靶向新生血管RGD-SDF-1 Nt干细胞归巢分子的设计及其在创面愈合血管再生中的作用

The vascular regeneration and repair is the first key step for injured tissues/organs repair and regeneration. Bone marrow mesenchymal stem cells or/and bone marrow derived stroma stem cells/endothelial progenitor cells (BMSCs/EPCs) contribute to postnatal vascular regeneration and tissue repair. Therefore, mobilization or transplantation of BMSCs/EPCs is a promising strategy for reconstructing vessels and tissues of injured tissues/organs. However, problems in targetting wounds and availability of stem cells exist, potentially, which result in poor therapeutic effect and limited application. Thus, enhancing the targeting potential of BMSCs/EPCs is the key for the effective therapy of stem cells. As CXCR4, the exclusive receptor of stromal cell-derived factor-1α (SDF-1α), is stably expressed on BMSCs/EPCs, so SDF-1α plays a crucial role in the mobilizing, trafficking and homing BMSCs/EPCs from bone marrow to the injury tissues/organs. The N-terminal region (Nt)of the SDF-1α protein is prerequisite and sufficient to bind and activate its receptor. A number of integrins (αvβ3、αvβ5 and α5β1) expressed on the surface of neonatal endothelial cells regulate cell migration, proliferation and differentiation during neovascularization, which have the ability to recognize the peptide motif Arg-Gly-Asp (RGD). RGD-containing polypeptides can ont only promote neovascularization via enhancing the αvβ3 and αvβ5-dependent vasculogenesis, but also be used as a vehicle in the design of targetting drugs. Therefore,the polypeptides containing RGD is usually used as a vehicle to target neonatal vessels in wound sites. On account of these views, we have designed a kind of fusion polypeptide that is composed of the receptor binding domain of SDF-1α (Nt) and short peptids containing RGD, which could then bind the receptor CXCR4 expressed on BMSCs, as well as intergrin receptors expressed on the surface of neonatal endothelial cells (EC) in wounds. Finally, we hypothesis that the fusion polypeptides/stem cells homing molecules can facilitate wound healing through enhancing BMSCs/EPCs recruitment and neovascularization in wounds. We will synthesis several different assembly polypeptides, then we are going to screen a polypeptide with best activity to identify in vitro its bridging effect that conbinating with the CXCR4 receptor of BMSCs/EPCs and the integrin of neonatal EC in wounds, respectively; After transplanting BMSCs/EPCs of pretreated with the homing molecule in vitro, we will establish the homing efficiency of stem cells in wound, and the biologic activity of the fusion polypeptide for promoting neovascularization and wound healing.

促创面血管再生重建微循环是组织/器官损伤修复的前提和基础。以骨髓基质干细胞/内皮祖细胞为基础的骨髓动员和干细胞移植治疗是促进血管生成和组织修复的希望策略。干细胞靶向性差、到达受损组织量少、利用率低，致使干细胞治疗应用受限和疗效不佳，故增强其靶向性是提高疗效的关键。SDF-1是干细胞迁移的关键趋化因子，其受体CXCR4表达于骨髓干细胞膜。本项目拟设计并合成一种既能与干细胞膜受体CXCR4结合，又能与损伤区新生内皮细胞表达的整合素结合，能引导干细胞靶向受损组织的归巢分子RGD-SDF-1 Nt多肽；体外鉴定归巢分子与干细胞和内皮细胞相互作用的桥连效应；经在体实验鉴定归巢分子处理的干细胞靶向创面的聚集效率、促进血管再生和组织修复疗效。最终获得一种能增强干细胞向受损组织靶向归巢和利用效率的新型干细胞归巢分子，为开发组织再生修复新药及新型治疗策略奠定基础。

我们设计合成了归巢分子融合多肽（RGD-SDF-1 Nt），分子的一端设计为SDF-1α与受体结合的结构域1～17个氨基酸序列，即N-末端（Nt）1～17（RPVSLSYRCPC RFESH）个氨基酸序列，此序列被证实具有CXCR4受体结合的功能。仅考虑归巢分子捕获循环干细胞的能力，将此片断中的Lys（K）置换为Arg(R)，让其丧失趋化活性，但受体的结合能力与SDF-1相当。因此，理论上，分子此端具有结合干细胞的功能；分子的另一端设计为不同长度的含有RGD序列的短肽，包括GRGDS（识别αvβ3, α5β1 and aIIbb3）和GRGDVY（识别αvβ3）。这些序列均能分别识别不同整合素受体，能与新生血管内皮细胞膜整合素受体（如αvβ3、αvβ5和a5b1 ）高亲和、特异性结合，如此，此归巢分子能靶向运载干细胞到达损伤区域血管，通过RGD介导与创面新生血管EC（αvβ3, αvβ5）高亲和力结合，将干细胞牢固定植于受损组织区域，从而增加干细胞的归巢效率。最终达到显著提高严重组织损伤血管再生和组织修复重建的目的。此外，还设计了两条阳性多肽RGD-SDF-1 Nt（SDF-1α N-末端1～17氨基酸）和GRGDVY（六肽，识别αvβ3）作为阳性对照多肽。.本研究通过IP实验筛选分子两端能分别能与CXCR4（干细胞）和整合素β3（创面新生血管）的高亲和力结合的归巢分子融合多肽（II）。通过实验证实此分子一端能高亲和力结合BMSC的膜受体CXCR4，分子另一端又能与受损组织区新生血管内皮细胞高表达的整合素受体β3结合；通过在体实验进一步证实，与未经处理BMSC移植比较，经归巢分子（RGD- SDF-1 Nt）和阳性多肽SDF-1Nt处理后BMSC移植，向创面募集的数量显著增多，由此证实归巢分子和SDF-1Nt能增强BMSC的创面归巢效率，而以归巢分子的作用最为显著；同时，经归巢分子处理的BMSC移植治疗创面，能显著增强受损组织区新生血管密度和CD31的表达、提高创面愈合率、增强创面组织α-SMA和胶原的表达，由此表明经归巢分子处理后的BMSC具有增强血管生成、加速创面愈合和提高修复质量的作用。.最终，本研究内容已按计划完成，此外，还增加完成了一些阳性多肽SDF-1 Nt的研究.数据。