本文選題：瞬時(shí)感受器電位香草素受體亞家族Ⅳ型(TRPV4) + 腦缺血再灌注損傷��； 參考：《南京醫(yī)科大學(xué)》2014年博士論文

【摘要】：隨著世界人口進(jìn)入老齡化的速度加快,腦卒中(缺血性腦血管病)的發(fā)病率逐年升高,現(xiàn)已成為威脅人類(lèi)生命的最主要疾病之一。因腦卒中具有發(fā)病率高、致殘率高和死亡率高的特點(diǎn),給個(gè)人、家庭和社會(huì)帶來(lái)巨大的精神壓力和經(jīng)濟(jì)負(fù)擔(dān)。雖然盡早溶栓治療通過(guò)恢復(fù)腦血氧供應(yīng)能保護(hù)神經(jīng)元,但是缺血后再灌注往往也會(huì)加重腦組織的損傷(再灌注損傷),導(dǎo)致神經(jīng)元壞死。因此,研究缺血誘導(dǎo)腦損傷的病理生理機(jī)制,尋找有效的治療靶點(diǎn),對(duì)提高腦卒中患者的治愈率具有重要意義。 缺血再灌注誘導(dǎo)腦損傷主要與谷氨酸興奮性毒作用、胞內(nèi)鈣(intracellular Ca2+concentration,[Ca2+]i)超載、自由基與一氧化氮的損傷、腦水腫等病理機(jī)制有關(guān)。其中,谷氨酸興奮性毒作用被認(rèn)為是腦缺血誘導(dǎo)神經(jīng)元死亡的重要機(jī)制。然而,臨床資料顯示阻斷谷氨酸受體對(duì)腦卒中并未能取得理想的療效。因此,探究非谷氨酸依賴(lài)性鈣離子超載誘導(dǎo)的神經(jīng)元損傷機(jī)制將非常有意義。 瞬時(shí)感受器電位(transient receptor potential, TRP)受體家族是一類(lèi)重要的非選擇性陽(yáng)離子通道。瞬時(shí)感受器電位香草素受體亞家族Ⅳ型(TRP vanilloid4, TRPV4)在中樞神經(jīng)系統(tǒng)廣泛分布,主要表達(dá)在海馬、大腦皮層、丘腦、小腦等腦區(qū)。TRPV4受體作為一種以鈣離子為主的陽(yáng)離子通道,被激活后能引起以Ca2+為主的內(nèi)向電流。近年來(lái),越來(lái)越多的研究報(bào)道了TRP受體家族成員參與腦缺血再灌注損傷的病理過(guò)程。TRP家族中的nelastati家族Ⅱ型、Ⅶ型和canonical家族Ⅵ型等成員已被報(bào)道介導(dǎo)了腦缺血引起的神經(jīng)元死亡。TRPV4受體自發(fā)現(xiàn)以來(lái),因其可被低滲、溫?zé)�、機(jī)械、花生四烯酸及其代謝物等多種刺激激活而越來(lái)越受到人們關(guān)注。腦缺血時(shí),由于能量代謝障礙所引起的細(xì)胞水腫可以通過(guò)改變細(xì)胞膜機(jī)械張力激活TRPV4受體；能量代謝障礙所產(chǎn)生的大量花生四烯酸也可以通過(guò)其代謝產(chǎn)物5,6-環(huán)氧二十碳三烯甘油酸等激活TRPV4受體。提示缺血再灌注引起腦損傷的病理過(guò)程有可能與TRPV4受體的激活有關(guān)。阻斷TRPV4受體可以減輕氧化應(yīng)激對(duì)海馬星形膠質(zhì)細(xì)胞的損傷,對(duì)氧糖剝奪導(dǎo)致的海馬CA1神經(jīng)元損傷也具有保護(hù)作用,提示阻斷TRPV4受體有可能減輕缺血導(dǎo)致的腦損傷。 我們前期在初級(jí)感覺(jué)神經(jīng)元上的研究發(fā)現(xiàn),TRPV4受體激活后通過(guò)影響其下游的信號(hào)通路(PKA、PKC、PKG等)調(diào)節(jié)細(xì)胞膜上的電壓依賴(lài)性離子通道和TRPV1受體的功能,提高神經(jīng)元的興奮性。在視網(wǎng)膜神經(jīng)節(jié)細(xì)胞上的研究發(fā)現(xiàn)TRPV4受體激動(dòng)劑能夠劑量依賴(lài)性增加[Ca2+]i,提高細(xì)胞的興奮性,誘導(dǎo)細(xì)胞凋亡。此外,激活TRPV4受體能夠增加培養(yǎng)的海馬神經(jīng)元之間的微小興奮性突觸后電流的頻率,提示腦缺血再灌注通過(guò)激活TRPV4受體有可能促進(jìn)谷氨酸的釋放。 因此,本課題將通過(guò)在體實(shí)驗(yàn)首先明確TRPV4受體是否參與腦缺血再灌注損傷的病理過(guò)程,然后重點(diǎn)研究阻斷TRPV4受體對(duì)腦缺血再灌注損傷是否有保護(hù)作用及其分子機(jī)制,為今后預(yù)防和治療腦缺血再灌注損傷提供新的靶點(diǎn)和思路。 研究目的 1.明確TRPV4受體是否參與腦缺血再灌注誘導(dǎo)的損傷； 2.闡明阻斷TRPV4受體保護(hù)腦缺血再灌注損傷的分子機(jī)制。 