促红细胞生成素（EPO）对类缺血神经元的保护作用
摘要
目的:建立原代培养神经元的类缺血模型,观察使用促红细胞生成素(EPO)后是否具有神经保护作用,并比较不同EPO剂量组的效应,从而探讨EPO的神经保护作用。
方法:取新生wistar大鼠进行原代神经元培养,在体外培养7天,倒置显微镜观察神经元形态,先进行神经元特异性烯醇化酶(NSE)鉴定。然后随机分为正常对照组、直接缺血组、不同浓度EPO干预组。分别进行相应处理:正常对照组进行正常培养,不做任何处理;直接缺血组进行缺血缺氧24h;低、中、高浓度EPO干预组在缺血缺氧前24h加入对应浓度的EPO,24h后进行缺血缺氧24h。最后先倒置显微镜观察神经元形态,然后进行相应检测:MTT(四噻唑蓝)比色法检测细胞存活率;流式细胞仪检测不同组别细胞的凋亡率;乳酸脱氢酶(LDH)法检测各组细胞的活性。
结果:培养出细胞后,倒置显微镜观察细胞形态,胞体增大并且具有折光性,呈典型的神经元形态,鉴定后证明培养出了可供实验用的原代神经元。缺血缺氧后细胞突起减少,细胞数减少。MTT(四噻唑蓝)比色法及乳酸脱氢酶(LDH)法检测细胞活性后发现组间神经元活性有统计差别(P＜0.05);其中EPO干预组的活性均分别高于直接缺血缺氧组(P＜0.05),其中1U/mlEPO组的细胞活性分别高于0.1U/ml、10U/ml组(P＜0.05)。流式细胞仪检测不同组别细胞的凋亡率发现组间神经元凋亡率有统计差别(P＜0.05);其中EPO干预组的凋亡率均分别低于直接缺血缺氧组(P＜0.05),其中1U/mlEPO组的细胞凋亡率分别低于0.1U/ml、10U/ml组(P＜0.05)。
结论:本实验经MTT法、LDH法及流式法分析,实验中加入中浓度的EPO可有效的减少缺血缺氧引起的神经元损伤,降低神经元的凋亡率,从而对损伤神经元具有保护作用;低、高浓度的EPO组也有保护作用,但不及中浓度组。
Objective:To investigate the damage of ischemic-like on the primary cultured rat's neurons,the protective effects of EPO and the optimal dosage.
Methods:Cortical neurons cultured for 7 days were randomly divided into five groups:control group,ischemic-like group and plus three kinds of concentration of EPO groups treated.Then morphology of neurons was observed under inverted microscope.The survival rates of neurons at different groups were measured with MTT reduction assay and LDH examination.The apoptosis of neurons was detected using flow cytometry.Statistical analysis were using SPSS 12.0 software.Statistical analysis fixed that P＜0.05 is significant.
Results:We established a experimented cultured rat's cortical neurons by using the commonculture method.The cortical neurons was detected by neuronal specific enolase(NSE) immunocyto-chemical staining.We established a model by the mothed of ischemic-like conditioning on the cortial neurons.There was a notable difference between the ischemic-like group and the groups plused EPO(P＜0.05).The medium concentration EPO could reduce apoptosis of neuron and increase survival rates of neuron.But the lower and the high concentration EPO groups exert less action although having neuroprotective effects also.
Conclusion:Through the flow cytometry,MTT reduction assay and LDH examination,we find that using the medium concentration EPO can reduce the damage of neurons caused by ischemic -like through reducing neuron apoptosis and increase the survival rate of neurons.What suggests that EPO has an effective neuroprotection.
