低氧诱导的大鼠嗅黏膜来源嗅鞘细胞自噬及其对细胞增殖能力的影响

doi:10.13481/j.1671-587x.20160303

摘要/Abstract

摘要：

目的: 探讨低氧诱导SD新生鼠嗅黏膜来源嗅鞘细胞(OECs)自噬的发生,分析自噬对细胞增殖能力的影响。方法: 采用酶消化法和改良的Nash差速贴壁法分离提纯培养SD新生鼠嗅黏膜来源OECs,利用免疫荧光法鉴定细胞属性和纯度。以低氧条件(氧浓度为2％)培养的SD新生鼠嗅黏膜来源OECs作为低氧处理组,以正常条件下培养的OECs作为对照组,采用自噬抑制剂3-MA处理后的OECs继续在低氧条件下培养作为3-MA低氧处理组。相差显微镜下观察各组细胞形态;Western blotting法检测对照组、4h低氧处理组和8h低氧处理组OECs中低氧诱导因子1α(HIF-1α)、自噬标志蛋白LC3Ⅰ/Ⅱ和自噬标志基因Beclin-1的蛋白表达水平;采用CCK-8法检测对照组、3-MA低氧处理组、低氧处理组和3-MA低氧处理组OECs的增殖能力。结果: 在对照组、低氧处理组和3-MA低氧处理组经分离提纯的OECs均呈梭形,表现为典型的双极、三极细胞形态,3组间无明显差异。OECs呈NGFRp75和GFAP阳性,纯度为87%;与对照组比较,低氧处理组OECs中HIF-1α表达水平升高(P<0.05),LC3Ⅰ/Ⅱ和Beclin-1表达水平升高(P<0.05);与低氧处理组比较,3-MA低氧处理组OECs增殖能力降低。结论: 低氧诱导的自噬可降低SD新生鼠嗅黏膜来源OECs的增殖能力。

关键词: 嗅鞘细胞, 低氧, 自噬, 细胞增殖

Abstract:

Objective: To discuss the occurrence of hypoxia-induced autophagy in the olfactory ensheathing cells (OECs) from olfactory mucosa in the newly-born SD rat models,and to evaluate the effect of autophagy on the cell proliferation. Methods: The OECs were obtained and cultivated by enzyme digestion and advanced Nash differential attachment,and the properties and purities of OECs were identified by immunofluorescent assay;the OECs cultured in hypoxia condition with 2% oxygen levels were used as hypoxia group,the cells cultured under the normal culture conditions were regarded as control group, and the OECs treated with autophagy inhibitor 3-MA and cultured under hypoxic conditions were regarded as 3-MA+hypoxia group.The morphology of cells in various groups was observed under phase contrast microscope; Western blotting method was used to measure the protein expression levels of hypoxia inducible factor-1(HIF-1α ),LC3Ⅰ/Ⅱ (autophagy marker protein) and Beclin-1 (autophagy marker gene) in the cells in control group,4 h hypoxia group,and 8 h hypoxia group;CCK-8 method was used to measure the influence of autophagy in cell proliferation in control group, 3-MA group,hypoxia group and 3-MA+hypoxia group, Results: After separated and purificated,the OECs in control group,hypoxia group and 3-MA+hypoxia group showed fusiform, typical bipolar,and tripolar cell morphology,and had no significant differences.In the cultivated OECs,the NGFRp 75 and GFAP were expressed positively through separation and purification,and the purity was 87%. Compared with control group,the expression level of HIF-1α in the OECs in hypoxia group was increased (P<0.05),and the expression levels of LC3I Ⅱ and Beclin-1 in the OECs in hypoxia group were increased (P<0.05).Compared with hypoxia group,the proliferation ability of OECs in 3-MA+hypoxia group was reduced. Conclusion: The autophagy induced by hypoxia can decrease the cell proliferation of OECs from olfactory mucosa in the newly-born SD rats.

