细胞缺氧损伤中TRPM7介导的凋亡机制及NGF对TRPM7调节机制研究
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摘要
第一部分
TRPM7介入氧糖剥夺／再氧合诱导细胞凋亡的机制研究
第一节
建立四环素调控性稳定表达全长、缺激酶区TRPM7的HEK293细胞系
TRPM7(transient receptorpotentialmelastatin 7)，也称ChaK1(Channel-Kinase1)，是一种独特的、既有非选择性阳离子通道又有蛋白激酶活性的双功能蛋白。本部分实验主要是建立四环素调控性稳定表达全长、缺激酶区TRPM7的HEK293细胞系，为进一步在细胞水平研究TRPM7及其激酶功能提供实验基础。
采用阳离子脂质体法将含小鼠TRPM7 cDNA的真核表达质粒pcDNA5／FRT／TO-HA-TRPM7／WT(表达全长TRPM7(TRPM7／WT))和pcDNA5／FRT／TO-HA-TRPM7／△Kin(表达缺激酶区TRPM7(TRPM7／△Kin))分别与Flp重组酶表达质粒pOG44共同转入Flp-In~(TM) T-REx~(TM) 293细胞，经过潮霉素筛选，收集抗药细胞克隆建立细胞系，分别命名为293-TRPM7／WT(转染pcDNA5／FRT／TO-HA-TRPM7／WT)和293-TRPM7／△Kin(转染pcDNA5／FRT／TO-HA-TRPM7／ΔKin)。采用免疫印迹和免疫荧光技术对细胞进行鉴定，结果显示：293-TRPM7／WT和293-TRPM7／△Kin细胞均在四环素诱导后才表达TRPM7蛋白(前者表达TRPM7／WT，后者表达TRPM7／△Kin)，且TRPM7蛋白表达水平均呈四环素浓度依赖性。综上所述，我们成功建立了四环素调控性稳定表达全长、缺激酶区TRPM7的HEK293细胞系。
第二节
TRPM7激酶区在氧糖剥夺／再氧合诱导细胞凋亡中的作用，及其与Annexin-1之间的关系
脑缺血后的神经元损伤是造成神经功能缺损的主要原因。最近的研究显示，TRPM7通道的活动与缺氧神经元的死亡密切相关，但是TRPM7激酶在缺血缺氧神经元损伤过程中作用尚未见报导。
本部分实验利用四环素调控性稳定表达全长和缺激酶区TRPM7的293-TRPM7／WT和293-TRPM7／△Kin细胞系，通过建立氧糖剥夺／再氧合(oxygen-glucose deprivation／reoxygenation，OGD／R)离体模型，即细胞在无糖无氧的环境中孵育1h(氧糖剥夺)后恢复正常培养一定时间(再氧合)，模拟在体缺血再灌注过程，从细胞水平初步探讨TRPM7激酶区在OGD／R引起的细胞损伤和细胞凋亡中的作用及其可能的机制。Annexin-1是TRPM7激酶区重要的底物蛋白，本研究通过观察Annexin-1的变化来讨论TRPM7激酶区的作用机制。
实验采用MTT法检测细胞活性，Annexin V-FITC、PI双染法结合流式细胞术检测、分析细胞凋亡率，应用免疫荧光和免疫印迹技术检测TRPM7、Annexin-1蛋白的分布和表达，利用免疫共沉淀法分析TRPM7与Annexin-1的相互作用。
结果显示：
1、TRPM7激酶区参与TRPM7促进OGD／R细胞损伤和细胞凋亡。
1)在正常情况下，四环素诱导和四环素非诱导的293-TRPM7／WT细胞和293-TRPM7／△Kin细胞活性之间没有显著性差异。经氧糖剥夺处理1h后，在再氧合Oh、6h、12h、24h过程中，四环素诱导293-TRPM7／WT细胞和293-TRPM7／△Kin细胞活性在每个时间点上都显著低于四环素非诱导293-TRPM7／WT细胞和293-TRPM7／△Kin细胞活性(P＜0．01)。而且从再氧合12h时开始，四环素诱导293-TRPM7／WT细胞活性明显低于四环素诱导293-TRPM7／△Kin细胞(P＜0．05)，到再氧合24h差异更加显著(P＜0．01)。
2)在正常情况下，四环素诱导、四环素非诱导的293-TRPM7／WT细胞和293-TRPM7／△Kin细胞凋亡率分别为：1．4％±0．16％、1．5％±0．12％、1．4％±0．13％、1．4％±0．15％，各组之间没有显著性差异；氧糖剥夺1h再氧合24h处理后，四环素诱导293-TRPM7／WT细胞凋亡率(34．1％±1．22％)显著高于四环素非诱导293-TRPM7／WT细胞(10．2％±1．05％，P＜0．05)和四环素诱导293-TRPM7／ΔKin细胞(13．5％±1．01％，P＜0．05)。
2、OGD／R过程中，TRPM7通过其激酶区增强与Annexin-1结合。
1)免疫荧光双标记发现，TRPM7／WT蛋白和Annexin-1共定位于四环素诱导293-TRPM7／WT细胞，TRPM7／WT蛋白主要分布在细胞膜上，Annexin-1分布在整个细胞中，两者在细胞内共存，共定位在细胞膜上。
2)免疫共沉淀发现，在正常情况下，四环素诱导293-TRPM7／WT细胞中有少量Annexin-1与TRPM7／WT结合；氧糖剥夺1h后，随着再氧合时间的延长，Annexin-1与TRPM7／WT的结合逐渐增多。再氧合24h时，用BAPTA-AM或EDTA螯合细胞内或细胞外钙离子不影响四环素诱导293-TRPM7／WT细胞中Annexin-1与TRPM7／WT的结合，而四环素诱导293-TRPM7／ΔKin细胞中未发现Annexin-1与TRPM7／ΔKin结合。
3、OGD／R诱导HEK293细胞出现TRPM7激酶区依赖的Annexin-1蛋白高表达和Annexin-1细胞核、细胞膜转位。
