关键词: 周细胞, 脊髓, 微血管, 细胞分离

Abstract: Objective To establish a method for selective culturing mouse spinal cord microvascular pericytes (SCMP) by Pericytes Cell Medium (PCM) with 2% FBS. To observe the differentiation and function of the cultured cells. Methods After decapitation of 10 of 3-week-old C57 mice, the intact spinal cord was removed from the spinal column. Meninges were cleared from the spinal cord. Tissues were cut into small pieces (approx.1 mm3). The capillary fragments were obtained through two-step enzymatic digestion and 20% bovine serum albumin (BSA) centrifugation. The microvessels were incubated initially under conditions optimized for endothelial cells, but after two passages switched to a medium optimized for pericyte growth. The morphology of pericytes was observed by inverted microscope. Platelet-derived growth factor receptor β，(PDGFRβ), neuron-glial antigen 2 (NG2)，von Willebrand factor (vWF), and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence．PDGFRβ,CD31,CD11b and GFAP were detected by Flow cytometry. When switching the cells from pericyte medium into DMEM containing 10% FBS, theα-SMA, a marker of pericyte differentiation, was detected by Immunofluorescence and Western blot. The matrigel pericyte-endothelial cell co-culture was used to verify the function of cultured cells. Results The cells migrated from the attached capillary fragments after 48h, and got confluence after 7 to 9 days. Primary cultures consisted endothelial cells with pericytes. After switching to PCM, the pericytes grew predominantly and showed the typical rhomboid morphology. Immunofluorescence revealed that PDGFRβ and NG2 were positive， vWF and GFAP were negative. Flow cytometry showed that cells were strongly positive for PDGFRβ(95.52%±2.55%), negative for CD31(0.80%±0.26%), CD11b(1.02%±0.35%) or GFAP(0.63%±0.26%). DMEM containing 10% FBS promoted α-SMA expression, demonstrating the cells could still differentiate. In matrigel co-culture experiments, pericytes aligned with endothelial cords, retaining the ability to associate with endothelial cell. Conclusion This method may successfully obtain the highly purified SCMP. The cells have morphological and functional characteristics of pericytes and the ability to differentiate.

Key words: pericytes, spinal cord, microvessel, cell isolation