华中科技大学学报(医学版)
華中科技大學學報(醫學版)
화중과기대학학보(의학판)
ACTA UNIVERSITATIS MEDICINAE TONGJI
2015年
1期
47-51
,共5页
罗振钊%王静%孔曼%胡绘%卢忠心%施静
囉振釗%王靜%孔曼%鬍繪%盧忠心%施靜
라진쇠%왕정%공만%호회%로충심%시정
小胶质细胞%细胞因子%神经元损伤度%表型
小膠質細胞%細胞因子%神經元損傷度%錶型
소효질세포%세포인자%신경원손상도%표형
microglia%cytokine%severity of neuronal injury%phenotype
目的:探讨不同的神经元损伤度对小胶质细胞表型的影响。方法将原代培养的神经元进行缺氧处理,不同的时长(0.5、1、2、4 h)后,复氧处理24 h ,收集神经元条件培养液(neuron‐conditioned media ,NCM ),然后将NCM刺激原代培养的小胶质细胞24 h(NCM∶小胶质细胞培养液=1∶1,V/V )。采用Western blot法检测小胶质细胞内的M2表型标记物精氨酸酶‐1(arginase‐1)和激活标记物离子钙结合衔接分子‐1(Iba‐1)的表达变化规律,采用免疫荧光法检测ar‐ginase‐1的表达变化规律。同时,采用 ELISA 法检测小胶质细胞培养液上清中的营养因子,如脑源性神经营养因子(BDNF)、胶质细胞源性神经营养因子(GDNF)和炎性因子,如肿瘤坏死因子‐α(TNF‐α)、白细胞介素‐1β(IL‐1β)、白细胞介素‐6(IL‐6)的分泌变化规律。最后,将不同损伤度的NCM 诱导小胶质细胞形成不同表型,将小胶质细胞和缺氧处理2 h后的神经元共培养24 h ,MTT法检测神经元的活力。结果轻微损伤(缺氧0.5、1 h)的NCM 明显下调M2型标记物arginase‐1的表达水平,中度(缺氧2 h)和重度(缺氧4 h)损伤的 NCM 能够上调arginase‐1的表达,各种损伤度的NCM都能上调Iba‐1的表达水平,提示其在一定程度上激活小胶质细胞。同时,轻微损伤的NCM明显上调小胶质细胞TNF‐α、IL‐1β和IL‐6的分泌,而中度和重度损伤的NCM 对这些促炎因子的释放没有影响,各种损伤度的NCM 都能明显上调营养因子的分泌。M T T法检测表明,轻微和重度损伤处理的NCM刺激的小胶质细胞进一步加重缺氧处理的神经元损伤,而中度损伤的NCM对缺氧处理后的神经元具有保护作用。结论神经元损伤度是决定小胶质细胞表型的重要因素,进而使小胶质细胞进一步发挥神经毒性或保护作用。
目的:探討不同的神經元損傷度對小膠質細胞錶型的影響。方法將原代培養的神經元進行缺氧處理,不同的時長(0.5、1、2、4 h)後,複氧處理24 h ,收集神經元條件培養液(neuron‐conditioned media ,NCM ),然後將NCM刺激原代培養的小膠質細胞24 h(NCM∶小膠質細胞培養液=1∶1,V/V )。採用Western blot法檢測小膠質細胞內的M2錶型標記物精氨痠酶‐1(arginase‐1)和激活標記物離子鈣結閤銜接分子‐1(Iba‐1)的錶達變化規律,採用免疫熒光法檢測ar‐ginase‐1的錶達變化規律。同時,採用 ELISA 法檢測小膠質細胞培養液上清中的營養因子,如腦源性神經營養因子(BDNF)、膠質細胞源性神經營養因子(GDNF)和炎性因子,如腫瘤壞死因子‐α(TNF‐α)、白細胞介素‐1β(IL‐1β)、白細胞介素‐6(IL‐6)的分泌變化規律。最後,將不同損傷度的NCM 誘導小膠質細胞形成不同錶型,將小膠質細胞和缺氧處理2 h後的神經元共培養24 h ,MTT法檢測神經元的活力。結果輕微損傷(缺氧0.5、1 h)的NCM 明顯下調M2型標記物arginase‐1的錶達水平,中度(缺氧2 h)和重度(缺氧4 h)損傷的 NCM 能夠上調arginase‐1的錶達,各種損傷度的NCM都能上調Iba‐1的錶達水平,提示其在一定程度上激活小膠質細胞。同時,輕微損傷的NCM明顯上調小膠質細胞TNF‐α、IL‐1β和IL‐6的分泌,而中度和重度損傷的NCM 對這些促炎因子的釋放沒有影響,各種損傷度的NCM 都能明顯上調營養因子的分泌。M T T法檢測錶明,輕微和重度損傷處理的NCM刺激的小膠質細胞進一步加重缺氧處理的神經元損傷,而中度損傷的NCM對缺氧處理後的神經元具有保護作用。結論神經元損傷度是決定小膠質細胞錶型的重要因素,進而使小膠質細胞進一步髮揮神經毒性或保護作用。
목적:탐토불동적신경원손상도대소효질세포표형적영향。방법장원대배양적신경원진행결양처리,불동적시장(0.5、1、2、4 h)후,복양처리24 h ,수집신경원조건배양액(neuron‐conditioned media ,NCM ),연후장NCM자격원대배양적소효질세포24 h(NCM∶소효질세포배양액=1∶1,V/V )。채용Western blot법검측소효질세포내적M2표형표기물정안산매‐1(arginase‐1)화격활표기물리자개결합함접분자‐1(Iba‐1)적표체변화규률,채용면역형광법검측ar‐ginase‐1적표체변화규률。동시,채용 ELISA 법검측소효질세포배양액상청중적영양인자,여뇌원성신경영양인자(BDNF)、효질세포원성신경영양인자(GDNF)화염성인자,여종류배사인자‐α(TNF‐α)、백세포개소‐1β(IL‐1β)、백세포개소‐6(IL‐6)적분비변화규률。최후,장불동손상도적NCM 유도소효질세포형성불동표형,장소효질세포화결양처리2 h후적신경원공배양24 h ,MTT법검측신경원적활력。결과경미손상(결양0.5、1 h)적NCM 명현하조M2형표기물arginase‐1적표체수평,중도(결양2 h)화중도(결양4 h)손상적 NCM 능구상조arginase‐1적표체,각충손상도적NCM도능상조Iba‐1적표체수평,제시기재일정정도상격활소효질세포。동시,경미손상적NCM명현상조소효질세포TNF‐α、IL‐1β화IL‐6적분비,이중도화중도손상적NCM 대저사촉염인자적석방몰유영향,각충손상도적NCM 도능명현상조영양인자적분비。M T T법검측표명,경미화중도손상처리적NCM자격적소효질세포진일보가중결양처리적신경원손상,이중도손상적NCM대결양처리후적신경원구유보호작용。결론신경원손상도시결정소효질세포표형적중요인소,진이사소효질세포진일보발휘신경독성혹보호작용。
