增强干细胞靶向归巢和抗凋亡活性融合多肽分子设计及创面愈合实验研究

Transplantation of BMSCs is a promising strategy for improving wound healing. However, BMSCs therapy has been hindered by both key problems, lack of targetting/poor cell engraftment and apoptosis of the implanted cells, resulting in poor therapeutic effects. Thus, enhancing the targeting and apoptosis-resistance of the implanted cells is the key for effective stem cell therapy. .Founded by the national natural science fund committee, we have designed stem cell homing molecule (SCHM) SDF-1 Nt-RGD composed of the receptor binding domain of SDF-1 (Nt) (no native SDF-1activity) and segments of RGD, which could then bind CXCR4 expressed on BMSCs via the Nt segment, and integrin receptors expressed on the surface of neonatal and activated endothelial cells in wounds via the RGD segments. Finally, we demonstrated that this SCHM can bind to the CXCR4 receptor of BMSC and integrin β3, β5 of wound tissue in vitro, respectively, and facilitate wound healing through enhancing BMSCs recruitment and neovascularization in wounds. Therefore, the potential of the implanted BMSCs homing to wounds can be markedly enhanced by pretreated cells with SCHM. .In the present study, to settle another major challenge to stem cell therapy, namely, survival of the implanted cell, we will redesign the SCHM. So far, preconditioning-induced protection in stem cell transplantation therapy is the best strategy to enhance stem cell survival, apoptosis resistance, and improving stem cell therapeutic potential. The SDF-1 preconditioned stem cells showed significantly enhanced cell survival and apoptosis-resistance potentiality during transplantation for wound healing. .In the present study, for the redesigned SCHM, we will only redesign the SDF-1 Nt part of this molecule, with the RGD-derived polypeptides unchanged, which is still the trafficking/targeting system of SCHM. The redesigned SDF-1 Nt motif is required and sufficient to bind and activate its receptor CXCR4, so that new SCHM will be assigned the activity of native SDF-1. We expect that this SCHM have the activity of native SDF-1. .we hypothesis that preconditioning stem cell with this SCHM, not only enhances the migration toward wounds, but also enhances survival and anti-apoptosis of transplanted BMSCs in vitro and boosts their therapeutic effects in wounds. We will synthesis and screen a polypeptide with the best activity to identify in vitro its bridging effect that conbine the CXCR4 and the integrin receptor, respectively; In vitro experiment, we will observe the migration ability, survival of BMSCs, and activation of the pro-survival signaling pathway after preconditioned with the SCHM. After transplanting BMSCs of preconditioned with the SCHM, we will observe the homing efficiency of stem cells, the rate of apoptosis in wounds, finally, we will demonstrate the improvement in therapeutic potential of transplanted cells, promoting angiogenesis and accelerating wound healing.

干细胞移植是严重、难愈创面治疗的希望策略，干细胞靶向性差、移植后大量凋亡是干细胞移植治疗疗效不佳的两大主要原因，故增强其靶向性和抗凋亡能力是关键。前一个自然基金的资助下，设计合成在干细胞归巢分子，证实其具有增强干细胞靶向归巢创面及促愈作用。但归巢定植创面的干细胞发生凋亡，故增强移植后干细胞存活是亟待解决的难题。SDF-1预处理是增强干细胞存活抵凋亡的重要策略。本项目欲在先前归巢分子基础上进行改造，不改变归巢体系，将其设计为具有SDF-1活性融合多肽。新的归巢分子不仅能促进干细胞归巢，还能增强干细胞的存活和抗凋亡能力；经其预处理的干细胞，观察其增殖、迁移和抗凋亡能力的改善及相关信号通路影响；经归巢分子预处理，观察干细胞靶向创面的归巢效率及在创面存活和凋亡率，对损伤组织修复重建的改善。最终获得增强干细胞靶向归巢和抗凋亡新型干细胞归巢分子。彻底改善干细胞移植治疗效率，为临床新药开发提供实验依据。

本研究通过设计合成归巢分子多肽（SDF-1-Nt RGD）数条，体外观察多肽预处理对小鼠骨髓间充质干细胞（BMSC）生物学活性的影响；观察多肽SRD预处理对MSC增殖、迁移、存活和抗凋亡功能的改善，以及对相关信号通路活化；移植经多肽SRD预处理的MSC，观察其靶向受损组织的聚集率和存活，及其对创面修复作用的改善。. 通过IP实验筛选分子获得活性最好的多肽SRD。功能性实验结果如下：（1）经多肽预处理对的BMSC的增殖活性、三系诱导分化活性以及表面标记物等生物学活性没有影响；（2）多肽SRD通过活化SDF-1/CXCR4/7的通路增强干细胞迁移；（3）此肽具有显著增强BMSC抗氧化应激性凋亡的作用，能显著降低高浓度H2O2诱导的BMSC凋亡，使其凋亡率下降5-6倍，从而发挥干细胞氧化应激保护作用；（4）多肽SRD活化PI3K/AKT/mTOR通路及抑制GSK-3β表达、上调抗氧化蛋白SIRT1/3和抗氧化转录因子Nrf2、FOXO1的表达可能是其增强BMSC抗氧化应激凋亡的关键机制；（5）此肽上调BMSC线粒体结构蛋白TOM20表达、增强抗氧化酶活性SOD2活性，以及稳定线粒体膜电位，由此能减少线粒体ROS产生。（6）通过建立小鼠皮肤损伤模型证实，多肽SRD预处理后的BMSC向创面募集的数量显著增多，且创面滞留时间延长，表明多肽具有增强干细胞靶向创面聚集和存活的作用，且通过增加创面新生血管密度、CD31、α-SMA和胶原的表达，显著提高创面愈合率，表明移植经多肽SRD预处理BMSC，能显著改善创面愈合质量和速度，提高干细胞移植治疗效率。（7）增加的研究观察了多肽预处理BMSC对LPS诱导的急性肺损伤的治疗作用。再次证实了多肽增强BMS靶向性和存活的作用，并发现多肽具有促进BMSC向肺泡I型和II型上皮细胞分化的作用，从而可能提高BMSC对损伤肺组织再生修复效应。. 本研究的科学意义在于为解决干细胞移植治疗临床面临的两大挑战，即干细胞靶向性差和移植后发生大量快速凋亡，多肽从根本上改善干细胞移植治疗效率低的状况。为临床治疗严重或难愈创面提供强有力的治疗手段。同时，由于多肽的强效抗氧化活性，可能对氧化损伤相关性疾病的防治也具有积极治疗作用。