自體尿源性干細胞結(jié)合3D多孔小腸粘膜下基質(zhì)用于兔尿道重建的實驗研究
發(fā)布時間：2018-12-18 00:41
【摘要】：目的:探索應用自體兔尿源干細胞細胞(rabbit urine-derived stem cells,rUSCs)復合小腸黏膜下基質(zhì)(Small intestinal submucosa,SIS)構(gòu)建兔組織工程尿道的可行性,為臨床構(gòu)建組織工程尿道,用于尿道下裂殘疾、長段尿道缺損等復雜、疑難情況尿道重建奠定基礎(chǔ)。方法:收集24只雄性新西蘭大白兔尿液及膀胱沖洗液,分離、培養(yǎng)得到rUSC,檢測其自我更新及體外多向分化(平滑肌和上皮方向)能力;用5%過氧乙酸(peracetic acid,PAA)處理SIS材料,對其微結(jié)構(gòu)、免疫原性、生物相容性進行研究,并將PKH67標記的rUSCs與SIS材料復合;隨機將24只雄性新西蘭大白兔分為兩組,并建立尿道缺損模型,缺損長度約為2.0cm,對照組應用單純5%PAA處理后的SIS材料修復(12只),實驗組應用5%PAA處理后并復合rUSCs的SIS材料修復(12只)。均于術(shù)后2周、3周、4周、12周行組織學和Western Blot檢測,12周行尿道造影。結(jié)果:原代培養(yǎng)第4天即可在鏡下觀察到rUSC單克隆細胞,在第6天可形成克隆團,在第11天出現(xiàn)細胞融合,形態(tài)成“米�！睜�,大小均一、排列緊湊;rUSCs經(jīng)體外誘導分化后,能高效的分化為平滑肌細胞和上皮細胞。經(jīng)5%PAA處理后,SIS材料表面變得疏松多孔,且DNA含量進一步降低(P0.05);組織學提示實驗組平滑肌束再生在各個時間點均優(yōu)于對照組(P0.05),且更有序;上皮層數(shù)和新生血管數(shù)量在早期(2、3、4周)好于對照組(P0.05),在晚期(12周)差異無統(tǒng)計學意義(P0.05);對照組基質(zhì)出現(xiàn)炎性細胞浸潤和纖維化;尿道造影顯示實驗組僅一只出現(xiàn)輕微尿道狹窄,而對照組三只均出現(xiàn)輕微尿道狹窄。免疫熒光顯示rUSCs在體內(nèi)可轉(zhuǎn)化為平滑肌和上皮細胞。結(jié)論:1)rUSCs能夠通過簡單無創(chuàng)的方法獲取,具有良好的自我更新能力,體外易于培養(yǎng)和擴增,并且具有分化成平滑肌細胞和上皮細胞的能力,是一種理想的組織工程種子細胞來源;2)SIS材料經(jīng)5%PAA處理后,SIS支架材料孔徑增大,孔隙率增高,且細胞成分進一步減少,可作為組織工程支架材料來源;3)復合rUSCs的3D多孔SIS材料能促進尿道組織再生,減少炎癥反應、尿道狹窄等并發(fā)癥。
[Abstract]:Objective: to explore the feasibility of using autologous rabbit urethral stem cells (rabbit urine-derived stem cells,rUSCs) combined with small intestinal submucosal matrix (Small intestinal submucosa,SIS) to construct tissue engineered urethra in rabbits. For hypospadias, long urethral defects, complex, difficult cases of urethral reconstruction laid the foundation. Methods: urine and bladder flushing fluid were collected from 24 male New Zealand white rabbits. RUSC, was obtained to detect the ability of self-renewal and differentiation (smooth muscle and epithelial direction) in vitro. SIS material was treated with 5% peracetic acid (peracetic acid,PAA), and its microstructure, immunogenicity and biocompatibility were studied. PKH67 labeled rUSCs was combined with SIS material. Twenty-four male New Zealand white rabbits were randomly divided into two groups, and the urethral defect model was established, the length of the defect was about 2.0 cm. The control group was repaired with SIS material treated with 5%PAA alone (12 rabbits). The experimental group was repaired with SIS material (12 rats) treated with 5%PAA and combined with rUSCs. Histology and Western Blot were detected at 2 weeks, 3 weeks, 4 weeks and 12 weeks after operation, and urethrography was performed at 12 weeks. Results: the rUSC monoclonal cells could be observed under microscope on the 4th day of primary culture, and the clones could be formed on the 6th day. On the 11th day, the cells were fused in the shape of "rice grain", uniform in size and compact in arrangement. RUSCs can effectively differentiate into smooth muscle cells and epithelial cells after differentiation in vitro. After treated with 5%PAA, the surface of SIS materials became loose and porous, and the content of DNA decreased further (P0.05). Histology showed that the regeneration of smooth muscle tract in the experimental group was better than that in the control group at all time points (P0.05), and the content of DNA in the experimental group was more orderly than that in the control group. The number of epithelial layer and the number of neovascularization in the early stage (2 ~ 3 weeks) was better than that in the control group (P0.05), but there was no significant difference in late (12 weeks) (P0.05), in the control group, inflammatory cell infiltration and fibrosis appeared in the matrix. Urethrography showed only one slight urethral stricture in the experimental group and slight urethral stricture in the control group. Immunofluorescence showed that rUSCs could be transformed into smooth muscle and epithelial cells in vivo. Conclusion: 1) rUSCs can be obtained by simple and non-invasive method. It has good self-renewal ability, easy to be cultured and expanded in vitro, and can differentiate into smooth muscle cells and epithelial cells. It is an ideal source of seed cells for tissue engineering. 2) after SIS was treated with 5%PAA, the pore size and porosity of SIS scaffold increased, and the cell composition decreased further, which could be used as the source of scaffold material for tissue engineering. 3) 3D porous SIS combined with rUSCs can promote urethral regeneration and reduce complications such as inflammation and urethral stricture.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R699