方法:取新生wistar大鼠进行原代神经元培养,在体外培养7天,倒置显微镜观察神经元形态,先进行神经元特异性烯醇化酶(NSE)鉴定。然后随机分为正常对照组、直接缺血组、不同浓度EPO干预组。分别进行相应处理:正常对照组进行正常培养,不做任何处理;直接缺血组进行缺血缺氧24h;低、中、高浓度EPO干预组在缺血缺氧前24h加入对应浓度的EPO,24h后进行缺血缺氧24h。最后先倒置显微镜观察神经元形态,然后进行相应检测:MTT(四噻唑蓝)比色法检测细胞存活率;流式细胞仪检测不同组别细胞的凋亡率;乳酸脱氢酶(LDH)法检测各组细胞的活性。
结果:培养出细胞后,倒置显微镜观察细胞形态,胞体增大并且具有折光性,呈典型的神经元形态,鉴定后证明培养出了可供实验用的原代神经元。缺血缺氧后细胞突起减少,细胞数减少。MTT(四噻唑蓝)比色法及乳酸脱氢酶(LDH)法检测细胞活性后发现组间神经元活性有统计差别(P＜0.05);其中EPO干预组的活性均分别高于直接缺血缺氧组(P＜0.05),其中1U/mlEPO组的细胞活性分别高于0.1U/ml、10U/ml组(P＜0.05)。流式细胞仪检测不同组别细胞的凋亡率发现组间神经元凋亡率有统计差别(P＜0.05);其中EPO干预组的凋亡率均分别低于直接缺血缺氧组(P＜0.05),其中1U/mlEPO组的细胞凋亡率分别低于0.1U/ml、10U/ml组(P＜0.05)。
结论:本实验经MTT法、LDH法及流式法分析,实验中加入中浓度的EPO可有效的减少缺血缺氧引起的神经元损伤,降低神经元的凋亡率,从而对损伤神经元具有保护作用;低、高浓度的EPO组也有保护作用,但不及中浓度组。
Objective:To investigate the damage of ischemic-like on the primary cultured rat's neurons,the protective effects of EPO and the optimal dosage.
Methods:Cortical neurons cultured for 7 days were randomly divided into five groups:control group,ischemic-like group and plus three kinds of concentration of EPO groups treated.Then morphology of neurons was observed under inverted microscope.The survival rates of neurons at different groups were measured with MTT reduction assay and LDH examination.The apoptosis of neurons was detected using flow cytometry.Statistical analysis were using SPSS 12.0 software.Statistical analysis fixed that P＜0.05 is significant.
Results:We established a experimented cultured rat's cortical neurons by using the commonculture method.The cortical neurons was detected by neuronal specific enolase(NSE) immunocyto-chemical staining.We established a model by the mothed of ischemic-like conditioning on the cortial neurons.There was a notable difference between the ischemic-like group and the groups plused EPO(P＜0.05).The medium concentration EPO could reduce apoptosis of neuron and increase survival rates of neuron.But the lower and the high concentration EPO groups exert less action although having neuroprotective effects also.
Conclusion:Through the flow cytometry,MTT reduction assay and LDH examination,we find that using the medium concentration EPO can reduce the damage of neurons caused by ischemic -like through reducing neuron apoptosis and increase the survival rate of neurons.What suggests that EPO has an effective neuroprotection.
引文
[1]Lee JM,Grabb MC,Zipfel GJ,et al.Brain tissue responses to ischemia.J Clininvest,2000,106(6):723.
[2]Hickenbottom SL,Grotta J.Neuroprotective therapy.Semin Neurol,1998,18(4):485.
[3]Dewar D,Yam P,MeCulloch J.Drug development for stroke:importance of protecting cerebral white matter.Eur J Pharmacol,1999,375:41-50.
[4]Wardlaw JM,Warlow CP,Sandercock PA.,et al.Neuroprotection disappointment yet Again.Lancent,2000,356-367.
[5]Brines ML,Ghezzi P,Keenan S,et al.Erythropoietin crosses the blood -brain barrier to protect against experimental brain injury[J].Proc Natl Acad Sci U S A,2000,97(19):230-251.
[6]Lacombe C,Mayeux P.Biology of erythropoietin[J].Haematologica,1998,83:724-732.
[7]Tan CC,Eckardt KU,Firth JD,etal.Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia[J].AmJ Physiol,1992,263:482-500.
[8]Masuda S,Okano M,Yamagishi K,et al.A novel site of erythropoietin production.Oxygen-dependent production in cultured rat astrocytes.J Biol Chem,1994,269:19488-19893.
[9]Marti HH,Gassmann M,Wenger RH.Deteetion of erythropoietin in human liquor intrinsic erythropoietin production in the brain.Kidney Int,1997,51:416-418.
[10]Sakanaka M,Wen TC,Matsuda S,et al.In vivo evidence that erythropoietin protects neurons from ischemic damage[J].Poc Natl Acad Sci,1998,95(8):463.
