尿源性干细胞在膀胱再生中的应用

Bladder diseases, one of the most comment disorders in urinary tract system, are seriously affect the patients' health and life. Bladder augmentation using patients' gastric or intestinal segment is one of main options for people with end-stage bladder diseases. Although this surgical approach increases or restores bladder capacity, it often causes several significant complications. Cell-based tissue engineering provides alternative for the patients with bladder end-stage diseases. Use of stem cell types originating from the patient holds promise. Our pervious studies demonstrated that a subpopulation of cells isolated from urine possess the characteristics similar to MSCs, i.e. clonogenicity, cell growth patterns, expansion capacity, cell surface marker expression profiles and multipotent differentiation capacity. Thus, we have designated these cells "urine-derived stem cells" (USCs). Our recent work showed two types of USCS, those that do or do not express telomerase activity (USCs-TA+ and USCs-TA-). USCs-TA+ have higher proliferative capacities and can be maintained for a greater number of passages than USCs-TA-. Now, we propose to explore the use of a porous biodegratable biomaterial that, based on our published work, may have significant advantages for bladder tissue engineering. We custom-designed a scaffold based on a 3D porous smal intestinal submucosa (SIS) and then grew urothelial and smooth muscle cells on the scaffold under dynamic culture conditions in vitro. This biomaterial possesses the appropriate macroporosity and interconnected microporosity to allow cell ingrowth, confer mechanical strength, and promote angiogenesis. Our overall hypothesis is that USCs-TA+ possess high differentiation capacity and can give rise more efficiently to functional urothelial, endothelial and smooth muscle cells. Further, 3D porous SIS scaffolds seeded with induced USCs are more suitable for engineering bladder tissue. To test this hypothesis, we will utilize cells differentiated from human USCs to construct bladder tissue with the modified SIS scaffold for implantation in an athymic rat model. To test the suitability of both the novel cell source and biomaterial for the production of bladder tissue, our Specific Aims are: Aim 1. Determine differentiation capacity and trophic effects of human USCs-TA+ in vitro. Aim 2. Develop a functional neo-bladder using differentiated human USCs-TA+ and porous SIS scaffolds, and assess their function and histology in vivol. The benefit of using USCs is that the cells can be obtained from patients' own urine via a simple, non-invasive and low-cost approach for this purpose. Our pilot work has poised us to successfully implement the proposed studies, which are necessary steps that must be optimized before further preclinical studies can begin. Success will bring us significantly closer to an improved treatment option for patients with bladder diseases who need bladder reconstruction.

膀胱疾病是泌尿系统最常见的疾病之一，且严重影响病人的身体健康. 常规的重建手术可能引起一系列严重的并发症。研究及使用来自泌尿系统的干细胞为组织工程膀胱及其重建提供了良好的细胞来源。我们前期的实验结果表明，尿液中存在一种具有显著干细胞特性的细胞，可通过简单、无损伤性方法获得。这种尿源性干细胞具有多种分化潜能，但对其在膀胱组织再生中的作用并不清楚。为了验证尿源性干细胞在膀胱组织再生中的作用，我们将分离出具有高端粒酶活性的尿源性干细胞，诱导为移行上皮，血管内皮和平滑肌细胞，测定其旁分泌因子,将细胞种植在多孔生物降解材料上，并移植动物体内用于膀胱扩大。采用细胞分子生物学，免疫组化、腺病毒载体转染标记，肌肉收缩实验、活体膀胱功能测定等手段, 探讨尿源性干细胞在膀胱再生中的重要作用。本研究将从来自泌尿系统的的干细胞这个新视点揭示干细胞在膀胱组织再生中的作用和机制，为膀胱及泌尿系统疾病的治疗提供新的思路

膀胱疾病是泌尿系统最常见的疾病之一，常规的重建手术可能引起一系列严重的并发症。本课题以兔来源尿源性干细胞作为种子细胞，以小肠粘膜下组织为生物材料，新西兰大白兔行膀胱切除及扩大术为膀胱损伤模型，将低氧处理后的兔尿源性干细胞与Hydrogel凝胶结合应用于兔子膀胱损伤模型中，探讨膀胱组织再生的情况。我们成功从兔子尿液及膀胱冲洗液中分离培养获得兔尿源性干细胞，鉴定其干细胞特性，诱导分化为移行上皮和平滑肌细胞，为模仿体内环境氧浓度，通过3%O2（低氧）体外培养，与常氧状态下培养的兔尿源性干细胞对比相关生物学性能。通过3%过氧乙酸处理新鲜小肠粘膜下组织，经100%胎牛血清浸泡，获得具有多孔结构，利于细胞向内生长的生物材料，通过检测残余DNA量、组织学、扫描电镜检测其制备效果。为防止抑制细胞在体内的脱落，将低氧预处理兔尿源性干细胞与Hydrogel凝胶结合应用于兔膀胱损伤模型。采用免疫组化、细胞膜标记、肌肉收缩实验、活体膀胱功能测定等手段, 检测膀胱再生过程中上皮组织、平滑肌及血管的再生情况，探讨尿源性干细胞在膀胱再生中的重要作用。实验结果显示，低氧环境可明显促进兔尿源性干细胞的增殖，抑制凋亡，提高细胞在体内的存活率。改良后的处理方式，使获得的小肠粘膜下组织具有良好的多孔结构，可以更好的保持生长因子、细胞因子和细胞外基质，为种植细胞提供更好的生长环境。体内实验部分，与对照组相比，治疗4周后，再生的膀胱组织扩张良好，移植物萎缩和组织变小。低氧组PKH-67标记细胞存活率明显高于常氧组；组织学检测提示实验组上皮组织、平滑肌肌束再生等亦好于对照组，且低氧组再生效果相对于常氧组显著提高。