人羊膜間充質(zhì)細胞結合靜電紡絲支架修復兔尿道缺損的研究
發(fā)布時間：2018-12-23 12:24
【摘要】：目的通過建立新西蘭大白兔尿道缺損模型，探討以人羊膜間充質(zhì)細胞（hAMCs）為種子細胞結合可降解聚乳酸/聚乙二醇（PLLA/PEG）靜電紡絲纖維支架修復兔尿道缺損的可行性。 方法采用胰蛋白酶-膠原酶聯(lián)合法從足月健康產(chǎn)婦剖宮產(chǎn)后的新鮮人羊膜組織中分離培養(yǎng)人羊膜間充質(zhì)細胞。將聚合物聚乳酸/聚乙二醇混合物采用靜電紡絲技術制成納米纖維支架。27只雄性新西蘭大白兔距尿道外口0.5cm處腹側尿道后壁向近端切除尿道2.0cm*1.5cm，制作尿道缺損模型。隨后隨機分成三組：A組（實驗組,n=9）應用負載人羊膜間充質(zhì)細胞的靜電紡絲納米纖維支架修復兔尿道缺損區(qū)；B組（對照組I,n=9）用靜電紡絲纖維支架直接修復兔尿道缺損區(qū)；C組（對照組II,n=9）將兔尿道缺損區(qū)直接行端端吻合。分別在手術后第4、8、12周切取含兔尿道修復區(qū)標本，HE染色和免疫組化檢查判斷修復段尿道組織再生情況；第12周行尿道造影，觀察尿道暢通情況，同時比較各組間尿道外形以及結石形成率、尿瘺和尿道狹窄等并發(fā)癥有無差異。 結果分離培養(yǎng)的人羊膜間充質(zhì)細胞呈不規(guī)則長梭形和多角形，成放射狀或漩渦狀分布，在特定誘導培養(yǎng)基下能向成脂、成骨和成軟骨細胞分化；免疫熒光法表型鑒定干細胞表面標記物Oct-4和NS在人羊膜間充質(zhì)細胞中廣泛的表達；流式細胞術鑒定，人羊膜間充質(zhì)細胞高表達造血干細胞標志CD29，CD90和CD105，不表達CD45。靜電紡絲技術制備的聚乳酸/聚乙二醇納米纖維支架通過細胞毒性測定證明對細胞無毒性。兔尿道缺損修復模型形態(tài)學觀察，A組尿道壁完整光滑，尿道缺損區(qū)與正常組織融合；組織學上A組隨著修復時間的延長，修復段尿道粘膜由單一的尿路上皮演變成多層尿路上皮結構，基本符合正常尿道組織的結構。B組修復段尿道無連續(xù)尿道上皮覆蓋，，C組尿道管腔結構完整，但組織結構紊亂。尿道造影結果顯示：A組尿道通暢，和術前造影結果對比無明顯差異，B組和C組均有不同程度的尿道狹窄。并發(fā)癥方面，B組和C組尿道狹窄、尿瘺和結石生成率均明顯高于A組，術后A組尿道狹窄(0/9)、尿瘺(0/9)和結石生成（1/9）,合并癥發(fā)生率為3.70%（1/27）；B組尿道狹窄(3/9)、尿瘺(1/9)和結石生成（8/9），合并癥發(fā)生率為44.44％(12/27)；C組尿道狹窄(9/9)、尿瘺(4/9)和結石生成（9/9），合并癥發(fā)生率為81.48％(22/27)，(P 0.05)。 結論1.人羊膜間充質(zhì)細胞具有向多種細胞類型分化的潛能，是組織工程尿道種子細胞的一種理想選擇；2.通過靜電紡絲技術制備的纖維聚乳酸/聚乙二醇支架組織相容性良好，是可行的組織工程支架。3.靜電紡絲纖維聚乳酸/聚乙二醇支架結合人羊膜間充質(zhì)細胞可以修復一定長度的新西蘭大白兔尿道缺損。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R695
本文編號：2389982
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R695
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