本文編號：2385025
[Abstract]:Objective: to explore the feasibility of using autologous rabbit urethral stem cells (rabbit urine-derived stem cells,rUSCs) combined with small intestinal submucosal matrix (Small intestinal submucosa,SIS) to construct tissue engineered urethra in rabbits. For hypospadias, long urethral defects, complex, difficult cases of urethral reconstruction laid the foundation. Methods: urine and bladder flushing fluid were collected from 24 male New Zealand white rabbits. RUSC, was obtained to detect the ability of self-renewal and differentiation (smooth muscle and epithelial direction) in vitro. SIS material was treated with 5% peracetic acid (peracetic acid,PAA), and its microstructure, immunogenicity and biocompatibility were studied. PKH67 labeled rUSCs was combined with SIS material. Twenty-four male New Zealand white rabbits were randomly divided into two groups, and the urethral defect model was established, the length of the defect was about 2.0 cm. The control group was repaired with SIS material treated with 5%PAA alone (12 rabbits). The experimental group was repaired with SIS material (12 rats) treated with 5%PAA and combined with rUSCs. Histology and Western Blot were detected at 2 weeks, 3 weeks, 4 weeks and 12 weeks after operation, and urethrography was performed at 12 weeks. Results: the rUSC monoclonal cells could be observed under microscope on the 4th day of primary culture, and the clones could be formed on the 6th day. On the 11th day, the cells were fused in the shape of "rice grain", uniform in size and compact in arrangement. RUSCs can effectively differentiate into smooth muscle cells and epithelial cells after differentiation in vitro. After treated with 5%PAA, the surface of SIS materials became loose and porous, and the content of DNA decreased further (P0.05). Histology showed that the regeneration of smooth muscle tract in the experimental group was better than that in the control group at all time points (P0.05), and the content of DNA in the experimental group was more orderly than that in the control group. The number of epithelial layer and the number of neovascularization in the early stage (2 ~ 3 weeks) was better than that in the control group (P0.05), but there was no significant difference in late (12 weeks) (P0.05), in the control group, inflammatory cell infiltration and fibrosis appeared in the matrix. Urethrography showed only one slight urethral stricture in the experimental group and slight urethral stricture in the control group. Immunofluorescence showed that rUSCs could be transformed into smooth muscle and epithelial cells in vivo. Conclusion: 1) rUSCs can be obtained by simple and non-invasive method. It has good self-renewal ability, easy to be cultured and expanded in vitro, and can differentiate into smooth muscle cells and epithelial cells. It is an ideal source of seed cells for tissue engineering. 2) after SIS was treated with 5%PAA, the pore size and porosity of SIS scaffold increased, and the cell composition decreased further, which could be used as the source of scaffold material for tissue engineering. 3) 3D porous SIS combined with rUSCs can promote urethral regeneration and reduce complications such as inflammation and urethral stricture.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R699
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相關(guān)期刊論文 前4條
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