hiPSC诱导分化及体内培育肾脏类器官的实验研究

Shortage of kidney is the current biggest obstacle encountered in clinical renal transplantation. Preparation of function good, portable artificial kidney is the important way to solve the clinical problem by regenerative medicine. The differentiation and assembly of iPSC on the kidney regeneration research has become a current research hot spot. In the previous work the applicant carried out kidney organoids culture in vitro and relevant appraisal work and found that there are two obvious defects in the kidney organoids cultured in vitro: the first is the less cell and the less nephron for nutrition supply , the second is the lower degree of cell differentiation. These two points are restricting the role of kidney organoids. The applicant intends to carry out research on these two points. The first, using of a variety of cells co culture in vitro and in vivo transplantation to promote the production of blood vessels of renal organoids, to solve the problem of organoids nutrition supply, which can increase the number of nephron. The second we intend to create a renal defect microenvironment by unilateral nephrectomy and drug induced chronic renal failure, which will be beneficial to the further differentiation of renal organoids related cells. In conclusion, this study intends to improve the experimental results, to obtain a good vascular and cell differentiation of kidney organoids,to lay the experimental foundation for the preparation of functional and portable kidney .

肾源短缺是当前临床肾脏移植中遇到的最大障碍。利用再生医学技术制备功能良好、可移植的人工肾脏是解决这一临床问题的重要途径。利用iPSC诱导分化组装进行肾脏再生研究已成为当前一大研究热点。申请人在前期工作中开展了肾脏类器官的体外培育及鉴定工作，发现体外培育的肾脏类器官存在两个明显的缺点：一是因营养供应问题，细胞数量受限导致肾单位数量少，二是细胞分化程度较低。这两点是制约肾脏类器官发挥功能的关键。申请人拟针对该两点开展研究。一、利用体外多种细胞共培养及体内移植培养的方式促进肾脏类器官产生血管，解决类器官营养供应问题，这样单个类器官可以拥有更多肾单位；二、我们拟通过单侧肾切除、药物诱导慢性肾衰等方式创造一个肾缺损微环境，后者将有利于肾脏类器官相关细胞的进一步分化。总之，本研究拟通过上述实验改进，获得血管化、细胞分化良好的肾脏类器官，为制备功能良好、可移植的人工肾脏奠定实验基础。

肾源短缺是制约当前临床肾脏移植的瓶颈。再生医学的发展为制备有功能、可移植的肾脏带来了希望。本项目追踪并优化了hiPSC诱导生成肾脏类器官的方法。同时，还尝试利用不同来源的hiPSC、不同方法诱导肾脏类器官并完成鉴定工作。为了解决肾脏类器官的营养供应问题，本项目通过体外和体内两种方式，探讨了肾脏类器官血管化问题。研究发现，体外肾脏祖细胞与间充质干细胞、脐静脉内皮细胞共培养可以形成紧密的类肾结构，并且这种共培养方式促进了类肾结构的增殖。在体内实验中，通过对比，发现肾被膜是肾脏类器官移植的最理想的移植位点。将肾脏类器官植入免疫缺陷鼠肾被膜下，肾脏类器官两周内体积迅速增大，四周左右可以看到小血管产生，从而解决了肾脏类器官营养供应问题。本项目制备了两种肾脏损伤模型。研究发现单侧肾切除创造的肾损伤微环境，能够促进对侧肾被膜下移植物的增殖及分化。研究还发现甘油诱导的急性肾损伤微环境能够促进后肾的进一步发育。此外，本项目发现脂肪干细胞在Wnt激动剂及FGF9信号的刺激下也能被诱导产生类间介中胚层细胞，后者灌注于肾脏脱细胞支架，得到了进一步分化。综上所述，本项目完成并优化了肾脏类器官的体外培养及鉴定工作，利用共培养及体内移植的方法促进了肾脏类器官的血管化。研究证明了肾脏损伤微环境对移植物增殖及分化的促进作用。证明肾脏脱细胞支架对种子细胞的促分化作用。以上结果为肾脏再生研究进一步研究奠定了基础并提供了借鉴。