Survival and migration of amniotic epithelial cells after transplantation into the injured spinal cord / 中国组织工程研究

ABSTRACT

BACKGROUND: It has been suggested that amniotic epithelial cells (AECs) express almost all the markers of neural cell and secrete biologically active neurotrophins such as brain derived neurotrophin factor (BDNF) and neurotrophin-3 (NT3).If AECs can substitute neural cells, its neurotrophic effect will bring expansive prospect in treating spinal cord injuries and degenerative neural disease.OBJECTIVE: To observe the survival, migration and secretory function of AECs after transplanted into the injured spinal cord.DESIGN: An observational experiment.SETTING: Department of Histology and Embryology, School of Basic Medical Science, Jilin University.MATERIALS Embryonic rat of 12-14 days (n =1) and adult Wistar rats (n =18, 300-350 g) were provided by the Experimental Animal Center of Jilin University. Immunohistochemical reagents Mouse anti-rat BrdU monoclonal antibody was bought from Sigma Company. Rabbit anti-rat NT3 polyclonal antibody and rabbit anti-rat BDNF polyclonal antibody were bought from Boster Company. SP immunohistochemistry reagents were purchased from Maixin Company.METHODS: The experiment was made in the Department of Histology and Embryology, Basic Medical Science of Jilin University from July to October 2005. ① Wistar rats were anesthetized by intraperitoneal injection of chloral hydrate, subcutaneous tissue and muscle were separated, spinous process and lamina of vertebra were removed by bone ribbing rongeur. to expose the spinal cord. The spinal cords were clamped at the twelfth thoracic vertebra (T12) for 3 minutes.After surgery, the wounds were smeared with penicillin G, then muscle and skin were sutured. The rats were anesthetized by inhaling ether if necessary. ② Obtaining and culture of AECs Amniotic membrane was peeled from the placenta of a pregnant Wistar rat of 12-14 days. The amnictic membrane was dissected into small pieces of 1 mm×1 mm×1 mm, then digested and cultured, and mechanically made into single cell suspension, finally plated in bottles. ③ Transplantation of AECs into injured spinal cord The initial wound was slit and injected with 5 μL Brdu labeled AECs (1×1012 L-1) to the exposed injured spinal cord at 3.0 mm anterior to the injured site. The injections were made at a rate of 5 μL per 3 minutes with a microsyringe. The syringe was slowly pulled out after 5 minutes, then muscle and skin were sutured. ④ Sampling and immunohistochemical analysis: Three animals were sacrificed at 1 week and the other three at 2 weeks postoperatively. The sections were fixed with 40 g/L paraformaldehyde in phosphate buffer solution (PBS) for 20 minutes at room temperature, followed by incubation with primary antibodies at 4 ℃ overnight. The samples were treated with secondary antibodies, biotinylated anti-mouse or rabbit immunoglobulin (IgG) at 37 ℃ for 20 minutes; Followed by incubation of horseradish peroxidase (HRP) labeled third antibodies at 37 ℃ for 20 minutes, then stained with 0.2 g/L diaminobenzidine (DAB) or AEC.MAIN OUTCOME MEASURES: Survival, migration and expression of AECs after transplanted into the injured spinal cord. RESULTS: After transplantation, most of the AECs gather beneath the pia mater of injured spinal cord at 1 week. But they migrated more extensively and many positive nuclear cells (brown) were observed in the center cannel and surrounding gray mater. Meantime, it was also detected that the transplanted AECs could express NT3 (positive cells stained as red) and BDNF in the injured spinal cord.CONCLUSION: AECs could survive for at least 3W after transplanted into the injured spinal cord of adult rats and could migrate widely; Furthermore, they could secrete neurotrophic factors such as NT-3 and BDNF.