脑去细胞生物支架的制备与鉴定

Preparation and Characterization of a Brain Decellularized Scaffold

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Trition X-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(d BECM)。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。

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（ d BECM） was prepared by chemical extraction combined with oscillation,successively with 3% Trition X-100,1% SDS,4% deoxycholic acid sodium and deionized water in 37℃. The microscopic morphology of d BECM 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 d BECM 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.