1)氧糖剥夺1h后，从再氧合6h开始，四环素诱导293-TRPM7／WT细胞中Annexin-1蛋白的表达水平随再氧合时间延长逐渐增加，到24h达到高峰，与氧糖剥夺1h再氧合24h后四环素非诱导293-TRPM7／WT、293-TRPM7／ΔKin细胞和四环素诱导293-TRPM7／ΔKin细胞相比，Annexin-1蛋白表达增加有显著性差异(P＜0．05)。而四环素非诱导293-TRPM7／WT、293-TRPM7／ΔKin细胞和四环素诱导293-TRPM7／ΔKin细胞之间比较无显著性差异。在再氧合6h、12h、24h过程中，四环素诱导293-TRPM7／WT细胞中Annexin-1的表达水平在各个时间点均显著高于四环素非诱导293-TRPM7／WT细胞(P＜0．05)。
2)氧糖剥夺1h再氧合24h时，免疫荧光结果显示：四环素诱导293-TRPM7／WT细胞中Annexin-1转位到细胞膜和细胞核，而四环素非诱导293-TRPM7／WT细胞和四环素诱导293-TRPM7／ΔKin细胞中Annexin-1未发生明显转位。进一步用免疫印迹分别检测胞膜、胞浆和胞核中Annexin-1的表达，发现：氧糖剥夺1h再氧合24h处理后，四环素诱导293-TRPM7／WT细胞胞浆中Annexin-1的表达水平比正常低，而胞膜特别是胞核中Annexin-1的表达水平明显高于正常；四环素非诱导293-TRPM7／WT细胞胞膜和胞核中Annexin-1的表达水平与正常相似，而胞浆中Annexin-1的表达水平高于正常。
4、Annexin-1参与TRPM7介导的OGD／R细胞损伤和细胞凋亡。
1)氧糖剥夺1h再氧合24h时，Annexin-1反义脱氧寡核苷酸(ASODN)显著下调四环素诱导293-TRPM7／WT细胞中Annexin-1的高表达至正常水平(P＜0．05)，同时显著增加细胞活性(P＜0．01)，而Annexin-1正义脱氧寡核苷酸(SODN)对Annexin-1的表达和细胞活性均没有影响。
2)氧糖剥夺1h再氧合24h时，Annexin-1 ASOND显著减少四环素诱导293-TRPM7／WT细胞凋亡率19．6％±1．01％(P＜0．05)；Annexin-1 SODN对细胞凋亡率没有影响，仍有高达33．7％±1．56％细胞凋亡。
综上所述，本实验首次验证了：TRPM7通过其激酶区上调Annexin-1的表达，促进Annexin-1的核、膜转位，加剧OGD／R引起的细胞损伤和细胞凋亡。本研究的发现为进一步研究TRPM7激酶介导脑缺血再灌注神经元凋亡提供了实验依据。
第二部分
NGF抑制Gd~(3+)敏感性钙内流，减少化学缺氧神经元死亡
本部分实验主要探讨急性缺氧神经元损伤过程中是否存在非谷氨酸受体通道和非电压门控钙通道依赖性的钙内流，及其与NGF神经元保护作用之间的关系。
以KCN作为诱导剂，在体外培养的新生大鼠海马神经元上建立急性缺氧的细胞模型，应用激光扫描共聚焦显微镜监测细胞内钙离子浓度变化，利用cFDA与PI染色法评价细胞的存活和死亡率。
结果显示：
1、给予原代培养的海马神经元KCN(3mM)处理15min后，细胞内钙离子浓度([Ca~(2+)]i)显著升高至缺氧前6．27±0．05倍(p＜0．05)，73．3％±12．1％细胞死亡(P＜0．05)。
2、20μM MK-801(NMDA受体拮抗剂)、40μM CNQX(AMPA受体拮抗剂)和5μM Nimodipine(电压门控钙通道拮抗剂)联用(简称MCN)显著降低KCN引起的[Ca~(2+)]_i升高和细胞死亡率(P＜0．05)，但[Ca~(2+)]_i为缺氧前4．02±0．04倍，仍有38．5％±4．4％细胞死亡，均显著高于正常(P＜0．05)。
3、Gd~(3+)(10μM)与MCN联用后几乎完全阻断KCN引起的[Ca~(2+)]_i升高，并进一步降低细胞死亡率。[Ca~(2+)]_i仅为缺氧前1．2±0．03倍，与正常相比差异无显著性，神经元死亡率降至8．2％±1．5％。
4、与Gd~(3+)的作用相似，NGF(100ng／m1)与MCN联用后显著减弱KCN引起的[Ca~(2+)]_i升高，[Ca~(2+)]_i仅为缺氧前1．42±0．06倍，与正常相比无统计学差异，只有9．1％±2．3％细胞死亡。NGF的作用可被磷脂酶C(PLC)拮抗剂U73122(10μM)逆转。
以上结果表明，在急性缺氧神经元损伤过程中存在非谷氨酸受体通道和非电压门控钙通道依赖性的Gd~(3+)敏感性钙内流和神经元死亡；NGF可通过激活PLC通路抑制Gd~(3+)敏感性钙内流，同时减少缺氧神经元死亡。
Part 1
Mechanism study for implication of TRPM7 in oxygen-glucosedeprivation/reoxygenation-induced apoptosis
Segment 1
Establishment of stable HEK293 cell lines with tetracycline-inducible expression offull-length or kinase domain truncated TRPM7
Transient receptor potential melastatin 7 (TRPM7),also named asChannel-Kinasel (ChaK1),is a unique bifunctional protein consisting of a proteinkinase domain fused to a non-selective cation channel.The aim of this study was toestablish stable HEK293 cell lines with tetracycline-inducible expression of full-lengthor kinase domain truncated TRPM7,so that we can investigate the functions of TRPM7and its kinase at cellular level.