[11]Yu X,Shacka JJ,Eells JB,et al.Erythropoietin receptor signaling is required for normal brain development[J].2002,129(2):520-525.
[12]Nakagawa E,Choi HB,et al.Erythropoietin and erythropoietin receptors in human CNS neurons[J].Poc Natl Acad Sci,2001,60(4):386-392.
[13]杨浩,王春婷.不同方法纯化体外培养脊髓神经元的比较[J].中国组织化学与细胞化学杂志,2000,9(3):258-260.
[14]王英,车宇新生大鼠皮层神经元原代培养方法的研究[J].Chin J Cell MolImmunol,2003,19(2):197-199.
[15]Siren AL,Fratelli M,Brines M,et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress.Natl AcadSci USA,2001,98(7):4044-4049.
[15]Siren AL,Fratelli M,Brines M,et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress.Natl AcadSci USA,2001,98(7):4044-4049.
[16] Gelik M, Gokmen N, Erbayraktar S, et al.Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinalcord ischemic injury.Natl Acad Sci USA, 2002, 99:2258-2263.
[17] Martinez-Estrada OM,Rodriguez-Millan E,Gonzalez-DeVicente E,et al.Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability [J].Eur J Neurosci ,2003,18:2538.
[18] Villa P,Bigini P,Mennini T,et al.Erythropoietin selectively aRenuates cytokine production and inflammation in cerebral isehemia by targeting neuronal apoptos [J].Exp Ned,2003,198:971.
[19] Morishita E,Masuda S,Nagao M,et al.Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons , and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience, 1997,76:105-116.
[20] Calapai G,Marciano MC,Corica F,et al. Erythropoietin protects against brain ischemic injury by inhibition of nitric oxide formation. Eur J Pharmacol,2000,401:349-356.
[21] Akimoto T,Kusano E,Muto S,et al.The effect of erythropoietin on interleukin-lbeta mediated increase in nitric oxide synthesis in vascular smooth muscle cells. J Hypertens ,1999,17:1249-1256.
[22] Siren AL,Knerlich F,Poser W,et al.Erythropoietin and erythropoietin receptor in human ischemic/ hypoxic brain. Acta Neuropathol (Berl), 2001,101:271-276.
[23] Kawakarni M,lwasaki S,Sato K,et al.Erythropoietin inhibits calcium-induced neurotransmitter release from clonal neuronal ceIIs[J ] . Biochem Biophys Res Commun, 2000, 279 :293.
[24] Sun Y.Zhou C,Polk P,et al.Mechanisms of erythropoietin-induced protection in neonatal hypoxia-ischemia rat mode[J ]. J Cereb Blood Metab,2004,24(2):259-270.
[1] Brines ML,Ghezzi P,Keenan S,et al.Erythropoietin crosses the blood -brain barrier to protect against experimental brain injury [J ].Proc Natl Acad Sci U S A ,2000,97(19): 230-251.
[2] Masuda S ,Okano M,Yamagishi K,et al.A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes [J ].J Biol Chem, 1994,269: 19488-19893.
[3] Marti HH, Gassmann M, Wenger RH.Deteetion of erythropoietin in human liquor intrinsic erythropoietin production in the brain[J ].Kidney Int, 1997, 51:416-418.
[4] Sakanaka M ,Wen TC ,Matsuda S,et al.In vivo evidence that erythropoietin protects neurons from ischemic damage[J ]. Poc Natl Acad Sci ,1998 ,95 (8) :4635
[5] Siren AL,Knerlich F,Poser W,et al.Erythropoietin and erythropoietin receptor in human ischemic/ hypoxic brain[J ]. Acta Neuropathol (Berl), 2001,101:271-276.
[6] Tan CC.Eckardt KU,Firth JD,etal.Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia[J ] .AmJ Physiol, 1992,263:482-580.
[7] Sadamoto YJgase K,Sakanaka M,et al.Erythropoietin prevents place navigation disability and cortical infarction in rats with permanent occlusion of the middle cerebral artery[J ] . Biochem Biophys Res Commun, 1998,253(1):26232.
[8] Siren AL, Fratelli M, Brines M, et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress[J].Natl AcadSci USA, 2001, 98 (7) :4044-4049.
