本文選題：明膠　切入點(diǎn)：細(xì)菌纖維素　出處：《上海交通大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分明膠-細(xì)菌纖維素復(fù)合舌黏膜上皮細(xì)胞重建兔尿道的實(shí)驗(yàn)研究目的:評(píng)估明膠-細(xì)菌纖維素復(fù)合舌黏膜上皮細(xì)胞重建兔尿道缺損的效果。材料及方法:制備明膠-細(xì)菌纖維素生物材料,通過掃描電鏡評(píng)估材料結(jié)構(gòu)特征。分離兔舌黏膜細(xì)胞,細(xì)胞培養(yǎng)擴(kuò)增至足夠數(shù)量后種植在明膠-細(xì)菌纖維素支架上。建立兔尿道缺損模型(2.0×0.8cm),單純明膠-細(xì)菌纖維素支架(對(duì)照組,N=10)和復(fù)合細(xì)胞的明膠-細(xì)菌纖維素支架(實(shí)驗(yàn)組,N=10)分別修復(fù)尿道缺損。在手術(shù)后的1和3個(gè)月,通過大體觀察、尿道造影及組織學(xué)分析評(píng)估修復(fù)尿道缺損的效果。結(jié)果:明膠-細(xì)菌纖維素由致密狀的上表面和孔隙狀的下表面的雙層結(jié)構(gòu)構(gòu)成。兔舌黏膜上皮細(xì)胞能被成功分離、培養(yǎng)和擴(kuò)增,在支架材料上生長(zhǎng)良好。在術(shù)后1個(gè)月,兩組修復(fù)段尿道均保持通暢。但在術(shù)后3個(gè)月,對(duì)照組尿道出現(xiàn)狹窄,而實(shí)驗(yàn)組尿道仍保持通暢。通過組織學(xué)分析發(fā)現(xiàn),在術(shù)后1個(gè)月,實(shí)驗(yàn)組修復(fù)段尿道表面有一層完整的上皮,而對(duì)照組尿道表面的上皮層不完整。但是,在術(shù)后3個(gè)月,兩組尿道表面均形成多層完整的上皮,兩者無統(tǒng)計(jì)學(xué)差異(p0.05)。在術(shù)后1和3個(gè)月,實(shí)驗(yàn)組尿道平滑肌和血管的再生明顯優(yōu)于對(duì)照組(p0.05)。另外,我們發(fā)現(xiàn)對(duì)照組尿道出現(xiàn)明顯的炎癥反應(yīng)和纖維化。結(jié)論:我們的研究表明復(fù)合舌黏膜上皮細(xì)胞的明膠-細(xì)菌纖維素能成功修復(fù)兔尿道缺損,促進(jìn)平滑肌和血管的再生,未發(fā)現(xiàn)明顯的炎癥反應(yīng)和纖維化表現(xiàn)。復(fù)合舌黏膜上皮細(xì)胞的明膠-細(xì)菌纖維素支架有望成為一種理想材料用于尿道修復(fù)重建。第二部分三維多孔膀胱脫細(xì)胞基質(zhì)在兔尿道修復(fù)重建中的應(yīng)用目的:評(píng)估三維多孔膀胱脫細(xì)胞基質(zhì)(BAM)修復(fù)兔尿道缺損的療效材料及方法:在30只兔中建立腹側(cè)尿道黏膜缺損(1.5×0.8 cm)模型。應(yīng)用5%過氧乙酸(PAA)處理過的BAM(三維多孔BAM,實(shí)驗(yàn)組,15只)和未處理過的BAM(對(duì)照組,15只)采用補(bǔ)片方式分別修復(fù)兔缺損尿道。手術(shù)后1,2和3個(gè)月兔行尿道造影和組織學(xué)分析。結(jié)果:在實(shí)驗(yàn)組中,13只兔的尿道通暢,無尿瘺和狹窄;而在對(duì)照組中,10只兔的尿道通暢。組織學(xué)檢測(cè)顯示,在術(shù)后1,2和3個(gè)月,實(shí)驗(yàn)組尿道上皮再生的速度快于對(duì)照組;實(shí)驗(yàn)組平滑肌與膠原比值和平滑肌含量高于對(duì)照組;同時(shí),血管再生優(yōu)于對(duì)照組。兩者比較均有統(tǒng)計(jì)學(xué)差異(p0.05)。結(jié)論:與未處理過的BAM相比,PAA處理過的BAM更能促進(jìn)尿道重建中上皮,平滑肌和血管的再生。三維多孔BAM可能作為一種優(yōu)化支架用于細(xì)胞種植的管狀或長(zhǎng)段尿道缺損的修復(fù)重建。第三部分明膠-細(xì)菌纖維素與三維多孔膀胱脫細(xì)胞基質(zhì)植入兔膀胱后的組織反應(yīng)研究目的:評(píng)估明膠-細(xì)菌纖維素(BC)與三維多孔膀胱脫細(xì)胞基質(zhì)(BAM)植入兔膀胱后的組織反應(yīng)。材料與方法:制備明膠-BC與三維多孔BAM支架材料,并評(píng)估它們的孔徑和孔隙率。將20只兔膀胱后壁的對(duì)應(yīng)兩側(cè)的黏膜與平滑肌剔除(1.5×2.0 cm)。然后,明膠-BC與三維多孔BAM支架材料分別植入膀胱后壁缺損處。手術(shù)后4、8周,動(dòng)物被處死(每個(gè)時(shí)間檢測(cè)點(diǎn)為10只),將植入處的膀胱組織進(jìn)行大體觀察,并行組織學(xué)檢測(cè)與肌肉收縮分析評(píng)估兩種材料植入處組織再生情況。結(jié)果:明膠-BA與三維多孔BAM支架材料的孔徑與孔隙率相似,兩者比較均無統(tǒng)計(jì)學(xué)差異(P0.05)。在術(shù)后4周,4只兔膀胱內(nèi)可見膀胱結(jié)石形成,但術(shù)后8周,膀胱內(nèi)未見有膀胱結(jié)石。組織分析顯示,在術(shù)后4、8周,明膠-BC支架材料植入處的尿路上皮、平滑肌及血管的再生情況與三維多孔BAM相比較,均無統(tǒng)計(jì)學(xué)差異(P0.05)。同樣,在材料植入處膀胱組織對(duì)卡巴膽堿、KCl及苯腎上腺素的收縮反應(yīng)方面,兩組比較均無統(tǒng)計(jì)學(xué)差異(P0.05)。另外,術(shù)后4、8周,兩種材料植入處均未見明顯的急、慢性炎癥反應(yīng)。結(jié)論:我們的結(jié)果提示明膠-BC與三維多孔BAM在促進(jìn)膀胱組織結(jié)構(gòu)和功能再生方面,具有相似的作用,均表現(xiàn)良好的生物相容性。明膠-BC與三維多孔BAM作為兩種優(yōu)化生物材料在尿道修復(fù)重建中可能具有相似的作用。