Eukaryotic expression plasmid pcDNA5/FRT/TO-HA-TRPM7/WT (expressingfull-length TRPM7 (TRPM7/WT)) or pcDNA5/FRT/TO-HA-TRPM7/△Kin(expressing kinase domain truncated TRPM7 (TRPM7/△Kin)) was cotransfected withthe Flp recombinase expression plasmid pOG44 into Flp-In~(TM) T-REx~(TM) 293 cells usingLipofectamine 2000~(TM).After selection with hygromycin,cell clones were pooled anddefined as 293-TRPM7/WT (transfected with pcDNA5/FRT/TO-HA-TRPM7/WT) and293-TRPM7/△Kin (transfected with pcDNA5/FRT/TO-HA-TRPM7/△Kin),respectively.The tetracycline-inducible expression of TRPM7/WT in 293- TRPM7/WTcells or TRPM7/△Kin in 293-TRPM7/△Kin cells was confirmed by western blot andimmunofluorescence analysis.The results showed that TRPM7/WT or TRPM7/△Kinexpression was switched on by application of tetracycline in a concentration dependent manner.In conclusion,we successfully established tetracycline-inducible HEK293stable cell lines expressing full-length or kinase domain truncated TRPM7.
Segment 2
The role of TRPM7 kinase domain in oxygen-glucosedeprivation/reoxygenation-induced apoptosis and the relationship betweenTRPM7 and Annexin-1
Neuronal death after cerebral ischemia is the main cause of neurologicimpairment.It is recently revealed that the activity of TRPM7 channel is closely relatedto anoxic neuronal death,however,the exact function of TRPM7 kinase in ischemicneuronal injury is unknown.
In this study,by using stable HEK293 cell lines with tetracycline (Tet)-inducibleexpression of full-length and kinase domain truncated TRPM7 (293-TRPM7/WT cellsand 293-TRPM7/△Kin cells) and establishing in vitro model of oxygen-glucosedeprivation followed by reoxygenation (OGD/R) mimicking in vivo ischemicreperfusion process,we investigated the role of TRPM7 kinase domain inOGD/R-induced cell damage and cell apoptosis and the possible mechanisms.SinceAnnexin-1 is an important substrate for TRPM7 kinase,changes of Annexin-1 wasobserved in this study to explored the mechanism of action of TRPM7 kinase domain.
Cell viability was determined by MTT assay.Apoptosis was quantitativelyanalyzed by Annexin V-FITC and PI double-staining followed by flow cytometry.Immunofluorescence and western blot were used to detect the localization andexpression of TRPM7 and Annexin-1,respectively.Interaction between TRPM7 andAnnexin-1 was measured by immunoprecipitation.
The results showed that:
1.TRPM7 kinase domain was implicated in OGD/R-induced,TRPM7-mediatedcell damage and apoptosis of HEK293 cells.
1) Under normal condition,cell viability among Tet-induced,non-Tet-induced293-TRPM7/WT and 293-TRPM7/△Kin cells were no statistic difference.After 1h ofOGD and 0h,6h,12h or 24h of reoxygenation,the viability of Tet-induced293-TRPM7/WT or 293-TRPM7/△Kin cells was significantly lower than that ofnon-Tet-induced 293-TRPM7/WT or 293-TRPM7/△Kin cells at each time point(P＜0.01).At 12h after reoxygenation,the viability of Tet-induced 293-TRPM7/WTcells was significantly lower than that of Tet-induced 293-TRPM7/△Kin cells (P＜0.05).The difference of cell viability between Tet-induced 293-TRPM7/WT cells and293-TRPM7/△Kin cells was more significant at 24h after reoxygenation (P＜0.01).
2) Under normal condition,the apoptotic rate of Tet-induced,non-Tet-induced293-TRPM7/WT and 293-TRPM7/△Kin cells were 1.4%±0.16%,1.5%±0.12%,1.4%±0.13%,1.4%±0.15%,respectively,and there was no statistic difference among them.However,after 1h of OGD and 24h of reoxygenation,the apoptotic rate of Tet-induced293-TRPM7/WT cells (34.1%±1.22%) was significantly higher than that ofnon-Tet-induced 293-TRPM7/WT cells (10.2%±1.05%,P＜0.05) and Tet-induced293-TRPM7/△Kin cells (13.5%±1.01%,P＜0.05).
2.OGD/R promoted the association between TRPM7 and Annexin-1 in acalcium-independent and TRPM7 kinase domain-dependent manner.
1) Double immunofluorescence staining assay showed colocalization ofTRPM7/WT protein with Annexin-1 in Tet-induced 293-TRPM7/WT cells.TRPM7/WT protein was mainly located at plasma membrane,while Annexin-1 wasevenly distributed throughout the cell.Colocalization of TRPM7/WT with Annexin-1was on plasma membrane.
2) Immunoprecipitation assay showed that under normal condition,a fewassociation of Annexin-1 with TRPM7/WT was detected in Tet-induced293-TRPM7/WT cells.After 1h of OGD,the association between Annexin-1 andTRPM7/WT was gradually increased with the increasing duration of reoxygenation.After 1 h of OGD and 24h of reoxygenation,chelating intracellular or extracellular Ca~(2+), using BAPTA-AM or EDTA,did not abrogate the association of Annexin-1 withTRPM7/WT in Tet-induced 293-TRPM7/WT cells,and no association betweenAnnexin-1 and TRPM7/△Kin was detected in Tet-induced 293-TRPM7/△Kin cells.
