组织工程化尿道的实验研究
摘要
对于尿道缺损、后尿道狭窄，临床上常以皮肤(粘膜)游离移植或局部带蒂皮瓣转移修复，但皮肤(粘膜)游离移植常因挛缩导致尿道狭窄，尿瘘发生率亦较高；局部带蒂皮瓣转移则常致局部臃肿，形态不佳，患者及家属难以接受。况且，自体组织移植是以牺牲正常组织为代价的“以手术创伤修复组织缺损”的治疗模式，既增加了新的手术创伤，又增加了手术并发症的机率，且自体组织来源有限，限制了其临床应用。
随着生命科学、材料科学以及相关物理、化学学科的发展，人们提出了一个新的概念——组织工程。它是应用细胞生物学和工程学的原理，研究和开发能修复损伤组织和改善其功能的具有活性的生物替代物的一门科学。虽然组织工程概念的提出只有十几年的时间，但它已得到了异常迅猛的发展，在许多方面已取得重大突破。
组织工程为整形外科医生解决临床问题提供了全新的思维模式。本实验研究、探讨哺乳类动物尿道粘膜上皮细胞的体外培养技术和方法，从仿生学角度出发，探索、制备可生物降解的胶原—壳聚糖复合材料支架，进而采用组织工程技术构建尿道。
实验中发现，以中性蛋白酶与胰蛋白酶联合消化尿道粘膜是比较理想的分离尿道粘膜上皮细胞的方法，并摸索出了获取细胞数量最多时的胰酶消化时间。采用此方法，成功地分离了新西兰幼兔、近交系Luis大鼠的尿道粘膜上皮细胞。
在国内首次报道了哺乳类动物尿道粘膜上皮细胞的体外培养技术和方法。分别采用以胶原为底物、以L-929细胞为滋养层、以胶原为底物同时以L-929细胞为滋养层的培养技术，在体外成功地培养了尿道粘膜上皮细胞，并进行了传代、冻存及复苏。对培养的尿道粘膜上皮细胞进行了HE染色、Giemsa染色、Feulgen染色、免疫组化染色，以透射电镜观察细胞超微结构，对培养的尿道粘膜上皮细胞进行了鉴定，并以流式细胞仪检测细胞周期时相和DNA合成情况。整个上皮细胞生长期间无成纤维细胞混杂生长，均为单一的二倍体上皮细胞。细胞可传11～13代，成活50～60天。第9代前的细胞形态良好，与原代无明显区别。细胞间富含桥粒等上皮细胞的典型结构，且没有出现永生化及恶性变。据此，可以认为我们所培养的角化细胞具有干细胞的部
Surgeons often apply free skin grafts or local flaps to reconstruct urethra in hypospadias, in which there are high complications, such as fistula, stenosis and s(?) on. Besides, the source of autografts limits their clinical application.
With the development of life science, material science, physics and chemistry, new science, namely tissue engineering arises, which studies and makes biologica substitute to repair damaged tissues applying the principles of cell biology an engineering.
In our experiment we reported the urethral epithelium culture techniques ar methods in vitro for the first time in the country. We have cultured, subculture frozen and revivified urethral epithelium in vitro successfully, using collagen substrate, L-929 cells as feed cells and collagen mixed with L-929 cell Immunohistochemical examination revealed that keratin staining was positive. lot of desmosomes were found under the transmission electron microscope. So (?) confirmed that the cells we had cultured were from epithelial source. During t epithelium growth period there were no fibroblasts to contaminate. The cells we all diploid epithelium, which can be subculured 11 to 13 generations, survivi
随着生命科学、材料科学以及相关物理、化学学科的发展，人们提出了一个新的概念——组织工程。它是应用细胞生物学和工程学的原理，研究和开发能修复损伤组织和改善其功能的具有活性的生物替代物的一门科学。虽然组织工程概念的提出只有十几年的时间，但它已得到了异常迅猛的发展，在许多方面已取得重大突破。
组织工程为整形外科医生解决临床问题提供了全新的思维模式。本实验研究、探讨哺乳类动物尿道粘膜上皮细胞的体外培养技术和方法，从仿生学角度出发，探索、制备可生物降解的胶原—壳聚糖复合材料支架，进而采用组织工程技术构建尿道。
实验中发现，以中性蛋白酶与胰蛋白酶联合消化尿道粘膜是比较理想的分离尿道粘膜上皮细胞的方法，并摸索出了获取细胞数量最多时的胰酶消化时间。采用此方法，成功地分离了新西兰幼兔、近交系Luis大鼠的尿道粘膜上皮细胞。
在国内首次报道了哺乳类动物尿道粘膜上皮细胞的体外培养技术和方法。分别采用以胶原为底物、以L-929细胞为滋养层、以胶原为底物同时以L-929细胞为滋养层的培养技术，在体外成功地培养了尿道粘膜上皮细胞，并进行了传代、冻存及复苏。对培养的尿道粘膜上皮细胞进行了HE染色、Giemsa染色、Feulgen染色、免疫组化染色，以透射电镜观察细胞超微结构，对培养的尿道粘膜上皮细胞进行了鉴定，并以流式细胞仪检测细胞周期时相和DNA合成情况。整个上皮细胞生长期间无成纤维细胞混杂生长，均为单一的二倍体上皮细胞。细胞可传11～13代，成活50～60天。第9代前的细胞形态良好，与原代无明显区别。细胞间富含桥粒等上皮细胞的典型结构，且没有出现永生化及恶性变。据此，可以认为我们所培养的角化细胞具有干细胞的部
Surgeons often apply free skin grafts or local flaps to reconstruct urethra in hypospadias, in which there are high complications, such as fistula, stenosis and s(?) on. Besides, the source of autografts limits their clinical application.
With the development of life science, material science, physics and chemistry, new science, namely tissue engineering arises, which studies and makes biologica substitute to repair damaged tissues applying the principles of cell biology an engineering.
In our experiment we reported the urethral epithelium culture techniques ar methods in vitro for the first time in the country. We have cultured, subculture frozen and revivified urethral epithelium in vitro successfully, using collagen substrate, L-929 cells as feed cells and collagen mixed with L-929 cell Immunohistochemical examination revealed that keratin staining was positive. lot of desmosomes were found under the transmission electron microscope. So (?) confirmed that the cells we had cultured were from epithelial source. During t epithelium growth period there were no fibroblasts to contaminate. The cells we all diploid epithelium, which can be subculured 11 to 13 generations, survivi
引文
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