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目的探讨脱细胞膀胱基质(urinary bladder matrix,UBM)复合大鼠骨髓间充质干细胞(bone marrow mesen-chymal stem cells,BMSCs)构建组织工程化吊带治疗压力性尿失禁的可行性。方法采用密度梯度离心法结合贴壁培养法体外分离、培养和扩增BMSCs,流式细胞术检测BMSCs免疫表型,将第3代BMSCs与UBM复合培养,7 d后通过光镜和电镜观察细胞在材料上的生长情况,进一步应用万能力学测试仪检测所构建吊带的最大负荷、杨氏模量、伸展性等机械特性,另以UBM负载大鼠膀胱平滑肌细胞株(smooth muscle cells,SMCs)构建SMCs吊带、单纯UBM及聚丙烯材料行效果比较。结果分离培养的BMSCs呈长梭形,折光性好,增殖旺盛,具有成体干细胞特性,细胞表面标志物CD29阳性,CD45和CD31阴性。UBM去细胞彻底,复合BMSCs或SMCs均可获得生长良好的细胞。机械性状检测表明,单纯UBM和复合BMSCs的UBM吊带与SMCs吊带及聚丙烯材料相比,最大负荷下降[(0.025±0.003)、(0.018±0.004)kN vs(0.030±0.003)、(0.143±0.034)kN,P<0.01];杨氏模量下降[(170.820±61.735)、(103.624±52.779)MPa vs(490.730±80.969)、(900.100±192.402)MPa,P<0.01];伸展性提高[(16.418±1.014)、(22.684±6.848)mm vs(9.498±1.384)、(2.975±0.401)mm,P<0.01]。结论成功分离、培养了大鼠BMSCs,并鉴定了其干性。构建的干细胞吊带与SMCs吊带及聚丙烯材料相比,其伸展性良好。
Objective To investigate the feasibility of using UBM composite bone marrow mesenchymal stem cells (BMSCs) to construct tissue engineering sling for stress urinary incontinence. Methods BMSCs were isolated, cultured and expanded in vitro using density gradient centrifugation combined with adherent culture. The immunophenotypes of BMSCs were detected by flow cytometry. The 3rd generation BMSCs were cultured with UBM. After 7 days, the cells were observed by light and electron microscope Furthermore, the mechanical properties of the maximal load, the Young’s modulus and the extensibility of the constructed sling were measured by using a universal mechanical tester. The UBM-loaded rat smooth muscle cells (SMCs) SMCs sling, simple UBM and polypropylene material row effect comparison. Results The BMSCs cultured in vitro showed a long fusiform shape with good refraction and proliferative properties. The characteristics of adult stem cells were CD29 positive, CD45 and CD31 negative. UBM to a thorough cell, BMSCs or SMCs composite can get well-growing cells. The results of mechanical tests showed that the maximum load of UBM slippage of pure UBM and composite BMSCs was significantly lower than that of SMCs sling and polypropylene [(0.025 ± 0.003), (0.018 ± 0.004) kN vs (0.030 ± 0.003), (0.143 ± 0.034 ) kN, P <0.01]; Young’s modulus decreased [(170.820 ± 61.735), (103.624 ± 52.779) MPa vs (490.730 ± 80.969), (900.100 ± 192.402) MPa, P <0.01] 16.418 ± 1.014), (22.684 ± 6.848) mm vs (9.498 ± 1.384), (2.975 ± 0.401) mm respectively, P <0.01]. Conclusion The rat BMSCs were successfully isolated and cultured, and their dryness was identified. The constructed stem cell sling has good extensibility compared to the SMCs sling and polypropylene material.