[9] Gelik M, Gokmen N, Erbayraktar S, et al.Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinalcord ischemic injury[J ].Natl Acad Sci USA, 2002, 99:2258-2263.
[10] Martinez-Estrada OM,Rodriguez-Millan E,Gonzalez-DeVicente E,et al.Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability [J].Eur J Neurosci ,2003,18:2538
[11] Villa P,Bigini P,Mennini T,et al.Erythropoietin selectively aRenuates cytokine production and inflammation in cerebral isehemia by targeting neuronal apoptos [J].Exp Ned,2003, 198 :971
[12] Morishita E,Masuda S,Nagao M,et al.Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons , and erythropoietin prevents in vitro glutamate-induced neuronal death[J ].Neuroscience, 1997,76:105-116.
[13] Calapai G,Marciano MC,Corica F,et al. Erythropoietin protects against brain ischemic injury by inhibition of nitric oxide formation[J ]. Eur J Pharmacol,2000,401:349-356.
[14] Akimoto T,Kusano E,Muto S,et al.The effect of erythropoietin on interleukin-lbeta mediated increase in nitric oxide synthesis in vascular smooth muscle cells[J]. J Hypertens ,1999,17:1249-1256.
[15] Kawakarni M,lwasaki S,Sato K,et al.Erythropoietin inhibits calcium-induced neurotransmitter release from clonal neuronal ceIIs[J ] . Biochem Biophys Res Commun , 2000,279 :293
[2]Hickenbottom SL,Grotta J.Neuroprotective therapy.Semin Neurol,1998,18(4):485.
[3]Dewar D,Yam P,MeCulloch J.Drug development for stroke:importance of protecting cerebral white matter.Eur J Pharmacol,1999,375:41-50.
[4]Wardlaw JM,Warlow CP,Sandercock PA.,et al.Neuroprotection disappointment yet Again.Lancent,2000,356-367.
[5]Brines ML,Ghezzi P,Keenan S,et al.Erythropoietin crosses the blood -brain barrier to protect against experimental brain injury[J].Proc Natl Acad Sci U S A,2000,97(19):230-251.
[6]Lacombe C,Mayeux P.Biology of erythropoietin[J].Haematologica,1998,83:724-732.
[7]Tan CC,Eckardt KU,Firth JD,etal.Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia[J].AmJ Physiol,1992,263:482-500.
[8]Masuda S,Okano M,Yamagishi K,et al.A novel site of erythropoietin production.Oxygen-dependent production in cultured rat astrocytes.J Biol Chem,1994,269:19488-19893.
[9]Marti HH,Gassmann M,Wenger RH.Deteetion of erythropoietin in human liquor intrinsic erythropoietin production in the brain.Kidney Int,1997,51:416-418.
[10]Sakanaka M,Wen TC,Matsuda S,et al.In vivo evidence that erythropoietin protects neurons from ischemic damage[J].Poc Natl Acad Sci,1998,95(8):463.
[11]Yu X,Shacka JJ,Eells JB,et al.Erythropoietin receptor signaling is required for normal brain development[J].2002,129(2):520-525.
[12]Nakagawa E,Choi HB,et al.Erythropoietin and erythropoietin receptors in human CNS neurons[J].Poc Natl Acad Sci,2001,60(4):386-392.
[13]杨浩,王春婷.不同方法纯化体外培养脊髓神经元的比较[J].中国组织化学与细胞化学杂志,2000,9(3):258-260.
[14]王英,车宇新生大鼠皮层神经元原代培养方法的研究[J].Chin J Cell MolImmunol,2003,19(2):197-199.
[15]Siren AL,Fratelli M,Brines M,et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress.Natl AcadSci USA,2001,98(7):4044-4049.
[15]Siren AL,Fratelli M,Brines M,et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress.Natl AcadSci USA,2001,98(7):4044-4049.
[16] Gelik M, Gokmen N, Erbayraktar S, et al.Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinalcord ischemic injury.Natl Acad Sci USA, 2002, 99:2258-2263.
[17] Martinez-Estrada OM,Rodriguez-Millan E,Gonzalez-DeVicente E,et al.Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability [J].Eur J Neurosci ,2003,18:2538.
[18] Villa P,Bigini P,Mennini T,et al.Erythropoietin selectively aRenuates cytokine production and inflammation in cerebral isehemia by targeting neuronal apoptos [J].Exp Ned,2003,198:971.
