基于IGF-1研究黑地黄丸调控5/6肾切除大鼠BMSCs归巢及RTECs凋亡的作用

Insulin-like growth factor -1 (IGF-1) is one of the cytokines secreted from bone marrow mesenchymal stem cells (BMSCs), not only can promote the BMSCs homing to the remnant kidney, but also has antagonistic effect on renal tubular epithelial cells (RTECs) apoptosis. It is known that RTECs apoptosis is the initial factor of renal interstitial fibrosis (RIF). Our previous research shows that the Hei Di-huang Bolus can inhibit RIF, reduce glomerular sclerosis, and vitro experiment indicated that the drug can promote BMSCs proliferation and secreting IGF-1. This study intends to verify the hypothesis that Hei Di-huang Bolus can promote BMSCs autocrine IGF-1 to improve the homing rate of itself to remnant kidney and can paracrine IGF-1 to inhibit RTECs apoptosis and delay RIF through both vivo and vitro experiments. The vivo experiment intends to observe the effect of Hei Di-huang Bolus on BMSCs distribution in renal tubules, homing of BMSCs and and expression of the RTECs apoptosis signals. The vitro experiment intends to observe the effect of Hei Di-huang Bolus on adhesion and migration of BMSCs and RTECs apoptosis. This research intends to further explore the mechanism of the protective effect of Hei Di-huang Bolus on kidney and find a new target for the treatment of CRF with traditional Chinese medicine.

胰岛素样生长因子-1（IGF-1）是骨髓间充质干细胞（BMSCs）分泌的细胞因子之一，不仅可以促进BMSCs归巢至残肾，而且具有抗肾小管上皮细胞（RTECs）凋亡的作用，而RTECs凋亡是肾间质纤维化（RIF）的始动因素。本课题组前期研究显示，黑地黄丸可以抑制RIF，减轻肾小球硬化，体外实验亦证实该药能够促进肾衰大鼠BMSCs增殖及分泌IGF-1。本研究拟通过体内实验观察黑地黄丸通过影响BMSCs分泌IGF-1干预BMSCs在肾小管中的分布、BMSC归巢、RTECs凋亡信号的表达；体外实验通过粘附实验、迁移实验和RTECs凋亡研究观察黑地黄丸的干预作用，验证黑地黄丸促进BMSCs自分泌IGF-1提高BMSCs至残肾的归巢率、通过旁分泌IGF-1抑制RTECs凋亡、延缓RIF的假说，进一步探索黑地黄丸肾保护作用的机理，为中医药治疗慢性肾衰寻找新靶点。

慢性肾衰竭是疑难病，缺乏有效治疗手段。BMSCs移植法为临床治疗慢性肾衰竭提供了新途径，但难点在于如何提高BMSCs移植后的增殖活性及归巢率。文献报道BMSCs可自分泌IGF-1促进自身增殖和迁移，而IGF-1又具有抗RTECs凋亡的作用。本课题组前期研究发现黑地黄丸不仅临床治疗慢性肾衰效果显著，基础研究显示黑地黄丸可提高肾衰大鼠BMSCs分泌IGF-1的能力。.课题组通过（1）体内实验建立5/6肾切除大鼠模型,分组给药后，取血清检测Scr、BUN、IGF-1水平；制作肾脏石蜡切片，进行HE、MASSON、PAS染色，观察组织病理学和超微结构的形态学变化，同时利用TUNEL染色和免疫组织化学染色技术，进行RTECs凋亡及肾小管间质纤维化指标的观察。抑制剂组验证机制。（2）体外实验提取原代BMSCs、RTECs并进行鉴定。构建HR-RTECs模型及EGFP-BMSCs细胞模型。建立BMSCs、RTECs的Transwell共培养系，分组给药干预后，检测各组BMSCs的迁移率，收集BMSCs检测CXCR4蛋白的表达，收集RTECs培养上清检测IGF-1、SDF-1的水平；收集下室RTECs，流式检测各组细胞凋亡率。抑制剂组验证机制。.研究结果显示，黑地黄丸可显著改善5/6肾切除大鼠的肾功能，延缓肾纤维化。黑地黄丸促进BMSCs自分泌IGF-1，促进BMSCs归巢，该过程可能由SDF-1/CXCR4轴介导。黑地黄丸促进BMSCs旁分泌IGF-1，发挥抗RTECs凋亡的作用，该过程与PI3K/AKT信号通路有关。.研究中发现，黑地黄丸在降低5/6肾切除大鼠体内IGF-1水平的同时伴有肾脏中klotho蛋白表达的增加。查阅文献后猜测这两者之间可能存在因果联系。针对这一情况，课题组将继续深入挖掘，为黑地黄丸的肾保护作用提供更多依据.