3.OGD/R induced TRPM7 kinase domain-dependent high expression ofAnnexin-1 and translocation of Annexin-1 to nucleus and cell membrane in HEK293cells.
1) After lh of OGD,the expression of Annexin-1 in Tet-induced293-TRPM7/WT cells was gradually upregulated during reoxygenation,which startedat 6h and reached a peak at 24h.After lh of OGD and 24h of reoxygenation,theexpression level of Annexin-1 in Tet-induced 293-TRPM7/△Kin cells was no statisticdifference from that in non-Tet-induced 293-TRPM7/△Kin cells and non-Tet-induced293-TRPM7/WT cells,but it was significantly lower than that in Tet-induced293-TRPM7/WT cells (P＜0.05).In addition,Tet-induced 293-TRPM7/WT cellsexpressed higher level of Annexin-1 protein than non-Tet-induced 293-TRPM7/WTcells at the time points of 6h,12h and 24h after reoxygenation (P＜0.05).
2) Immunofluorescence staining assay showed that Annexin-1 in Tet-induced293-TRPM7/WT cells was translocated to cell membrane and nucleus after 1h of OGDand 24h of reoxygenation,whereas Annexin-1 in Tet-induced 293-TRPM7/△Kin ornon-Tet-induced 293-TRPM7/WT cells did not.Expression of Annexin-1 in cellmembrane,cytoplasm and nucleus fractions was further measured by immunoblot.Itshowed that after 1h of OGD and 24h of reoxygenation,expression level of Annexin-1in cytosolic fraction in Tet-induced 293-TRPM7/WT cells was lower than normal,however,expression level of Annexin-1 in membrane,especially in nuclear fraction wasobviously higher than normal.On the other hand,Annexin-1 expression level in cellmembrane or nuclear fraction in non-Tet-induced 293-TRPM7/WT cells was nodifference from normal,while Annexin-1 expression level in cytosolic fraction washigher than normal.
4.Annexin-1 was involved in OGD/R-induced,TRPM7-mediated cell damage and apoptosis of HEK293 cells.
1) After 1h of OGD and 24h of reoxygenation,Annexin-1 antisenseoligodeoxynucleotides (ASODN) significantly downregulated OGD/R-induced highexpression of Annexin-1 in Tet-induced 293-TRPM7/WT cells to a level similar tonormal (P＜0.05),and markedly increased cell viability (P＜0.01).However,Annexin-1sense oligodeoxynucleotides (SODN) affected neither Annexin-1 expression inTet-induced 293-TRPM7/WT cells nor cell viability.
2) After 1h of OGD and 24h of reoxygenation,Annexin-1 ASODN significantlyreduced OGD/R-induced high apoptotic rate of Tet-induced 293-TRPM7/WT cells by19.6%±1.01% (P＜0.05).However,Annexin-1 SODN did not affect the apoptotic rateof Tet-induced 293-TRPM7/WT cells,the percentage of apoptotic cells was 33.7%±1.56%.
In conclusion,in this study,we firstly verify that TRPM7,through its kinasedomain,is implicated in upregulating Annexin-1 expression and promoting Annexin-1translocation from cytoplasm to nucleus and cell membrane in response to OGD/Rtreatment,and serves to aggravate OGD/R-induced cell damage and apoptosis inHEK293 cells.These findings provide theoretical evidence for further investigating therole of TRPM7 kinase in ischemic reperfusion-induced neuronal cell apoptosis.
Part 2
NGF inhibits Gd~(3+)-sensitive calcium influx and reduces chemical anoxic neuronal death
The aim of this study was to investigate whether glutamate receptor channels andvoltage-gated calcium channels independent calcium influx arises in the process ofacute anoxic neuronal damage and its possible relationship to neuronal protectivefunction of NGF.
In in vitro model of acute anoxia,hippocampal cultures from newborn rats wereexposed to KCN.Changes of intracellular Ca~(2+) concentration ([Ca~(2+)]_i) were monitoredby laser scanning confocal microscope and cell viability was assayed by PI and cFDAstaining.
The results showed that:
1.After treatment with 3mM KCN for 15min,[Ca~(2+)]_i in hippocampal neuronswas significantly increased 6.27±0.05-fold compared with pre-anoxia level(P＜0.05) and 73.3%±12.1% of the cells died (P＜0.05).
2.When combination of 20μM MK-801 (glutamate receptor antagonist),40μMCNQX (AMPA receptor antagonist) and 5μM nimodipine (voltage-gated calciumchannel antagonist) (hereafter denoted as MCN) were administrated to hippocampalneurons,KCN-induced increase of [Ca~(2+)]_i and cell death were significantlyreduced (P＜0.05).Levels of [Ca~(2+)]_i was 4.02±0.04-fold of pre-anoxia and cell deathrate was 38.5%±4.4%,both of which were still markedly higher than normal (P＜0.05).
3.Gd~(3+) (10μM) completely blocked KCN-induced elevation of [Ca~(2+)]_i and furtherreduced cell death in the presence of MCN.Levels of [Ca~(2+)]_i was 1.2±0.03-fold ofpre-anoxia,which was no statistic difference from normal.Cell death rate was only8.2%±1.5%.
4.Similar to Gd~(3+),NGF (100ng/ml),used in combination with MCN,significantly inhibited KCN-induced increase of [Ca~(2+)]_i and cell death.Levels of [Ca~(2+)]_iwas 1.42±0.06-fold of pre-anoxia,which was also no statistic difference from normal. Only 9.1%±2.3% of the cells died.The effects of NGF were abolished by PLCinhibitor U73122 (10μM).
It is concluded that Gd~(3+)-sensitive calcium influx and cell death,which areglutamate receptor channels and voltage-gated calcium channels independent,arise inthe process of acute anoxic neuronal damage.NGF can inhibit Gd~(3+)-sensitive calciuminflux and reduce anoxic neuronal death through activating PLC pathway.
