脑去细胞生物支架的制备与鉴定

通过化学萃取加震荡法制备脑去细胞外基质,并对其进行形态学分析和成分鉴定。健康成年SD大鼠20只,分成正常对照组和去细胞组。去细胞组大鼠经心脏灌流后取脑,通过化学萃取加震荡法,依次用3%的TritionX-100、1%的SDS、4%的脱氧胆酸钠震荡萃取,无菌蒸馏水漂洗制备脑去细胞外基质(dBECM)。通过扫描电镜观察dBECM微观形态,用HE染色和荧光DAPI染色分析其去细胞化的效果,进一步通过Masson染色和免疫荧光染色进行成分的鉴定。通过HE染色及荧光DAPI染色可以发现,该方法去细胞化程度完全(去细胞程度大于99%),仅保留大量细胞外基质,未见明显的细胞及细胞核成分残留;Masson染色和免疫荧光染色发现,dBECM保留弹性蛋白(4.0%±1.1%)、层粘连蛋白(19.0%±1.6%)、纤维连接蛋白(9.0%±2.1%)、IV型胶原蛋白(16.0%±1.9%)等成分。化学萃取加震荡法可有效地去除大鼠脑组织内的细胞成分,制备得到的脑去细胞外基质较好地维持宏观和微观的三维结构,保留细胞外基质支架的蛋白成分,是一种简便且理想的脑去细胞外基质制备技术。

Preparation and Characterization of a Brain Decellularized Scaffold

The aim of this work is to prepare a brain decellularized scaffold by chemical extraction combined with oscillation, and to perform preliminary characterization. Twenty SD rats were divided into decellularization and control group. In the decellularization group, the brain extracellular matrix (dBECM) was prepared by chemical extraction combined with oscillation, successively with 3% TritionX-100, 1%SDS,4% deoxycholic acid sodium and deionized water in 37℃. The microscopic morphology of dBECM was observed by scanning electron microscopy. The decellularization was analyzed by HE staining and DAPI staining. The components were identified by Masson staining and immunofluorescence staining. A lot of collagen fibers could be seen with HE and immunohistochemistry stain but no visible cell nuclei remained after decellularization. The degree of decellularization was about 99%. Masson staining and immunofluorescence staining revealed that dBECM retained elastin (4±1.1%), laminin (19±1.6%), fibronectin (9±2.1%) and collagen IV (16±1.9%). In conclusion, the method of chemical extraction combined with oscillation can effectively remove all cellular components while retain the extracellular matrix and three-dimensional structure. It is a convenient and ideal preparation method on decellularized brain scaffold for tissue engineering.