人参皂苷Rg1联合HIF-1α基因修饰的人羊膜间充质干细胞对小鼠急性肾小管坏死模型协同治疗效果及机制探讨

Human amnion-derived mesenchymal stem cells (hAD-MSCs) transplantation in the treatment of acute tubular necrosis(ATN) has a good application prospect, but hAD-MSCs survival and homing rate is low,the finite differentiation rate, transplantation effect is not ideal limits its application. Ginsenoside Rg1 as inducer of hAD-MSCs can promote it specialized to renal tubular cells, and also promote it the migration, homing to the kidney; at the same time, Rg1 as a small molecule substance, easily through the glomerular filtration membrane, has anti-inflammatory, antioxidant, free radical scavenging, inhibition of apoptosis, reduce the number of renal tubular necrosis and promoting angiogenesis and so on, so as to provide a good microenvironment for the engraftment and proliferation,differentiation, homing of hAD-MSCs in the kidney.This project carry out In vitro and in vivo two aspects, under the intervention of Rg1, the Regulation of CaR as a target,To observe the effect of Rg1 on the differentiation of hAD-MSCs into renal tubular cells and the protective effect of Rg1 on the injury of renal tubular cells in vitro;To observe the effect of Rg1 on the proliferation and differentiation,homing of hAD-MSCs ,and synergistic therapeutic effect of renal injury in vivo ,and to explore the role of Rg1 in renal tubular repair and mechanism, which is expected to improve stem cell homing rate and treatment of ATN to explore new avenues, drug and target.

人羊膜间充质干细胞(hAD-MSCs)移植在防治急性肾小管坏死(ATN)方面具有良好应用前景，但hAD-MSCs移植成活归巢率低、有限分化率、移植效果不理想限制了它的应用。Rg1作为hAD-MSCs的诱导剂可以促进hAD-MSCs特化为肾小管样细胞，并促进其迁移、归巢到肾脏；同时，Rg1作为小分子物质，易于通过肾小球滤过膜，具有抗炎、抗氧化、清除自由基、抑制细胞凋亡、减少肾小管坏死数目及促进血管生成等作用，从而为hAD-MSCs在肾脏内的植活及增殖分化、归巢提供良好的微环境。本项目采用体内外两方面，在Rg1干预下，以调节CaSR为靶点，在体外观察Rg1对hAD-MSCs向肾小管样细胞分化及对损伤肾小管细胞的保护作用的影响；体内观察Rg1对hAD-MSCs的增殖分化、归巢及协同治疗肾损伤效果，并探讨其在肾小管修复中作用与机制，这有望为提高干细胞归巢率和治疗ATN探索新的途径、药物及作用靶点。

急性肾损伤（AKI）是一种常见、多发性疾病，临床上缺乏有效治疗手段，仅以支持为主，其中很多AKI患者因未及时恢复转为慢性肾病，甚至发展为终末期肾病。本课题组已成功分离、培养、鉴定且获得了较高纯度和增殖分化能力强的人羊膜间充质干细胞（hAD-MSCs）的基础上，主要是采用人胎盘来源的hAD-MSCs和转染低氧诱导因子-1α基因的hAD-MSCs移植治疗急性肾损伤小鼠模型，且经人参皂苷Rg1药物干预下，评价Rg1、hAD-MSCs、Rg1+HIF-1α-hAD-MSCs修复AKI疗效与机制。. 研究结果表明：①hAD-MSCs 供源便利、可体外扩增分化、不牵涉伦理学问题、免疫排斥反应较低等优势，此外，hAD-MSCs 还具有向受损组织、炎症区域迁移、归巢的特性，且利用慢病毒转染HIF-1α基因后，并不改变其基本生物学特性；②在体内实验中，经过Rg1、hAD-MSCs、HIF-1α-hAD-MSCs移植治疗急性肾损伤模型小鼠均能改善修复肾损伤，且Rg1+HIF-1α-hAD-MSCs移植治疗效果更佳，优于单用Rg1或和hAD-MSCs，其机制可能与Rg1促进hAD-MSCs在肾脏的定植、存活以及自身旁分泌HIF-1α因子、降低NAG及Kim-1表达有关；③移植后第7天电镜检查发现：AKI组线粒体损伤严重，Rg1、hAD-MSCs、Rg1+hAD-MSCs及Rg1+HIF-1α基因修饰hAD-MSCs组较AKI组损伤程度明显减轻，Rg1+HIF-1α-hAD-MSCs组损伤最轻，而Rg1、hAD-MSCs组间无明显差异；④control、AKI组与Rg1组肾组织未见MAB281阳性的绿色荧光，第3、7天时，Rg1联合hAD-MSCs组绿色荧光细胞数明显多于hAD-MSCs组；⑤小动物活体成像显示：hAD-MSCs、Rg1+hAD-MSCs及Rg1+HIF-1α-hAD-MSCs组脾脏、肝脏、肺脏红色荧光信号最强，肾脏次之，且Rg1联合hAD-MSCs组荧光强度高于hAD-MSCs组。以上研究结果有望为中药单体人参皂苷Rg1联合HIF-1α基因修饰hAD-MSCs靶向基因治疗临床进展期AKI患者预后奠定基础，为临床AKI治疗提供新思路、靶点药物和基因生物疗法，同时拓展hAD-MSCs在再生医学领域广阔的应用前景。