TRPM7介入氧糖剥夺／再氧合诱导细胞凋亡的机制研究
第一节
建立四环素调控性稳定表达全长、缺激酶区TRPM7的HEK293细胞系
TRPM7(transient receptorpotentialmelastatin 7)，也称ChaK1(Channel-Kinase1)，是一种独特的、既有非选择性阳离子通道又有蛋白激酶活性的双功能蛋白。本部分实验主要是建立四环素调控性稳定表达全长、缺激酶区TRPM7的HEK293细胞系，为进一步在细胞水平研究TRPM7及其激酶功能提供实验基础。
采用阳离子脂质体法将含小鼠TRPM7 cDNA的真核表达质粒pcDNA5／FRT／TO-HA-TRPM7／WT(表达全长TRPM7(TRPM7／WT))和pcDNA5／FRT／TO-HA-TRPM7／△Kin(表达缺激酶区TRPM7(TRPM7／△Kin))分别与Flp重组酶表达质粒pOG44共同转入Flp-In~(TM) T-REx~(TM) 293细胞，经过潮霉素筛选，收集抗药细胞克隆建立细胞系，分别命名为293-TRPM7／WT(转染pcDNA5／FRT／TO-HA-TRPM7／WT)和293-TRPM7／△Kin(转染pcDNA5／FRT／TO-HA-TRPM7／ΔKin)。采用免疫印迹和免疫荧光技术对细胞进行鉴定，结果显示：293-TRPM7／WT和293-TRPM7／△Kin细胞均在四环素诱导后才表达TRPM7蛋白(前者表达TRPM7／WT，后者表达TRPM7／△Kin)，且TRPM7蛋白表达水平均呈四环素浓度依赖性。综上所述，我们成功建立了四环素调控性稳定表达全长、缺激酶区TRPM7的HEK293细胞系。
第二节
TRPM7激酶区在氧糖剥夺／再氧合诱导细胞凋亡中的作用，及其与Annexin-1之间的关系
脑缺血后的神经元损伤是造成神经功能缺损的主要原因。最近的研究显示，TRPM7通道的活动与缺氧神经元的死亡密切相关，但是TRPM7激酶在缺血缺氧神经元损伤过程中作用尚未见报导。
本部分实验利用四环素调控性稳定表达全长和缺激酶区TRPM7的293-TRPM7／WT和293-TRPM7／△Kin细胞系，通过建立氧糖剥夺／再氧合(oxygen-glucose deprivation／reoxygenation，OGD／R)离体模型，即细胞在无糖无氧的环境中孵育1h(氧糖剥夺)后恢复正常培养一定时间(再氧合)，模拟在体缺血再灌注过程，从细胞水平初步探讨TRPM7激酶区在OGD／R引起的细胞损伤和细胞凋亡中的作用及其可能的机制。Annexin-1是TRPM7激酶区重要的底物蛋白，本研究通过观察Annexin-1的变化来讨论TRPM7激酶区的作用机制。
实验采用MTT法检测细胞活性，Annexin V-FITC、PI双染法结合流式细胞术检测、分析细胞凋亡率，应用免疫荧光和免疫印迹技术检测TRPM7、Annexin-1蛋白的分布和表达，利用免疫共沉淀法分析TRPM7与Annexin-1的相互作用。
结果显示：
1、TRPM7激酶区参与TRPM7促进OGD／R细胞损伤和细胞凋亡。
1)在正常情况下，四环素诱导和四环素非诱导的293-TRPM7／WT细胞和293-TRPM7／△Kin细胞活性之间没有显著性差异。经氧糖剥夺处理1h后，在再氧合Oh、6h、12h、24h过程中，四环素诱导293-TRPM7／WT细胞和293-TRPM7／△Kin细胞活性在每个时间点上都显著低于四环素非诱导293-TRPM7／WT细胞和293-TRPM7／△Kin细胞活性(P＜0．01)。而且从再氧合12h时开始，四环素诱导293-TRPM7／WT细胞活性明显低于四环素诱导293-TRPM7／△Kin细胞(P＜0．05)，到再氧合24h差异更加显著(P＜0．01)。
2)在正常情况下，四环素诱导、四环素非诱导的293-TRPM7／WT细胞和293-TRPM7／△Kin细胞凋亡率分别为：1．4％±0．16％、1．5％±0．12％、1．4％±0．13％、1．4％±0．15％，各组之间没有显著性差异；氧糖剥夺1h再氧合24h处理后，四环素诱导293-TRPM7／WT细胞凋亡率(34．1％±1．22％)显著高于四环素非诱导293-TRPM7／WT细胞(10．2％±1．05％，P＜0．05)和四环素诱导293-TRPM7／ΔKin细胞(13．5％±1．01％，P＜0．05)。
2、OGD／R过程中，TRPM7通过其激酶区增强与Annexin-1结合。
1)免疫荧光双标记发现，TRPM7／WT蛋白和Annexin-1共定位于四环素诱导293-TRPM7／WT细胞，TRPM7／WT蛋白主要分布在细胞膜上，Annexin-1分布在整个细胞中，两者在细胞内共存，共定位在细胞膜上。
2)免疫共沉淀发现，在正常情况下，四环素诱导293-TRPM7／WT细胞中有少量Annexin-1与TRPM7／WT结合；氧糖剥夺1h后，随着再氧合时间的延长，Annexin-1与TRPM7／WT的结合逐渐增多。再氧合24h时，用BAPTA-AM或EDTA螯合细胞内或细胞外钙离子不影响四环素诱导293-TRPM7／WT细胞中Annexin-1与TRPM7／WT的结合，而四环素诱导293-TRPM7／ΔKin细胞中未发现Annexin-1与TRPM7／ΔKin结合。
3、OGD／R诱导HEK293细胞出现TRPM7激酶区依赖的Annexin-1蛋白高表达和Annexin-1细胞核、细胞膜转位。