[Abstract]:The first part of the experiment study on gelatin - bacterial cellulose composite tongue epithelial cells of rabbit urethra reconstruction Objective: To evaluate the gelatin bacterial cellulose composite tongue epithelial cells to reconstruct rabbit urethral defect effect. Materials and methods: preparation of gelatin bacterial cellulose biological materials by scanning electron microscopy evaluation of material structure. Isolated rabbit tongue mucosa cells, cells after a sufficient number of cultured to grow in gelatin bacterial cellulose bracket. A rabbit model of urethral defect (2 * 0.8cm), pure bacterial cellulose gelatin scaffold (control group, N=10) and composite cell gelatin bacterial cellulose scaffolds (experimental group, N=10) respectively. The defects were repaired in 1 after surgery and 3 months by gross observation, histological analysis evaluation of urethral defect repair effect of urethrography and tissue. Results: gelatin bacterial cellulose by dense on the surface and pore shape under the table A surface of the bilayer. Rabbit lingual epithelial cells can be successfully isolated, cultured and expanded, the scaffold grew well. In 1 months after operation, two groups of repair urethra were unobstructed. But 3 months after operation, the control group and the experimental group appear urethral stricture, urethral smooth. The histological analysis showed that in the 1 months after surgery, the experimental group to repair urethra surface has a layer of intact epithelium, while the control group of the urethral epithelial layer surface is not complete. However, 3 months after operation, two groups were formed multilayer complete urethral surface epithelium, both no statistical differences (P0.05). After 1 and 3 months, the experimental group of urethral smooth muscle and vascular regeneration was significantly better than the control group (P0.05). In addition, we found that the control group appears urethral inflammation and fibrosis significantly. Conclusion: our study showed that the composite tongue epithelial cells on gelatin bacterial cellulose Can successfully repair the rabbit urethral defect, promote regeneration of smooth muscle and vascular inflammation and fibrosis, no obvious findings. Composite tongue epithelial cells on gelatin scaffold of bacterial cellulose is a kind of ideal material for urethral reconstruction. The second part application of three-dimensional porous bladder acellular matrix in rabbit urethral reconstruction. Evaluation of three-dimensional porous bladder acellular matrix (BAM) clinical materials and methods for repairing rabbit urethral defect: the establishment of ventral urethral mucosal defect in 30 rabbits (1.5 x 0.8 cm) model. Application of 5% peracetic acid (PAA) treated BAM (3D porous BAM, experimental group, 15 rats) and untreated the BAM (control group, 15 rats) by patch were used to repair defects in rabbits urethra. Analysis of 1,2 and 3 rabbit urethra angiography and tissue after surgery. Results: in the experimental group, 13 rabbits of urethral patency, no fistula and narrow Narrow; in the control group, 10 rabbits of urethral patency. Histological examination revealed that at 1,2 and 3 months postoperatively, the experimental