iPS细胞转染抗菌肽LL-37为种子细胞构建泌尿系组织工程移植物的研究

Tissue engineering is currently one of the most promising physiological repair techniques in urological reconstruction. Efficient induction and differentiation of seed cells, rapid vascularization of the graft and anti-infective ability in the host body are the key of urological tissue engineering. In our previous studies, we have identified the cathelicidin LL-37 can accelerate the angiogenesis and anti-infective capacity of urological tissue-engineered grafts and promote the differentiation of stem cells. Induced pluripotent stem cells (iPS) are reprogramed and induced by somatic cells, with the advantages of multidirectional differentiation and elimination of autoimmune rejection. In this study, we would transfect iPS with the cathelicidin LL-37 in order to evaluate the anti-infective and pro-angiogenic ability of the transfected cells which secrets LL-37, the effects and the regulation mechanism of LL-37 on differentiation of iPS into urothelial cells and smooth muscle cells. At last, we would construct a new-style urological tissue engineered graft composited with the above transgenic seed cells and extracellular matrix to repair urethral and bladder defects, and we would also establish the transformation process of the grafts in vivo. In conclusion, we may provide innovative ideas and methods for the clinical application of urological tissue engineering.

组织工程是目前泌尿外科重建术中最有前景的生理性修复技术，种子细胞的高效诱导分化、移植物的快速血管化及在体内的抗感染能力是泌尿系组织工程的关键。我们前期研究已证实抗菌肽LL-37可加速泌尿工程组织的血管生成和提高抗感染能力，并能促进干细胞的诱导分化。诱导多能干细胞（iPS）由体细胞重新编程诱导产生，具有多向分化及消除免疫排斥等优点。本研究拟将iPS细胞转染抗菌肽LL-37，评价转基因iPS细胞分泌LL-37的抗感染及促血管化能力，并研究LL-37对iPS细胞分别向尿路上皮和平滑肌细胞的促分化作用及调控机制；将转基因种子细胞与支架材料复合构建泌尿工程组织，观察组织工程移植物在体内环境中的变化和转归情况，以期为泌尿系组织工程的临床应用提供新的思路与方法。

组织工程是目前泌尿外科重建术中最有前景的生理性修复技术，种子细胞的高效诱导分化、移植物的快速血管化是泌尿系组织工程的关键。我们在前期实验的基础上，对鼠源性多能诱导干细胞（miPSC）培养过程中添加GSK3β抑制剂CHIR99021、激活素A、FGF10和全反式维甲酸建立了“两步法”分化诱导体系，并验证了抗菌肽LL37转染miPSC后可适量分泌LL37，血管形成实验、小鼠下肢缺血实验等体内外实验证实了LL37促血管形成的作用。后由于miPSC培养困难、分化稳定性差等问题，我们及时更换为人源性多能诱导干细胞（hiPSC），并通过“两步法”成功分化为尿路上皮细胞。通过高通量测序、PCR筛选了下游差异表达基因circPLCE1，行PCR、western blot等实验初步验证了高表达circPLCE1的hiPSC分化后尿路上皮细胞标志物表达明显升高，而低表达分化circPLCE1的hiPSC分化后尿路上皮细胞标志物表达明显降低，这进一步说明了circPLCE1可促进hiPSC向尿路上皮细胞分化过程中发挥了促进作用。并且我们成功提取了hiPSC来源的外泌体，通过血管形成实验、增殖实验和小鼠下肢缺血实验等体内外实验验证了其促血管生成作用，通过高通量测序及PCR实验寻找外泌体内的差异表达基因。高表达mir-103a-3p可明显促进HUVEC的血管生成作用，而低表达mir-103a-3p的HUVEC血管生成能力下降，这说明了外泌体内mir-103a-3p的促血管生成机制。本研究为临床膀胱功能重建提供了稳定且高效的种子细胞，通过与外泌体结合构建多层生物复合结构，可为泌尿系组织工程的临床应用提供新的思路与方法。