靶向Notch信号通路调控骨髓间充质干细胞促进肾缺血再灌注损伤修复的研究

Bone marrow mesenchymal stem cells (MSCs) transplantation in the treatment of renal ischemia-reperfusion injury (IRI) largely depends on the survival, proliferation and differentiation ability of cells. Our previous research work shows that MSCs transplantation can protect renal IRI through the paracrine mechanism and differentiation mechanism, how to enhance the paracrine and differentiation mechanism is a problem to be solved. Notch signal pathway which is the regulator is involved in proliferation and differentiation of stem cell.This research will establish expression system of Notch intracellular domain (NICD) in rats MSCs induced by tetracycline, then transplanting the cells to treat renal IRI in rats. In order to enhance the survival and proliferation efficiency of MSCs, NICD expression is up-regulated; then, to promote MSCs differentiation to renal tubular cells, NICD expression is down-regulated; Finally, NICD activated again, to render the proliferation and differentiation of MSCs, in order to achieve the aim of long-term treatment. In the course, to monitor survival, proliferation, migration and the differentiation state of MSCs via immunohistochemical assay, PCR and Western blot. To evaluate the effect of Notch signal in MSCs transplantation dynamically.

骨髓间充质干细胞（MSCs）移植治疗肾缺血再灌注损伤（IRI）的疗效很大程度取决于植入细胞的成活、增殖和分化能力。我们前期研究工作中表明MSCs移植可以通过旁分泌机制和分化机制参与肾IRI的修复，如何增强MSCs的旁分泌和分化机制是亟待解决的问题。Notch信号通路参与干细胞增殖和分化的调控，控制着移植细胞的转归取向从而影响治疗效果。本研究将在大鼠MSCs中建立四环素可诱导的Notch胞内区（NICD)表达系统，再将该细胞移植治疗肾IRI大鼠。在移植早期诱导NICD高表达以期增强MSCs成活和增殖效率；在移植后期降低NICD表达便于MSCs向肾小管细胞分化；最后再次激活NICD使沉寂于肾脏内的MSCs增殖分化，以期达到长效的治疗目的。在此过程中用免疫组化、PCR、免疫印迹等方法监测植入细胞成活、增殖、迁移和分化状态，动态地评价Notch信号对移植MSCs转归和疗效的调控。

骨髓间充质干细胞（MSCs）用于治疗肾缺血再灌注损伤是近年来研究的热点课题，Notch 信号通路参与干细胞增殖和分化的调控，控制着移植细胞的转归取向从而影响治疗效果。本研究通过体外分离培养大鼠MSCs，构建Tet-On系统调控 Notch 胞内区（NICD)稳定表达的NICD-Tet-MSCs细胞，然后将此细胞移植至肾缺血再灌注损伤大鼠体内，诱导 NICD 表达变化达到调控MSCs 成活和增殖；在此过程中采用组织病理技术、PCR、免疫印迹等方法检测相关基因和蛋白表达，检测肾缺血损伤前后血清肌酐变化以及肾组织中外源性MSCs分布。动态地评价 Notch 信号对移植 MSCs 转归和疗效的调控。主要研究结果：1. 经MigR1质粒构建的外源性MigR1-NICD-MSCs细胞移植入大鼠，该组大鼠经肾IRI后24h、48h、72h血清肌酐及尿素氮水平明显低于对照组（MigR1-MSCs组）（P<0.05），进一步肾组织H&E染色结果显示肾小管上皮细胞坏死减轻，肾小管病理损伤评分更低，差异有统计学意义（P<0.05）；2. Tet-on 3G系统构建NICD诱导表达的骨髓间充质干细胞NICD-Tet-MSCs中稳定表达NICD并受多西环素（Dox）调控；3.与对照组相比，NICD-Tet-MSCs移植组大鼠肾IRI术后血清肌酐水平明显降低(P<0.05)；4.外源性MSCs分布在NICD-Tet-MSCs移植组大鼠肾组织中增殖持续时间更长；5.NICD-Tet-MSCs移植组大鼠肾组织中血管内皮生长因子蛋白表达升高，肿瘤坏死因子α蛋白表达降低，差异有显著性意义(P<0.05)。上述结果表明，体外能够成功构建Tet-On系统调控NICD稳定表达的大鼠MSCs，此细胞可促进大鼠肾缺血再灌注损伤的修复。