欢迎访问《解剖学报》官方网站！今天是
English
改良的体外中枢神经系统损伤后胶质瘢痕模型的建立
An improved glial scar model after central nervous system injury in vitro
目的 建立胶质疤痕体外模型，观察其形态及细胞外基质的表达情况。方法 体外分别培养来自大脑皮层的星形胶质细胞和脑膜成纤维细胞，经胶质纤维酸性蛋白（GFAP）和纤连蛋白（FN）抗体免疫细胞化学染色鉴定后，将两种细胞混合共培养，2d后添加转化生长因子-β1（TGF-β1），以未添加TGF-β1为对照组；GFAP和FN免疫细胞化学染色观察胶质疤痕的形态；免疫细胞化学染色和Western blotting观察促红素肝细胞受体B2（EphB2）、Eph受体作用配体B2（ephrinB2）、神经蛋白聚糖（neurocan）和神经抗原2（NG2）表达情况。 结果 疤痕样细胞团簇结构主要是由星形胶质细胞和成纤维细胞共同组成；实验组EphB2、ephrinB2和neurocan、NG2表达量较对照组明显增加（P＜0.05）。 结论 体外混合培养星形胶质细胞与成纤维细胞并添加TGFβ1后成功建立体外中枢神经系统损伤后疤痕模型。
Objective To establish a scar model in vitro and observe the morphology and extracellular matrix of the scar. Methods Astrocytes and meningeal fibroblasts were cultured from the cerebral cortex in vitroand identified by glial fibrillary acidic protein (GFAP) and fibronectin(FN) immunofluorescence cell staining respectively. Astrocytes and meningeal fibroblasts were co-cultured and treated by TGF-β1 after 2 days. The group without TGF-β1 treating was used as the control. The morphology of scar was observed by GFAP and FN immunofluorescence cell staining. Immunofluorescence cell staining and Western blotting were used to examine the expression of EphB2, ephrinB2, neurocan and NG2 in the scar. Results The scar-like cell cluster was formed from astrocytes and fibroblasts. In the TGF-β1 group, the expressions of EphB2 and ephrinB2, neurocan and NG2 were significantly increased when compared with the control group (P<0.05). Conclusion A scar model after central nervous system injury has bean established by co-culturing astrocytes and fibroblasts treate by TGF-β1 in vitro.
［1］Fawcett JW, Asher RA. The glial scar and central nervous system repair ［J］. Brain Res Bull, 1999, 49(6): 377-391.
［2］Asher RA, Morgenstern DA, Moon LD, et al. Chondroitin sulphate proteoglycans: inhibitory components of the glial scar［J］. Prog Brain Res,2001, 132:611-619.
［3］Moon LD, Asher RA, Rhodes KE, et al. Regeneration of CNS axons back to their target following treatment of adult rat brain with chondroitinase ABC［J］. Nat Neurosci,2001, 4(5):465-466.
［4］Sicotte M, Tsatas O, Jeong SY, et al. Immunization with myelin or recombinant Nogo-66/MAG in alum promotes axon regeneration and sprouting after corticospinal tract lesions in the spinal cord ［J］. Mol Cell Neurosci, 2003, 23(2): 251-263.
［5］Fitch MT, Silver J. CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure ［J］. Exp Neurol, 2008, 209(2): 294-301.
［6］Kawano H, Kimura-Kuroda J, Komuta Y, et al. Role of the lesion scar in the response to damage and repair of the central nervous system ［J］. Cell Tissue Res, 2012, 349(1): 169-180.
［7］Bundesen LQ, Scheel TA, Bregman BS, et al. Ephrin-B2 and EphB2 regulation of astrocyte-meningeal fibroblast interactions in response to spinal cord lesions in adult rats ［J］. J Neurosci, 2003, 23(21): 7789-7800.
［8］Kimura-Kuroda J, Teng X, Komuta Y, et al. An in vitro model of the inhibition of axon growth in the lesion scar formed after central nervous system injury［J］. Mol Cell Neurosci, 2010,43(2): 177-187.
［9］Rolls A,Shechter R, Schwartz M. The bright side of the glial scar in CNS repair［J］. Nat Rev Neurosci,2009, 10(3):235-241.
［10］Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation［J］. Trends Neurosci,2009, 32(12):638-647.
［11］Yiu G, He Z. Glial inhibition of CNS axon regeneration［J］. Nat Rev Neurosci. 2006, 7(8):617-627.
［12］Faulkner JR, Herrmann JE, Woo MJ, et al. Reactive astrocytes protect tissue and preserve function after spinal cord injury ［J］. J Neurosci, 2004, 24(9): 2143-2155.
［13］Yoshioka N, Kimura-Kuroda J, Saito T,et al. Small molecule inhibitor of type I transforming growth factor-β receptor kinase ameliorates the inhibitory milieu in injured brain and promotes regeneration of nigrostriatal dopaminergic axons［J］.J Neurosci Res, 2011, 89(3):381-393.
［14］Lagord C, Berry M,Logan A. Expression of TGFbeta2 but not TGFbeta1 correlates with the deposition of scar tissue in the lesioned spinal cord ［J］. Mol Cell Neurosci, 2002, 20(1): 69-92.
［15］Goldshmit Y, McLenachan S, Turnley A. Roles of Eph receptors and ephrins in the normal and damaged adult CNS［J］. Brain Res Rev, 2006, 52(2):327-345.
［16］Rhodes KE, Fawcett JW. Chondroitin sulphate proteoglycans: preventing plasticity or protecting the CNS ［J］? J Anat,2004, 204(1):33-48.
［17］Shen LH, Li Y, Gao Q, et al. Down-regulation of neurocan expression in reactive astrocytes promotes axonal regeneration and facilitates the neurorestorative effects of bone marrow stromal cells in the ischemic rat brain ［J］. Glia, 2008, 56(16): 1747-1754.
［18］Naima J, Sari SH. Transforming growth factor β-induced expression of chondroitin sulfate proteoglycans is mediated through non-Smad signaling pathways ［J］. Exp Neurol, 2015,263:372-384.
［19］Davies JE, Tang X, Denning JW, et al. Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries［J］. Eur J Neurosci, 2004, 19(5):1226-1242.
［20］Alonso G. NG2 proteoglycan-expressing cells of the adult rat brain: possible involvement in the formation of glial scar astrocytes following stab wound［J］. Glia, 2005,49(3):318-338.
［21］Buss A, Pech K, Kakulas BA, et al. NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury ［J］. BMC Neurol, 2009, 9: 32.
［22］Smith GM, Rutishauser U, Silver J, et al. Maturation of astrocytes in vitro alters the extent and molecular basis of neurite outgrowth ［J］. Dev Biol, 1990, 138(2): 377-390.
siRNA干扰EphB2抑制胶质疤痕形成及组织工程修复脊髓损伤
/
| 〈 |
|
〉 |
