携带神经干细胞肌基膜管组织工程支架中神经干细胞的存活分化

摘要 背景：多项研究已证实神经干细胞能促进脊髓损伤大鼠神经功能的恢复，肌基膜管具有良好的细胞、组织相容性和降解性，那么能否将二者结合起来构建一个新的神经组织工程支架？ 目的：以神经干细胞为种子细胞，以肌基膜管为支架，观察携带神经干细胞的肌基膜管组织工程支架中神经干细胞的存活与分化情况。 方法：体外分离培养大鼠神经干细胞，并进行鉴定。用化学萃取方法制作去细胞骨骼肌基膜管支架，将神经干细胞移植入肌基膜管支架培养7 d后，用免疫组织化学方法检测神经干细胞的存活及分化情况，扫描电镜观察其超微结构。 结果与结论：神经干细胞分离培养第5天，Nestin免疫荧光染色可见大量神经球。加血清诱导神经干细胞分化至7 d，进行抗NF、抗GFAP免疫荧光染色，镜下可见NF、GFAP阳性细胞，证明培养的神经干细胞具有多项分化潜能。苏木精-伊红染色法显示肌基膜管中肌细胞成分已消失，肌基膜管支架内主要是大致平行的管道。携带神经干细胞的肌基膜管组织工程支架免疫荧光染色证明，神经干细胞在支架内仍具有干细胞特性，并可分化为神经元和神经胶质细胞。扫描电镜显示神经干细胞可以稳固地贴附在肌基膜管内，提示制备的神经组织工程支架具有良好的生物相容性，可以进行体内移植治疗脊髓损伤等神经系统疾病。 关键词：肌基膜管；组织工程支架；神经干细胞；移植；脊髓损伤 doi:10.3969/j.issn.1673-8225.2010.47.003

Survival and differentiation of neural stem cells adhered to muscle basal lamina tissue engineering scaffolds

Abstract BACKGROUND: Many researches have shown that neural stem cell can promote the rat neural function recover of spinal injury. Muscle basal lamina has a good biocompatibility and degradability. Therefore the combination of muscle and neural stem cells to be a new nerve tissue engineering scaffold has been considered. OBJECTIVE: To observe the survival and differentiation of neural stem cells as seed cells adhered to the muscle basal lamina as a scaffold. METHODS: Rat neural stem cells were isolated, cultured and then identified in vitro. Following preparation of acellular bone muscle basal lamina scaffolds by chemical extraction method, the neural stem cells were implanted into the scaffolds. After seven days of cultivation, the survival and differentiation of neural stem cells were detected by immunohistochemical technique, and the ultra-structure was observed under a scanning electron microscope. RESULTS AND CONCLUTION: On the fifth day of cultivation, a great amount of neurospheres were seen by Nestin immunofluorescence. Following 7-day culture and differentiation in serum medium, the neural stem cells were used for anti-NF and anti-GFAP immunofluorescent staining. NF and GFAP positive cells were found under the microscope, which indicated that the cultured neural stem cells owned multi-lineage differentiation potential. Hemotoxylin and eosin (HE) staining showed that myocytes disappeared from the muscle basal lamina and there were roughly parallel tunnels inside the muscle basal lamina scaffolds. Immunofluorescent staining showed that the neural stem cells adhered to the scaffolds still had the characteristics of stem cells and could differentiate into neurons and neurogliocytes. The neural stem cells were firmly attached to the muscle basal lamina under the scanning electron microscope. These findings indicated that nerve tissue engineering scaffold has a good biocompatibility and can be used for in vivo transplantation to treat nervous system diseases, such as spinal cord injury.