【作者】 史建国；

【导师】 王晓雄； 符伟军；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2012， 博士

【摘要】 研究背景及目的：输尿管损伤后常常导致输尿管缺损或狭窄,继发患侧尿路梗阻、感染、尿外渗、尿性囊肿等并发症,严重威胁患者的健康及生活质量。对于损伤程度较重、缺损段较长的损伤,临床上常以胃肠道替代输尿管的方法处理。然而,肠代输尿管后常伴随各种并发症的发生,如尿路结石,慢性感染,肠粘连等。有研究者尝试使用人工合成材料或者异体移植的方法进行处理,但是治疗效果均不满意。因此,输尿管损伤后缺损的治疗已经成为泌尿外科的亟待解决的问题之一。组织工程科学的发展为输尿管损伤及缺损的修复重建提供了新的途径。组织工程的方法进行输尿管重建必须解决两个关键问题：支架材料及种子细胞。本研究以人脂肪干细胞为种子细胞,以聚乳酸/胶原为基本材料制作一种新型输尿管支架材料,拟为输尿管组织工程重建提供一种新的可行的治疗方法。研究的主要目的为：1、探索人脂肪干细胞的分离、培养及鉴定的方法,为以脂肪干细胞为种子细胞的研究提供实践基础；2、探索人脂肪干细胞诱导分化为尿路上皮的可行性；为输尿管组织工程研究提供新的种子细胞来源；3、制作一种新型组织工程输尿管支架,并评估这种新型输尿管支架是否利于人脂肪干细胞的生长、增殖；4、将携带细胞的新型输尿管支架置入裸鼠体内,评价材料的生物相容性,观察支架材料内细胞存活状态、诱导分化后的细胞表型能否保持,以初步评估利用这种新型输尿管支架材料进行组织工程输尿管的可行性。方法：1.取吸脂术患者皮下脂肪组织,采用酶消化法分离人脂肪干细胞,通过流式细胞学的方法鉴定其表型特征,通过成脂、成骨诱导初步鉴定其分化潜能。2.通过Transwell间接共培养法进行脂肪干细胞向尿路上皮细胞的诱导分化,采用RT-PCR、western blot、免疫荧光的方法对诱导后的细胞进行尿路上皮标志物的(CK-18,UP2)表达鉴定。3.以聚乳酸及Ⅰ型胶原作为基本原料,采用溶剂挥发法及电纺丝方法制作一种新型生物可降解输尿管支架材料,将脂肪干细胞与支架材料进行体外复合培养,通过电镜、LIVE/DEAD、H&E染色、MTT等方法评估新型输尿管支架是否利于人脂肪干细胞的生长、增殖。4.将携带人脂肪干细胞的新型输尿管支架材料植入裸小鼠(4周龄雌性)背部皮下,生长14天后取材,进行细胞径际分析、H&E染色、尿路上皮标志物的免疫荧光鉴定。结果：1.采用酶消化法分离的脂肪干细胞在细胞培养瓶内呈贴壁生长,倒置光学显微镜下观察呈长梭形,流式细胞学检测表明,细胞表达间充质干细胞的标志物(CD29,CD44,CD90以及CD105),不表达造血干细胞标志物CD31及组织相容性抗原标志HLA-DR。成脂诱导14天后细胞内可见大量脂滴,并且油红O染色呈阳性；成骨诱导21天后,茜素红染色后观察可见钙结节形成。2.通过与尿路上皮的间接共培养7天后,诱导后的细胞形态呈现尿路上皮细胞样,培养14天后,RT-PCR及western blot可检测到尿路上皮标志物(CK-18、UP2)在基因转录及蛋白表达水平上调。免疫荧光检测表明,40%-50%的诱导后的干细胞表达尿路上皮标志物(CK-18及UP2)。3.电镜观察聚乳酸/Ⅰ型胶原输尿管支架材料的表面具有三维空间结构,材料由直径约3微米(3.44±0.94μm)的纤维构成,纤维间形成孔径约10微米(10.54±3.18μ m)的孔隙。细胞支架体外孵育培养后3天,电镜观察发现支架材料表面细胞呈铺展状态,均匀分布于支架表面；MTT实验对细胞种植在支架后第1,3,5,7天进行连续观察发现携带胶原的支架材料细胞呈现持续增殖的趋势；细胞种植7天后H&E染色发现细胞均匀分布于支架材料表面,部分细胞浸润生长进入材料的内部；Live/dead检测发现材料上生长的细胞90%以上为活细胞。4.所有16只动物在携带细胞的新型输尿管支架材料植入体内14天的实验期间均安全存活,伤口愈合良好,无感染、炎症反应发生,诱导后的人脂肪干细胞能够在裸鼠体内存活达14天,浸润入支架材料的内部,并能够表达尿路上皮的标志物。结论：1、采用酶消化法提取人脂肪干细胞是可行的,提取的细胞能够表达间充质细胞的标志物,具有良好的增殖及分化潜能。2、采用间接共培养法能够诱导人脂肪干细胞向尿路上皮细胞分化,诱导分化后的细胞形态发生改变,并且可表达尿路上皮的标志物。3、采用聚乳酸/Ⅰ型胶原材料制作的新型输尿管支架材料,能够适合人脂肪干细胞的生长增殖。4、体内植入实验证实携带细胞的新型输尿管支架材料具有良好的生物学性能,诱导分化后的人脂肪干细胞在随支架植入体内后能够存活、生长并保持其分化特征。本研究为输尿管组织工程重建提供了新的有希望的治疗方案。更多还原