group of urethral epithelial regeneration was faster than the control group; the experimental group and the ratio of collagen and smooth muscle smooth muscle was higher than that in control group; at the same time, angiogenesis is better than that of the control group. The two there were statistically significant differences (P0.05). Conclusion: compared with untreated BAM, PAA treated BAM can promote epithelial urethral reconstruction, and vascular smooth muscle regeneration. Three dimensional porous BAM may serve as a scaffold for the repair and reconstruction of optimal cell seeding or long tubular urethral defect. The third part of gelatin bacteria cellulose and three-dimensional porous bladder to study tissue reaction after implantation of rabbit bladder stromal cells: evaluation of gelatin bacterial cellulose (BC) and three-dimensional porous bladder acellular matrix (BAM) implanted in rabbit bladder after tissue reaction. Materials and methods Preparation of gelatin -BC and porous BAM scaffolds, and evaluate their pore size and porosity. The corresponding 20 rabbits on both sides of the posterior wall of the bladder mucosa and smooth muscle removed (1.5 x 2 cm). Then, the gelatin -BC and 3-D porous BAM scaffolds were implanted into the posterior wall of bladder defect 4,8 weeks after the operation. That animal was killed (each time the detecting point is 10), gross observation will be implanted at the bladder tissue, histological detection and muscle contraction analysis evaluation of two kinds of materials were implanted into the tissue regeneration. Results: the pore size and porosity of gelatin -BA and three-dimensional porous BAM scaffold is similar to that of the two were no statistically significant difference (P0.05). After 4 weeks, 4 rabbits were found in the bladder bladder stone formation, but 8 weeks after operation, there was no bladder bladder stones. Structure analysis showed that in 4,8 weeks after operation, -BC gelatin scaffold was implanted into the urinary tract epithelial and smooth muscle. The regeneration of blood vessels and three-dimensional porous BAM, there was no significant difference (P0.05). Similarly, in the material implanted bladder tissue of Kaba choline, KCl and contractile responses to phenylephrine, the two groups were not statistically significant (P0.05). In addition, 4,8 weeks after operation, two materials were implanted there was no obvious acute and chronic inflammation. Conclusion: Our results suggest that the gelatin -BC and three-dimensional porous structure and function of BAM in promoting the regeneration of bladder tissue, had similar effects, showed good biocompatibility. Gelatin -BC and three-dimensional porous BAM as two kinds of optimization of biological materials may have similar effects on urethral repair reconstruction.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R318.08;R699

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