新型全肝脏去细胞化流程对细胞外基质及细胞组分残留量影响的研究

The Effect of a Novel Whole Liver Decellularization Protocol on the Extracellular Matrix and Cell Components Residuals

组织工程中去细胞化支架的构建,存在着保留细胞外基质和完全去除供体细胞成分的矛盾,如何评价去细胞化流程的效果是解决的关键。本研究采用以TritonX-100为主,辅助以SDS、酶学法、物理冻融法、高低渗法等方法的新型全肝脏去细胞化流程制作去细胞支架,通过免疫组化及电镜观察支架形态结构,ELISA法检测细胞外基质各组分含量,吸光度及Western-blotting法检测供体细胞组分残留量,免疫荧光法检测支架内三维循环培养诱导WBF-344细胞分化。结果表明,该流程很好的保留了胶原蛋白、层粘连蛋白、纤粘连蛋白等细胞外三维结构;氨基聚糖、HGF、VEGF等细胞外基质保存量分别达正常组织的90%及40%;基本去除残留DNA的同时,相比SF.Badylak et al.法,细胞残留组分量如GAPDH、MCH-I及HMGB-1明显下降,仅为正常组织的10%;WBF-344细胞在支架培养系统内生长良好,白蛋白表达逐渐增强。本研究成功构建了新的、较完善的去细胞化支架制作与评价体系,为干细胞分化在肝脏疾病治疗找到一种新的应用模式,

There has been a conflict between preservation of extracellular matrix and reduction of donor＇ s cell components on decellularized scaffold production in tissue engineering. Therefore a protocol for decellularization is the key to solve this problem. We developed a novel decellularization protocol which used TritonX - 100 as a major agent for decellularizing assisted by SDS, enzymes, freeze-thaw processing, hypotonic and hypertonic solutions etc. After morphological and histological observation through immunohistochemistry and scanning electron microscope, evaluation of ECM content by ELISA, using absorbance detection and Western-blotting to test cell components residuals, and immunofluorescence after 3D perfusion culture of WBF -344 ceils in the scaffold, the results showed that, by using this new protocol, three dimensional structure like collagen, laminin, fibronectin appeared well protected, and the extracellular matrix contents of decellularized scaffold such as GAG, HGF and VEGF was 90% and 40% of normal tissue. Besides a DNA elimination, MCH-I and HMGB - 1 were only 10% of normal tissue. WBF - 344 cells grew well in the scaffold and the albumin level was rising. In conclusion, this study built a novel decellularization protocol and evaluation system, and proposed a promising application of the acellular liver scaffold in stem cell differentiation and liver disease therapy.