多样化运用UTMD在慢性肝损动物模型中促进骨髓间充质干细胞归巢及调控定向分化的实验研究

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are ideal seed cells for the cell transplantation therapy of end-stage liver diseases. The bottleneck problem of this research currently includes: the undefined proliferation of BM-MSCs in vivo and the difficulty of homing into injured liver. The keys to solve the problem are①the stable concentration of regulating the oriented differentiation around the stem cells②the cooperative reinforcement of homing ability of injured liver and stem cells. The applicant previously demonstrated that enhancement effect and stable expression of exogenous genes in pluripotent stem cell was achieved by the ultrasound targeted microbubble destruction combining recombinant adeno-associated virus. Based on our previous original results, we will carry on further study to solve above-mentioned problems. We will establish the animal models of chronic liver injury, and then employ UTMD technique to promote the transfection of rAAV encoding hepatic growth factor(HGF) into BM-MSCs. The oriented differentiation of BM-MSCs into hepatic cells will be continuously stimulated by the stable expressing HGF. Meanwhile, the homing ability of BM-MSCs themselves will be enhanced by the upregulation of chemotatic factors and the corresponding receptors on the cell surface which through the irradiation of UTMD and secreted HGF. In addition, the application of UTMD irradiation on liver will increase the potential of attracting BM-MSCs from circulation by the occurrence of "molecular zip code" on the vessel wall in the injured livers, which will further promote the homing effect of BM-MSCs. The applicant will deeply investigate the mechanism of UTMD inducing the change of biological characteristics of BM-MSCs, through observing the involvement of Wnt/β-Catenin signaling pathway in relation with the proliferation and the differention of BM-MSCs, in order to provide a new method for the therapy of end-stage liver diseases by BM-MSCs transplantation.

骨髓间充质干细胞是治疗终末期肝病的理想种子细胞，目前制约其发展的难题是在持续肝损状态下细胞归巢困难及分化方向不确定。解决此问题的关键是①干细胞周围具备定向分化因子的稳定浓度②协同增强肝组织和干细胞归巢能力。申请人前期利用超声靶向破坏微泡(UTMD)联合重组腺相关病毒(rAAV)实现了基因在多潜能干细胞的稳定表达；本课题将以此为基础，对上述难题展开研究：建立慢性肝损动物模型；利用UTMD增强携带肝细胞生长因子（HGF）的rAAV转染骨髓间充质干细胞，HGF稳定表达使干细胞持续向肝细胞定向分化；借助UTMD辐照及HGF联合作用使骨髓间充质干细胞表面趋化因子/受体表达增多，细胞主动归巢能力增强；采用UTMD定位辐照进一步诱导肝组织血管壁出现"分子邮政编码"，增加干细胞归巢的吸引力；同时探讨UTMD促进干细胞分化及归巢中Wnt信号通路的参与机制；从而为骨髓间充质干细胞治疗终末期肝病探索出新的思路。

骨髓间充质干细胞（BMSCs）在治疗肝损伤中显示出极大潜力，但在体内外应用中仍存在分化效率低和归巢率低等缺陷。本研究探索应用超声技术来促进BMSCs的分化及归巢，增强其体内治疗效果，并探讨相应机制。结果发现，在体外实验中，采用超声辐照联合肝细胞生长因子（HGF）处理BMSCs后，在一周内，超声加速了BMSCs向肝样细胞的分化，且这种加速分化与Wnt/β-catenin信号通路的激活相关；进一步重复超声辐照联合高浓度HGF处理BMSCs后，在3周内，BMSCs稳定向肝样细胞分化，且细胞表达趋化因子SDF-1α和CXCR4显著增加。本研究成功建立急性肝损大鼠动物模型，利用超声辐照微泡（UTMD）促进BMSCs归巢，持续观察2周，UTMD组肝脏功能改善程度较其他组更为显著；单独使用BMSCs亦可改善急性肝损动物模型的肝功能，但其修复肝脏的过程伴随纤维化程度的增加，而采用UTMD不仅加速肝功能改善，且不带来明显纤维化；探索其相关机制发现，UTMD可使受损肝脏表达更多趋化因子，吸引干细胞归巢，而更多BMSCs归巢后使受损肝脏的细胞更多处于增殖状态，凋亡程度亦显著减低。本研究继之成功建立慢性肝损动物模型，为了维持BMSCs细胞归巢至肝脏后稳定向肝样细胞分化，我们构建携带HGF的重组腺相关病毒载体转染BMSCs，使用UTMD联合这种稳定表达HGF的BMSCs进行治疗，持续观察10周，结果发现，UTMD不仅明显提高BMSCs的归巢率，肝功能指标恢复较其他组更为明显，且UTMD组中纤维化程度较单独BMSCs及单独使用超声介导BMSCs的方法明显减轻。本研究通过体内外研究证实了超声辐照技术促进BMSCs移植的效果，为BMSC的移植治疗提供了新的思路，且为未来向临床方向转化提供实验基础。