1)氧糖剥夺1h后，从再氧合6h开始，四环素诱导293-TRPM7／WT细胞中Annexin-1蛋白的表达水平随再氧合时间延长逐渐增加，到24h达到高峰，与氧糖剥夺1h再氧合24h后四环素非诱导293-TRPM7／WT、293-TRPM7／ΔKin细胞和四环素诱导293-TRPM7／ΔKin细胞相比，Annexin-1蛋白表达增加有显著性差异(P＜0．05)。而四环素非诱导293-TRPM7／WT、293-TRPM7／ΔKin细胞和四环素诱导293-TRPM7／ΔKin细胞之间比较无显著性差异。在再氧合6h、12h、24h过程中，四环素诱导293-TRPM7／WT细胞中Annexin-1的表达水平在各个时间点均显著高于四环素非诱导293-TRPM7／WT细胞(P＜0．05)。
2)氧糖剥夺1h再氧合24h时，免疫荧光结果显示：四环素诱导293-TRPM7／WT细胞中Annexin-1转位到细胞膜和细胞核，而四环素非诱导293-TRPM7／WT细胞和四环素诱导293-TRPM7／ΔKin细胞中Annexin-1未发生明显转位。进一步用免疫印迹分别检测胞膜、胞浆和胞核中Annexin-1的表达，发现：氧糖剥夺1h再氧合24h处理后，四环素诱导293-TRPM7／WT细胞胞浆中Annexin-1的表达水平比正常低，而胞膜特别是胞核中Annexin-1的表达水平明显高于正常；四环素非诱导293-TRPM7／WT细胞胞膜和胞核中Annexin-1的表达水平与正常相似，而胞浆中Annexin-1的表达水平高于正常。
4、Annexin-1参与TRPM7介导的OGD／R细胞损伤和细胞凋亡。
1)氧糖剥夺1h再氧合24h时，Annexin-1反义脱氧寡核苷酸(ASODN)显著下调四环素诱导293-TRPM7／WT细胞中Annexin-1的高表达至正常水平(P＜0．05)，同时显著增加细胞活性(P＜0．01)，而Annexin-1正义脱氧寡核苷酸(SODN)对Annexin-1的表达和细胞活性均没有影响。
2)氧糖剥夺1h再氧合24h时，Annexin-1 ASOND显著减少四环素诱导293-TRPM7／WT细胞凋亡率19．6％±1．01％(P＜0．05)；Annexin-1 SODN对细胞凋亡率没有影响，仍有高达33．7％±1．56％细胞凋亡。
综上所述，本实验首次验证了：TRPM7通过其激酶区上调Annexin-1的表达，促进Annexin-1的核、膜转位，加剧OGD／R引起的细胞损伤和细胞凋亡。本研究的发现为进一步研究TRPM7激酶介导脑缺血再灌注神经元凋亡提供了实验依据。
第二部分
NGF抑制Gd~(3+)敏感性钙内流，减少化学缺氧神经元死亡
本部分实验主要探讨急性缺氧神经元损伤过程中是否存在非谷氨酸受体通道和非电压门控钙通道依赖性的钙内流，及其与NGF神经元保护作用之间的关系。
以KCN作为诱导剂，在体外培养的新生大鼠海马神经元上建立急性缺氧的细胞模型，应用激光扫描共聚焦显微镜监测细胞内钙离子浓度变化，利用cFDA与PI染色法评价细胞的存活和死亡率。
结果显示：
1、给予原代培养的海马神经元KCN(3mM)处理15min后，细胞内钙离子浓度([Ca~(2+)]i)显著升高至缺氧前6．27±0．05倍(p＜0．05)，73．3％±12．1％细胞死亡(P＜0．05)。
2、20μM MK-801(NMDA受体拮抗剂)、40μM CNQX(AMPA受体拮抗剂)和5μM Nimodipine(电压门控钙通道拮抗剂)联用(简称MCN)显著降低KCN引起的[Ca~(2+)]_i升高和细胞死亡率(P＜0．05)，但[Ca~(2+)]_i为缺氧前4．02±0．04倍，仍有38．5％±4．4％细胞死亡，均显著高于正常(P＜0．05)。
3、Gd~(3+)(10μM)与MCN联用后几乎完全阻断KCN引起的[Ca~(2+)]_i升高，并进一步降低细胞死亡率。[Ca~(2+)]_i仅为缺氧前1．2±0．03倍，与正常相比差异无显著性，神经元死亡率降至8．2％±1．5％。
4、与Gd~(3+)的作用相似，NGF(100ng／m1)与MCN联用后显著减弱KCN引起的[Ca~(2+)]_i升高，[Ca~(2+)]_i仅为缺氧前1．42±0．06倍，与正常相比无统计学差异，只有9．1％±2．3％细胞死亡。NGF的作用可被磷脂酶C(PLC)拮抗剂U73122(10μM)逆转。
以上结果表明，在急性缺氧神经元损伤过程中存在非谷氨酸受体通道和非电压门控钙通道依赖性的Gd~(3+)敏感性钙内流和神经元死亡；NGF可通过激活PLC通路抑制Gd~(3+)敏感性钙内流，同时减少缺氧神经元死亡。
Part 1
Mechanism study for implication of TRPM7 in oxygen-glucosedeprivation/reoxygenation-induced apoptosis
Segment 1
Establishment of stable HEK293 cell lines with tetracycline-inducible expression offull-length or kinase domain truncated TRPM7
Transient receptor potential melastatin 7 (TRPM7),also named asChannel-Kinasel (ChaK1),is a unique bifunctional protein consisting of a proteinkinase domain fused to a non-selective cation channel.The aim of this study was toestablish stable HEK293 cell lines with tetracycline-inducible expression of full-lengthor kinase domain truncated TRPM7,so that we can investigate the functions of TRPM7and its kinase at cellular level.