【Abstract】 Background and ObjectivesUreteral injuries often resulted in ureteral defect or stricture, and lead to secondaryurinary tract obstruction, infection, urinary extravasation, and urinoma, etc, whichdeeply impaired the wellbeing and life quality of the patients. Under the condition ofureteral major injuries or large defect, the commonly used material for ureteralreplacement is gastrointestinal tract. However, replacement with gastrointestinal isassociated with lots of complications including urinary calculi, chronic infections,intestinal adhesions, etc. Some researchers have tried the synthetic materials orallotransplantation to reconstructing the ureter with disappointing effect. Therefore,the treatment of ureteral injuries and defects has been an urgent problem to be solved.The development of tissue engineering offers a new way for reconstructing theureteral injuries and defects. The two key factors of tissue engineering the ureter arescaffold and seeding cell. In this research, we took the isolated human adipose derivedstem cells (ADSCs) as the seeding cells. In addition, we constructed a new typeureteral scaffold with PLA/collagen biomaterial in that providing a new method forureteral tissue reconstruction.The objectives of our research were as follows:1. To explore the method of isolation, culture and characterization of humanADSCs, in order to provide practical foundation for the research about using thehuman ADSCs as the seeded cells.2. To test the possibility of transdifferentiation of human ADSCs into urothelialcells, in order to provide new resource of seeding cells for ureteral engineering.3. To construct a new type of tissue engineering ureteral scaffold and to testwhether the scaffold is good for the growth and proliferation of human ADSCs.4. The seeded scaffold was implanted into the body of athymic mice to evaluatethe biocompatibility. Besides, the growth and the expression of urothelial markers from the seeded cells within the scaffold were also assessed for in vivo study, in orderto evaluate the feasible of ureteral tissue engineering with the novel ureteral scaffold.Methods1. Samples of human subcutaneous adipose tissues were obtained from patientsundergoing liposuction procedures. Enzyme digestion was used to primary isolate thehuman ADSCs. Flow cytometry assays were used to confirm the phenotype. Thedifferentiation potential was initially testified by adipogenic and osteogenicdifferentiation protocol.2. HADSCs were indirectly cocultured with urothelial cells in a transwellcoculture system for urothelial differentiation. Then, the differentiation was evaluatedby detecting urothelial lineage markers (cytokeratin-18and uroplakin2. throughRT-PCR, western blot, and immunofluorescence.3. A new type of PLA/collagen biodegradable ureteral scaffold was constructedthrough solvent evolution and electrospinning method. Then, the human ADSCs werecultured with the constructed ureteral scaffold in vitro. Whether the scaffold was goodfor the growth and proliferation of human ADSCs was evaluated by scanning electronmicroscopy (SEM), hematoxylin and eosin staining, LIVE/DEAD, and MTT analysis.4. The scaffolds, which were pretreated by seeding the urothelial differentiatedhuman ADSCs, were then implanted subcutaneously into4-week-old female athymicmice in the back.14days later, the grafted scaffold was harvested and evaluated bycell tracking, H&E staining, immunofluorescence.Results1. The human ADSCs isolated by Enzyme digestion displayed fusiform shapeand adherent to the bottom of the cell flask, observing under the inverted opticmicroscope. Flow cytometry demonstrated the isolated cells positive expression ofmesenchymal stem cell markers (CD29, CD44, CD90, and CD105), negativeexpression of hemopoietic stem cell marker CD31and histocompatibility antigensHLA-DR. After14days adipogenic induction, the formation of intracellularmicrodroplets was noted and stained positive for Oil Red O. After21days osteogenicinduction, the presence of calcium deposits in cultures was stained with Alizarin Red S.2. After cocultured with urothelial cells for7days, the morphology of the humanADSCs changed into urothelial-like. After14days coculture, the induced cellsdisplayed up-regulation of urothelial markers (CK-18, UP2) both in mRNA andprotein level testified by RT-PCR and Western blot. In addition, about40%-50%induced cells express urothelial markers (CK-18, UP2) according toimmunofluorescence.3. Three dimensional structure was observed on outer surface of PLA/collagen Rureteral scaffold according to SEM. The scaffold was constructed by fibers about3m(3.44±0.94m) in diameter and the pole size between the fibers about10m (10.54±3.18m). After the cell seeded onto the ureteral scaffold and cultured over a period of3days, the cells displayed a spreading appearance and uniform distributed on thescaffold, according to the SEM. MTT assays confirmed that the cells seeded onto thescaffold showed continual proliferation according to the measurement in1,3,5,7days after cell seeding. After cell seeding onto the scaffold for7days, the cells spreadevenly on the surface of scaffold and some of cells infiltrating into the inside of thescaffolds, according to the H&E staining. More than90%of the cells grown on thescaffold were alive according to Live/dead analysis.4. All the16surgical animals lived normally during the14days follow-upobservations after the novel cell seeded ureteral scaffold implantation in vivo. Inaddition, the wound healing was fine and no infection and inflammation was observed.The differentiated human ADSCs survived, infiltrated into the grafts and maintainedspecific urothelial markers after14days implantation in the body of athymic mice.Conclusions1. Enzyme digestion method was feasible for isolation of human ADSCs. Theisolated cells expressed mesenchymal markers and possess the good proliferation anddifferentiation potential.2. The human ADSCs can be induced into urothelial lineage differentiation byindirect coculture method. The differentiated human ADSCs changed their morphology and expressed urothelial markers.3. The novel ureteral scaffold, constructed with PLA/collagen, was suitable forgrowth and proliferation of human ADSCs.4. For in vivo study, it was demonstrated that the novel seeded ureteral scaffoldhad good biological function. The differentiated human ADSCs could survive, growand maintain differentiation marker after implantation with the scaffold. This researchprovided a new way for ureteral reconstruction and tissue engineering.更多还原