Objective To explore the effects of different severities of neuronal injury on microglial phenotype. Methods Primary neurons were treated by hypoxia for 0.5 ,1 ,2 ,and 4h,and then reoxygenated for 24 h.Neuron‐conditioned media (NCM)were collected and added to microglial cultures(1∶1 volume).Twenty‐four h later ,Western blot was used to detect the expressions of the microglial M 2 phenotype marker arginase‐1 and the activation marker ionized calcium binding adapter mole‐cule 1(Iba‐1).Immunofluorescence was performed to detect the expression of arginase‐1.Additionally ,microglial trophic mole‐cules brain‐derived neurotrophic factor(BDNF) ,glial cell line‐derived neurotrophic factor(GDNF)and proinflammatory cyto‐kines TNF‐α,IL‐1β,IL‐6 were determined by ELISA. Finally ,different phenotypes of primary microglia were formed after cells were treated with different NCM.MTT assays were used to determine the viability of neurons which were treated by hypoxia for 2 h and then co‐cultured with microglia for 24 h.Results NCM from mild injuries(treated by hypoxia for 0.5 or 1 h)signifi‐cantly down‐regulated the expression of the microglial M 2 phenotype marker arginase‐1 ,and those from moderate and severe in‐juries up‐regulated the arginase‐1 expression. Iba‐1 was upregulated in all injury conditions ,suggesting that all the injury condi‐tions can induce microglial activation to some degree.Moreover ,NCM from mild injuries up‐regulated the secretion of microglial TNF‐α,IL‐1βand IL‐6 ,but NCM from moderate(hypoxia 2 h)and severe(hypoxia 4 h)injuries had no effects on these proin‐flammatory cytokines. NCM of different injury severities significantly up‐regulated the release of these trophic molecules. MTT assays demonstrated that microglia incubated with NCM of mild and severe injuries further exacerbated the injury of neurons treated by hypoxia.Microglia incubated with NCM of moderate injuries protected neurons from hypoxia injury.Conclusion The severity of neuronal injury is an important factor in determining microglial phenotypes ,which has neurotoxic or neuroprotective effects.