Eukaryotic expression plasmid pcDNA5/FRT/TO-HA-TRPM7/WT (expressingfull-length TRPM7 (TRPM7/WT)) or pcDNA5/FRT/TO-HA-TRPM7/△Kin(expressing kinase domain truncated TRPM7 (TRPM7/△Kin)) was cotransfected withthe Flp recombinase expression plasmid pOG44 into Flp-In~(TM) T-REx~(TM) 293 cells usingLipofectamine 2000~(TM).After selection with hygromycin,cell clones were pooled anddefined as 293-TRPM7/WT (transfected with pcDNA5/FRT/TO-HA-TRPM7/WT) and293-TRPM7/△Kin (transfected with pcDNA5/FRT/TO-HA-TRPM7/△Kin),respectively.The tetracycline-inducible expression of TRPM7/WT in 293- TRPM7/WTcells or TRPM7/△Kin in 293-TRPM7/△Kin cells was confirmed by western blot andimmunofluorescence analysis.The results showed that TRPM7/WT or TRPM7/△Kinexpression was switched on by application of tetracycline in a concentration dependent manner.In conclusion,we successfully established tetracycline-inducible HEK293stable cell lines expressing full-length or kinase domain truncated TRPM7.
Segment 2
The role of TRPM7 kinase domain in oxygen-glucosedeprivation/reoxygenation-induced apoptosis and the relationship betweenTRPM7 and Annexin-1
Neuronal death after cerebral ischemia is the main cause of neurologicimpairment.It is recently revealed that the activity of TRPM7 channel is closely relatedto anoxic neuronal death,however,the exact function of TRPM7 kinase in ischemicneuronal injury is unknown.
In this study,by using stable HEK293 cell lines with tetracycline (Tet)-inducibleexpression of full-length and kinase domain truncated TRPM7 (293-TRPM7/WT cellsand 293-TRPM7/△Kin cells) and establishing in vitro model of oxygen-glucosedeprivation followed by reoxygenation (OGD/R) mimicking in vivo ischemicreperfusion process,we investigated the role of TRPM7 kinase domain inOGD/R-induced cell damage and cell apoptosis and the possible mechanisms.SinceAnnexin-1 is an important substrate for TRPM7 kinase,changes of Annexin-1 wasobserved in this study to explored the mechanism of action of TRPM7 kinase domain.
Cell viability was determined by MTT assay.Apoptosis was quantitativelyanalyzed by Annexin V-FITC and PI double-staining followed by flow cytometry.Immunofluorescence and western blot were used to detect the localization andexpression of TRPM7 and Annexin-1,respectively.Interaction between TRPM7 andAnnexin-1 was measured by immunoprecipitation.
The results showed that:
1.TRPM7 kinase domain was implicated in OGD/R-induced,TRPM7-mediatedcell damage and apoptosis of HEK293 cells.
1) Under normal condition,cell viability among Tet-induced,non-Tet-induced293-TRPM7/WT and 293-TRPM7/△Kin cells were no statistic difference.After 1h ofOGD and 0h,6h,12h or 24h of reoxygenation,the viability of Tet-induced293-TRPM7/WT or 293-TRPM7/△Kin cells was significantly lower than that ofnon-Tet-induced 293-TRPM7/WT or 293-TRPM7/△Kin cells at each time point(P＜0.01).At 12h after reoxygenation,the viability of Tet-induced 293-TRPM7/WTcells was significantly lower than that of Tet-induced 293-TRPM7/△Kin cells (P＜0.05).The difference of cell viability between Tet-induced 293-TRPM7/WT cells and293-TRPM7/△Kin cells was more significant at 24h after reoxygenation (P＜0.01).
2) Under normal condition,the apoptotic rate of Tet-induced,non-Tet-induced293-TRPM7/WT and 293-TRPM7/△Kin cells were 1.4%±0.16%,1.5%±0.12%,1.4%±0.13%,1.4%±0.15%,respectively,and there was no statistic difference among them.However,after 1h of OGD and 24h of reoxygenation,the apoptotic rate of Tet-induced293-TRPM7/WT cells (34.1%±1.22%) was significantly higher than that ofnon-Tet-induced 293-TRPM7/WT cells (10.2%±1.05%,P＜0.05) and Tet-induced293-TRPM7/△Kin cells (13.5%±1.01%,P＜0.05).
2.OGD/R promoted the association between TRPM7 and Annexin-1 in acalcium-independent and TRPM7 kinase domain-dependent manner.
1) Double immunofluorescence staining assay showed colocalization ofTRPM7/WT protein with Annexin-1 in Tet-induced 293-TRPM7/WT cells.TRPM7/WT protein was mainly located at plasma membrane,while Annexin-1 wasevenly distributed throughout the cell.Colocalization of TRPM7/WT with Annexin-1was on plasma membrane.
2) Immunoprecipitation assay showed that under normal condition,a fewassociation of Annexin-1 with TRPM7/WT was detected in Tet-induced293-TRPM7/WT cells.After 1h of OGD,the association between Annexin-1 andTRPM7/WT was gradually increased with the increasing duration of reoxygenation.After 1 h of OGD and 24h of reoxygenation,chelating intracellular or extracellular Ca~(2+), using BAPTA-AM or EDTA,did not abrogate the association of Annexin-1 withTRPM7/WT in Tet-induced 293-TRPM7/WT cells,and no association betweenAnnexin-1 and TRPM7/△Kin was detected in Tet-induced 293-TRPM7/△Kin cells.
3.OGD/R induced TRPM7 kinase domain-dependent high expression ofAnnexin-1 and translocation of Annexin-1 to nucleus and cell membrane in HEK293cells.