[19] Morishita E,Masuda S,Nagao M,et al.Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons , and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience, 1997,76:105-116.
[20] Calapai G,Marciano MC,Corica F,et al. Erythropoietin protects against brain ischemic injury by inhibition of nitric oxide formation. Eur J Pharmacol,2000,401:349-356.
[21] Akimoto T,Kusano E,Muto S,et al.The effect of erythropoietin on interleukin-lbeta mediated increase in nitric oxide synthesis in vascular smooth muscle cells. J Hypertens ,1999,17:1249-1256.
[22] Siren AL,Knerlich F,Poser W,et al.Erythropoietin and erythropoietin receptor in human ischemic/ hypoxic brain. Acta Neuropathol (Berl), 2001,101:271-276.
[23] Kawakarni M,lwasaki S,Sato K,et al.Erythropoietin inhibits calcium-induced neurotransmitter release from clonal neuronal ceIIs[J ] . Biochem Biophys Res Commun, 2000, 279 :293.
[24] Sun Y.Zhou C,Polk P,et al.Mechanisms of erythropoietin-induced protection in neonatal hypoxia-ischemia rat mode[J ]. J Cereb Blood Metab,2004,24(2):259-270.
[1] Brines ML,Ghezzi P,Keenan S,et al.Erythropoietin crosses the blood -brain barrier to protect against experimental brain injury [J ].Proc Natl Acad Sci U S A ,2000,97(19): 230-251.
[2] Masuda S ,Okano M,Yamagishi K,et al.A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes [J ].J Biol Chem, 1994,269: 19488-19893.
[3] Marti HH, Gassmann M, Wenger RH.Deteetion of erythropoietin in human liquor intrinsic erythropoietin production in the brain[J ].Kidney Int, 1997, 51:416-418.
[4] Sakanaka M ,Wen TC ,Matsuda S,et al.In vivo evidence that erythropoietin protects neurons from ischemic damage[J ]. Poc Natl Acad Sci ,1998 ,95 (8) :4635
[5] Siren AL,Knerlich F,Poser W,et al.Erythropoietin and erythropoietin receptor in human ischemic/ hypoxic brain[J ]. Acta Neuropathol (Berl), 2001,101:271-276.
[6] Tan CC.Eckardt KU,Firth JD,etal.Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia[J ] .AmJ Physiol, 1992,263:482-580.
[7] Sadamoto YJgase K,Sakanaka M,et al.Erythropoietin prevents place navigation disability and cortical infarction in rats with permanent occlusion of the middle cerebral artery[J ] . Biochem Biophys Res Commun, 1998,253(1):26232.
[8] Siren AL, Fratelli M, Brines M, et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress[J].Natl AcadSci USA, 2001, 98 (7) :4044-4049.
[9] Gelik M, Gokmen N, Erbayraktar S, et al.Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinalcord ischemic injury[J ].Natl Acad Sci USA, 2002, 99:2258-2263.
[10] Martinez-Estrada OM,Rodriguez-Millan E,Gonzalez-DeVicente E,et al.Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability [J].Eur J Neurosci ,2003,18:2538
[11] Villa P,Bigini P,Mennini T,et al.Erythropoietin selectively aRenuates cytokine production and inflammation in cerebral isehemia by targeting neuronal apoptos [J].Exp Ned,2003, 198 :971
[12] Morishita E,Masuda S,Nagao M,et al.Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons , and erythropoietin prevents in vitro glutamate-induced neuronal death[J ].Neuroscience, 1997,76:105-116.
[13] Calapai G,Marciano MC,Corica F,et al. Erythropoietin protects against brain ischemic injury by inhibition of nitric oxide formation[J ]. Eur J Pharmacol,2000,401:349-356.
[14] Akimoto T,Kusano E,Muto S,et al.The effect of erythropoietin on interleukin-lbeta mediated increase in nitric oxide synthesis in vascular smooth muscle cells[J]. J Hypertens ,1999,17:1249-1256.
[15] Kawakarni M,lwasaki S,Sato K,et al.Erythropoietin inhibits calcium-induced neurotransmitter release from clonal neuronal ceIIs[J ] . Biochem Biophys Res Commun , 2000,279 :293