly underestimated the influences of transplantation strategy and microenvironment regulation of stem cell niche, and finally affected the migration, homing and differentiation of hAECs. As we known, extracellular matrix (ECM) is a major factor of stem cell microenvironment. Hyaluronan (HA), as a major component of extracelluar matrix, could also modulate the proliferation & differentiation of stem cell, and are looked as potential factor reducing the inflammatory reaction and immune response of insulitis in T1DM model. To hAECs, it can differentiate into IPCs, and enhance the immunotolerance of IPCs in T1DM model under a good microenvironment condition. Therefore, we presume that a combination therapty of hAECs and HA for T1DM rat may exert synergy to rebulid the immune balance, and restore or/and remodel the new IPCs system in T1DM rat, in which the FoxP3 signal pathway will play a vital role. To verify the hypothesis, a series of experimental techniques, such as immunoblotting, RT-PCR, RNAi, gene knock-out and PCR array, will be employed to investigate the molecular regulating networks of hAECS proliferation after HA treatment, and mainly observe the synergy effects of a combination therapty of hAECs and HA on T1DM rat and then elucidate their mechanisms of action at different levels ranged from molecule, cell to overall accordingly. These findings will replenish and enrich the regulatory network of proliferation & differentiation of hAECs in vivo/vitro, and provide a new therapeutic approach for T1DM by hAECs transplantation at the same time.

人羊膜上皮细胞(hAECs)是细胞移植与再生医学领域最有潜力的干细胞资源，然而其在移植治疗1型糖尿病（T1DM）却未得到预想的结果。基于前期研究及文献报道，可能是移植策略和微环境影响了干细胞的迁移归巢与分化。胞外基质成分透明质酸(HA)是干细胞增殖分化先天调节子，也是减轻T1DM胰岛炎性反应与免疫应答的潜在因子，而hAECs具有分化胰岛β细胞与增强机体免疫耐受的潜力。为此提出hAECs联合HA移植治疗T1DM有协同效应，将有利于重建免疫平衡，修复与重塑胰岛组织，且FoxP3+信号通路发挥了积极作用。围绕这一假说，采用免疫印迹、RT-PCR、RNA干扰、基因敲除、PCR芯片等方法，拟在揭示HA促hAECs增殖的分子调控网络基础上，从分子、细胞及个体水平上阐明hAECs与HA治疗T1DM的协同效应及机制，为完善hAECs增殖分化的调控网络体系奠定基础，也为hAECs移植治疗T1DM提供新思路。

人羊膜上皮细胞(human amniotic epithelial cells,hAECs)是细胞移植与再生医学领域理想的种子细胞资源。干细胞的增殖和定向分化是临床应用亟待解决的关键技术问题，而hAECs的增殖及移植治疗1型糖尿病(type 1 diabetes,T1D)却达到预想结果。胞外基质成分透明质酸(hyaluronic acid, HA)是重要免疫调节因子，是干细胞增殖分化潜在调节因子。本项目提出HA有利于促进hAECs增殖，可协同hAECs重建T1D的免疫平衡，修复与重塑胰岛组织的假说。重点研究了HA对hAECs增殖及其向胰岛分泌细胞分化的影响，以及HA协同hAECs移植治疗T1D的效应与机制。研究发现分子量为300 KDa的HA，在1mg/mL剂量下能显著促进hAECs增殖，且不影响其细胞表型、旁分泌及多系分化能力，有利于增强其干性因子和免疫抑制因子的表达水平，并揭示了HA促进hAECs增殖的机制涉及TGF-β/BMP信号通路；构建的HA基础三阶段hAECs分化方案能明显提高胰岛素分泌细胞分化效率。HA主要能促进定型内胚层标志物及部分胰岛祖细胞标志物的表达，但不能诱导胰岛分泌细胞发育关键转录因子Pdx1的表达。不过，其与常用诱导剂联用后，能显著协同促进Pdx-1表达，大幅度提高胰岛素分泌细胞的分化效率；HA能明显增强hAECs移植治疗STZ诱导的T1D模型小鼠的降血糖效果，改善腹腔糖耐量能力，通过改善免疫微环境，以及促进α细胞向β细胞转分化，显著减轻胰岛组织损伤，提高胰岛素分泌水平；进一步的研究表明，HA能协同增强hAECs抑制人外周血单核细胞（PBMCs）的增殖，促进人PBMCs中Treg细胞的关键转录因子Foxp3的表达，并显著降低人PBMCs中Th1、Tc1细胞的比例。因此，hAECs联合HA移植治疗T1D模型，主要是通过改善机体免疫微环境发挥其疗效。这些发现开辟了HA的新用途，为加快hAECs临床应用，大幅提升hAECs移植治疗T1D的疗效提供了新思路，具有重要的学术价值和现实意义。