1) After lh of OGD,the expression of Annexin-1 in Tet-induced293-TRPM7/WT cells was gradually upregulated during reoxygenation,which startedat 6h and reached a peak at 24h.After lh of OGD and 24h of reoxygenation,theexpression level of Annexin-1 in Tet-induced 293-TRPM7/△Kin cells was no statisticdifference from that in non-Tet-induced 293-TRPM7/△Kin cells and non-Tet-induced293-TRPM7/WT cells,but it was significantly lower than that in Tet-induced293-TRPM7/WT cells (P＜0.05).In addition,Tet-induced 293-TRPM7/WT cellsexpressed higher level of Annexin-1 protein than non-Tet-induced 293-TRPM7/WTcells at the time points of 6h,12h and 24h after reoxygenation (P＜0.05).
2) Immunofluorescence staining assay showed that Annexin-1 in Tet-induced293-TRPM7/WT cells was translocated to cell membrane and nucleus after 1h of OGDand 24h of reoxygenation,whereas Annexin-1 in Tet-induced 293-TRPM7/△Kin ornon-Tet-induced 293-TRPM7/WT cells did not.Expression of Annexin-1 in cellmembrane,cytoplasm and nucleus fractions was further measured by immunoblot.Itshowed that after 1h of OGD and 24h of reoxygenation,expression level of Annexin-1in cytosolic fraction in Tet-induced 293-TRPM7/WT cells was lower than normal,however,expression level of Annexin-1 in membrane,especially in nuclear fraction wasobviously higher than normal.On the other hand,Annexin-1 expression level in cellmembrane or nuclear fraction in non-Tet-induced 293-TRPM7/WT cells was nodifference from normal,while Annexin-1 expression level in cytosolic fraction washigher than normal.
4.Annexin-1 was involved in OGD/R-induced,TRPM7-mediated cell damage and apoptosis of HEK293 cells.
1) After 1h of OGD and 24h of reoxygenation,Annexin-1 antisenseoligodeoxynucleotides (ASODN) significantly downregulated OGD/R-induced highexpression of Annexin-1 in Tet-induced 293-TRPM7/WT cells to a level similar tonormal (P＜0.05),and markedly increased cell viability (P＜0.01).However,Annexin-1sense oligodeoxynucleotides (SODN) affected neither Annexin-1 expression inTet-induced 293-TRPM7/WT cells nor cell viability.
2) After 1h of OGD and 24h of reoxygenation,Annexin-1 ASODN significantlyreduced OGD/R-induced high apoptotic rate of Tet-induced 293-TRPM7/WT cells by19.6%±1.01% (P＜0.05).However,Annexin-1 SODN did not affect the apoptotic rateof Tet-induced 293-TRPM7/WT cells,the percentage of apoptotic cells was 33.7%±1.56%.
In conclusion,in this study,we firstly verify that TRPM7,through its kinasedomain,is implicated in upregulating Annexin-1 expression and promoting Annexin-1translocation from cytoplasm to nucleus and cell membrane in response to OGD/Rtreatment,and serves to aggravate OGD/R-induced cell damage and apoptosis inHEK293 cells.These findings provide theoretical evidence for further investigating therole of TRPM7 kinase in ischemic reperfusion-induced neuronal cell apoptosis.
Part 2
NGF inhibits Gd~(3+)-sensitive calcium influx and reduces chemical anoxic neuronal death
The aim of this study was to investigate whether glutamate receptor channels andvoltage-gated calcium channels independent calcium influx arises in the process ofacute anoxic neuronal damage and its possible relationship to neuronal protectivefunction of NGF.
In in vitro model of acute anoxia,hippocampal cultures from newborn rats wereexposed to KCN.Changes of intracellular Ca~(2+) concentration ([Ca~(2+)]_i) were monitoredby laser scanning confocal microscope and cell viability was assayed by PI and cFDAstaining.
The results showed that:
1.After treatment with 3mM KCN for 15min,[Ca~(2+)]_i in hippocampal neuronswas significantly increased 6.27±0.05-fold compared with pre-anoxia level(P＜0.05) and 73.3%±12.1% of the cells died (P＜0.05).
2.When combination of 20μM MK-801 (glutamate receptor antagonist),40μMCNQX (AMPA receptor antagonist) and 5μM nimodipine (voltage-gated calciumchannel antagonist) (hereafter denoted as MCN) were administrated to hippocampalneurons,KCN-induced increase of [Ca~(2+)]_i and cell death were significantlyreduced (P＜0.05).Levels of [Ca~(2+)]_i was 4.02±0.04-fold of pre-anoxia and cell deathrate was 38.5%±4.4%,both of which were still markedly higher than normal (P＜0.05).
3.Gd~(3+) (10μM) completely blocked KCN-induced elevation of [Ca~(2+)]_i and furtherreduced cell death in the presence of MCN.Levels of [Ca~(2+)]_i was 1.2±0.03-fold ofpre-anoxia,which was no statistic difference from normal.Cell death rate was only8.2%±1.5%.
4.Similar to Gd~(3+),NGF (100ng/ml),used in combination with MCN,significantly inhibited KCN-induced increase of [Ca~(2+)]_i and cell death.Levels of [Ca~(2+)]_iwas 1.42±0.06-fold of pre-anoxia,which was also no statistic difference from normal. Only 9.1%±2.3% of the cells died.The effects of NGF were abolished by PLCinhibitor U73122 (10μM).
It is concluded that Gd~(3+)-sensitive calcium influx and cell death,which areglutamate receptor channels and voltage-gated calcium channels independent,arise inthe process of acute anoxic neuronal damage.NGF can inhibit Gd~(3+)-sensitive calciuminflux and reduce anoxic neuronal death through activating PLC pathway.
引文
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