人羊膜间充质细胞结合静电纺丝支架修复兔尿道缺损的研究

发布时间：2018-12-23 12:24

【摘要】：目的通过建立新西兰大白兔尿道缺损模型，探讨以人羊膜间充质细胞（hAMCs）为种子细胞结合可降解聚乳酸/聚乙二醇（PLLA/PEG）静电纺丝纤维支架修复兔尿道缺损的可行性。方法采用胰蛋白酶-胶原酶联合法从足月健康产妇剖宫产后的新鲜人羊膜组织中分离培养人羊膜间充质细胞。将聚合物聚乳酸/聚乙二醇混合物采用静电纺丝技术制成纳米纤维支架。27只雄性新西兰大白兔距尿道外口0.5cm处腹侧尿道后壁向近端切除尿道2.0cm*1.5cm，制作尿道缺损模型。随后随机分成三组：A组（实验组,n=9）应用负载人羊膜间充质细胞的静电纺丝纳米纤维支架修复兔尿道缺损区；B组（对照组I,n=9）用静电纺丝纤维支架直接修复兔尿道缺损区；C组（对照组II,n=9）将兔尿道缺损区直接行端端吻合。分别在手术后第4、8、12周切取含兔尿道修复区标本，HE染色和免疫组化检查判断修复段尿道组织再生情况；第12周行尿道造影，观察尿道畅通情况，同时比较各组间尿道外形以及结石形成率、尿瘘和尿道狭窄等并发症有无差异。结果分离培养的人羊膜间充质细胞呈不规则长梭形和多角形，成放射状或漩涡状分布，在特定诱导培养基下能向成脂、成骨和成软骨细胞分化；免疫荧光法表型鉴定干细胞表面标记物Oct-4和NS在人羊膜间充质细胞中广泛的表达；流式细胞术鉴定，人羊膜间充质细胞高表达造血干细胞标志CD29，CD90和CD105，不表达CD45。静电纺丝技术制备的聚乳酸/聚乙二醇纳米纤维支架通过细胞毒性测定证明对细胞无毒性。兔尿道缺损修复模型形态学观察，A组尿道壁完整光滑，尿道缺损区与正常组织融合；组织学上A组随着修复时间的延长，修复段尿道粘膜由单一的尿路上皮演变成多层尿路上皮结构，基本符合正常尿道组织的结构。B组修复段尿道无连续尿道上皮覆盖，，C组尿道管腔结构完整，但组织结构紊乱。尿道造影结果显示：A组尿道通畅，和术前造影结果对比无明显差异，B组和C组均有不同程度的尿道狭窄。并发症方面，B组和C组尿道狭窄、尿瘘和结石生成率均明显高于A组，术后A组尿道狭窄(0/9)、尿瘘(0/9)和结石生成（1/9）,合并症发生率为3.70%（1/27）；B组尿道狭窄(3/9)、尿瘘(1/9)和结石生成（8/9），合并症发生率为44.44％(12/27)；C组尿道狭窄(9/9)、尿瘘(4/9)和结石生成（9/9），合并症发生率为81.48％(22/27)，(P 0.05)。结论1.人羊膜间充质细胞具有向多种细胞类型分化的潜能，是组织工程尿道种子细胞的一种理想选择；2.通过静电纺丝技术制备的纤维聚乳酸/聚乙二醇支架组织相容性良好，是可行的组织工程支架。3.静电纺丝纤维聚乳酸/聚乙二醇支架结合人羊膜间充质细胞可以修复一定长度的新西兰大白兔尿道缺损。
[Abstract]:Objective to establish the urethral defect model of New Zealand white rabbits and to explore the feasibility of repairing urethral defect with (hAMCs) as seed cells combined with degradable poly (lactic acid) / polyethylene glycol (PLLA/PEG) electrospun fiber scaffold. Methods Human amniotic mesenchymal cells were isolated from fresh human amniotic membrane tissue after cesarean section by trypsin-collagenase-linked assay. The polymer poly (lactic acid) / polyethylene glycol (PEG) mixture was used to fabricate nanofiber scaffolds by electrostatic spinning. 27 male New Zealand rabbits were removed from the posterior wall of the ventral urethra from the external urethral orifice (0.5cm) to the proximal end of the urethra, and the urethra was removed from 2.0 cm to 1.5 cm. The model of urethral defect was made. Then it was randomly divided into three groups: group A (experimental group, nong9) treated with electrostatic spinning nanofiber scaffold loaded with human amniotic mesenchymal cells to repair urethral defect in rabbits; In group B (control group), the urethral defect was repaired directly with electrostatic spinning fiber scaffold, and in group C (II,n=9) the urethral defect was directly end-to-end anastomosed. The specimens of the urethral repair area of rabbits were cut at 812 weeks after operation, and the regeneration of urethra was evaluated by HE staining and immunohistochemical examination. At the 12th week, urethrography was performed to observe the unobstructed urethra. The appearance of urethra, the rate of stone formation, the complications of urinary fistula and urethral stricture were compared. Results the cultured human amniotic mesenchymal cells were irregular fusiform and polygonal, radial or swirling, and could differentiate into adipogenic, osteoblast and chondroblast under specific induction medium. The expression of stem cell surface markers Oct-4 and NS in human amniotic mesenchymal cells was identified by immunofluorescence method, and the high expression of hematopoietic stem cell markers CD29,CD90 and CD105, in human amniotic mesenchymal cells was detected by flow cytometry. Poly (lactic acid) / polyethylene glycol nanofiber scaffolds prepared by electrostatic spinning technique were proved to be non-toxic to cells by cytotoxicity test. Morphological observation of rabbit urethral defect repair model showed that the urethral wall in group A was intact and smooth, and the urethral defect area was fused with normal tissue. Histologically, with the extension of repair time, the urethral mucosa in group A changed from a single urethral epithelium to a multilayer urethral epithelium, which basically accorded with the structure of normal urethra. In group B, there was no continuous urethral epithelium covering the urethra. In group C, the urethral lumen was complete, but the structure of the urethra was disordered. The results of urethrography showed that the urethral patency in group A was not different from that before operation, and there were different degrees of urethral stricture in group B and group C. The incidence of urethral stricture, fistula and stone formation in group B and C were significantly higher than those in group A (0 / 9), urinary fistula (0 / 9) and stone formation (1 / 9). The incidence of complications was 3.70% (1 / 27). Group B had urethral stricture (3 / 9), urinary fistula (1 / 9) and stone formation (8 / 9). The incidence of complications was 44.44% (12 / 27). Group C had urethral stricture (9 / 9), urinary fistula (4 / 9) and stone formation (9 / 9). The incidence of complications was 81.48% (22 / 27), (P). Conclusion 1. Human amniotic mesenchymal cells have the potential to differentiate into a variety of cell types, which is an ideal choice for tissue engineering urethral seed cells. 2. The fiber polylactic acid / polyethylene glycol scaffold prepared by electrostatic spinning has good histocompatibility and is a feasible scaffold for tissue engineering. Electrostatic spinning fiber polylactic acid / polyethylene glycol scaffolds combined with human amniotic membrane mesenchymal cells can repair urethral defects in New Zealand white rabbits.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R695

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