Key words: olfactory ensheathing cells, hypoxia, autophagy, cell proliferation

中图分类号:

R329.28

王珺, 张利剑, 夏鹤春, 梁雪云, 王亚平. 低氧诱导的大鼠嗅黏膜来源嗅鞘细胞自噬及其对细胞增殖能力的影响[J]. 吉林大学学报(医学版), 2016, 42(03): 430-434.

WANG Jun, ZHANG Lijian, XIA Hechun, LIANG Xueyun, WANG Yaping. Effect of autophagy inducedby hypoxia on cell proliferation in olfactory ensheathing cells from olfactory mucosa of rats[J]. Journal of Jilin University Medicine Edition, 2016, 42(03): 430-434.

参考文献

[1] Tabakow P,Jarmundowicz W,Czapiga B,et al. Transplantation of autologous olfactory ensheathing cells in complete human spinal cord injury [J]. Cell Transplant,2013.doi:10.3727/096368913×663532.

[2] Dlouhy BJ,Awe O,Rao RC,et al.Autograft-derived spinal cord mass following olfactory mucosal cell transplantation in a spinal cord injury patient:Case report [J].J Neurosurg Spine,2014,21(4):618-622.

[3] Sethi R,Sethi R,Redomod A,et al.Olfactory ensheathing cells promote differerntiation of neural stem cells and robust neurite extension [J].Stem Cell Rev,2014,10(6):772-785.

[4] Brenner C,Kroemer G.Apoptosis.Mitochondria-the death signal integrators [J].Science,2000,289(5482):1150-1151.

[5] Cao L,Liu L,Chen ZY,et al.Olfactory ensheathing cells genetically modified to secrete GDNF to promote spinal cord repair [J].Brain,2004,127(Pt 3):535-549.

[6] Cui Q,Pollett MA,Symons NA,et al.A new approach to CNS repair using chimeric peripheral nerve grafts [J].J Neurotrauma,2003,20(1):17-31.

[7] Takami T,Oudega M,Bates ML,et al.Schwann cell but not olfactory ensheathing glia transplants improve hindlimb locomotor performance in the moderately contused adult rat thoracic spinal cord[J].J Neurosci,2002,22(15):6670-6681.

[8] Kachramanoglou C,Law S,Andrews P,et al.Culture of olfactory ensheathing cells for central nerve repair:the limitations and potential of endoscopic olfactory mucosal biopsy [J]. Neurosurgery,2013,72(2):170-179.

[9] 潘文明.大鼠脊髓损伤后细胞自噬的表达及作用的实验研究 [D].苏州:苏州大学,2010.

[10] Nash HH,Borke RC,Anders JJ.New method of purification for establishing primary cultures of ensheathing cells from the adult olfactory bulb [J]. Glia,2001,34(2):81-87.

[11] Roet KC,Verhaagen J.Understanding the neural repair-promoting properties of olfactory ensheathing cells [J].Exp Neurol,2014,261:549-609.

[12] Engel DC,Slemmer JE,Vlug AS,et al.Combined effects of mechanical and ischemic injury to cortical cells:secondary ischemia increases damage and decreases effects of neuroprotective agents [J].Neuropharmacology,2005,49(7):985-995.

[13] Paspala SA,Vishwakarma SK,Murthy TV,et al.Potential role of stem cells in severe spinal cord injury:current perspectives and clinical data [J].Stem Cells Cloning,2012,5:15-27.

[14] Sun C,Meng Q,Zhang L,et al.Glutamate attenuates IGF-1 receptor tyrosine phosphorylation in mouse brain:possible significance in ischemic brain damage [J].Neurosci Res,2012,74(3/4):290-297.

[15] Su Z,He C.Olfactory ensheathing cells:biology in neural development and regeneration [J].Prog Neurobiol,2010,92(4):517-532.

[16] Diskin T,Tal-Or P,Erlich S,et al.Closed head injury induces upregulation of Beclin 1 at the cortical site of injury [J].J Neurotrauma,2005,22(7):750-762.

[17] Adhami F,Liao G,Morozov YM,et al.Cerebral ischemia-hypoxia induces intravascular coagulation and autophagy [J].Am J Pathol,2006, 169(2):566-583.