第一部分TRPV4受體在腦缺血再灌注損傷中的作用 材料與方法 1.腦缺血再灌注小鼠模型制備：用魚(yú)線(xiàn)阻塞右側(cè)大腦中動(dòng)脈60min,制備大腦中動(dòng)脈栓塞(middle cerebral artery occlusion, MCAO)再灌注小鼠模型。 2.實(shí)時(shí)定量RT-PCR和Western blot:MCAO再灌注后不同時(shí)間點(diǎn)取出海馬組 織提取mRNA和蛋白,檢測(cè)TRPV4受體的nRNA和蛋白水平。 3.TTC染色：MCAO再灌注24h的腦組織,冠狀切片后置TTC溶液中反應(yīng),正常腦組織呈紅色而梗死組織呈白色,白色腦組織體積即為MCAO再灌注后梗死體積。 4.甲苯胺藍(lán)染色：腦組織用多聚甲醛固定后石蠟包埋切片,用甲苯胺藍(lán)染色,進(jìn)行海馬CA1錐體神經(jīng)細(xì)胞的計(jì)數(shù)。 結(jié)果 1. MCAO再灌注后2-72hTRPV4受體的mRNA的表達(dá)增加；MCAO再灌注4-48hTRPV4受體蛋白水平增加,MCAO再灌注后18hTRPV4受體蛋白水平達(dá)到峰值,之后逐漸下降,MCAO再灌注后72h恢復(fù)到正常水平。 2.側(cè)腦室給予TRPV4受體特異性阻斷劑HC-067047,在0.1-30μM/2μL/只的劑量范圍內(nèi),能夠濃度依賴(lài)性地減小MCAO再灌注后24h腦梗死的體積；HC-067047(10μM/2μL/只)對(duì)腦缺血再灌注損傷神經(jīng)保護(hù)作用的有效時(shí)間窗長(zhǎng)達(dá)12h。側(cè)腦室給予TRPV4受體非特異性阻斷劑釕紅也能夠有效減小MCAO再灌注后24h腦梗死的體積。 3.與對(duì)照組小鼠相比,側(cè)腦室注射TRPV4受體激動(dòng)劑4a-PDD能夠引起海馬CA1神經(jīng)元的死亡。 結(jié)論 腦缺血再灌注后4-48h海馬TRPV4受體表達(dá)明顯增加。阻斷TRPV4受體對(duì)腦缺血再灌注損傷有保護(hù)作用。 第二部分阻斷TRPV4受體保護(hù)腦缺血再灌注損傷的分子機(jī)制研究 材料與方法 1.腦缺血再灌注損傷的細(xì)胞模型建立：腦缺血時(shí),由于能量代謝障礙會(huì)引起腦水腫。我們的前期研究已證明,細(xì)胞水腫可以激活TRPV4受體。為了研究腦缺血后細(xì)胞水腫激活TRPV4受體導(dǎo)致細(xì)胞死亡的機(jī)制,本研究采用將細(xì)胞外液滲透壓降低到240mOsm/kg(低滲刺激)模擬腦水腫細(xì)胞模型。 2.場(chǎng)電位記錄：制備海馬冠狀切片,檢測(cè)海馬Schaffer支-CA1突觸的興奮性突觸后電位(excitatory post-synaptic potentiation, EPSP)和雙脈沖易化(paired-pulse facilitation, PPF)。 3.全細(xì)胞膜片鉗記錄：制備海馬冠狀切片,檢測(cè)腦片海馬CA1錐體神經(jīng)元微小興奮性突觸后電流(miniature excitatory postsynaptic current, mEPSC)、NMDA受體介導(dǎo)的電流(NMDA-indcued current,INMDA)和海馬突觸前錐體神經(jīng)元高電壓激活鈣電流(high voltage-gated calcium current,Ica)。 4.甲苯胺藍(lán)染色：同第一部分。 結(jié)果 1.場(chǎng)電位記錄結(jié)果顯示,TRPV4受體激動(dòng)齊4α-PDD進(jìn)行海馬腦片灌流能引起海馬Schaffer側(cè)支-CA1突觸EPSP的斜率增加伴有PPF的比值降低,提示TRPV4受體活化能增加突觸前神經(jīng)遞質(zhì)的釋放。當(dāng)降低海馬腦片細(xì)胞外灌流液的滲透壓到240mOsm/kg(低滲刺激),海馬Schaffer側(cè)支-CA1突觸EPSP斜率顯著增加,而PPF的比值降低。TRPV4受體阻斷劑(HC-067047)能夠阻斷低滲突觸前神經(jīng)遞質(zhì)的釋放。 2.全細(xì)胞膜片鉗記錄結(jié)果顯示,低滲增加海馬腦片CA1錐體細(xì)胞mEPSC的頻率和幅度,提示低滲刺激不僅能增加突觸前神經(jīng)遞質(zhì)的釋放,還能增加突觸后膜谷氨酸受體的活性。 3.低滲刺激對(duì)海馬CA1突觸前錐體神經(jīng)元的電壓門(mén)控性鈣電流(Ica)的幅度、電流—電壓曲線(xiàn)、激活曲線(xiàn)和失活曲線(xiàn)無(wú)明顯作用。給予N型或P/Q型電壓門(mén)控性鈣通道阻斷劑不能影響低滲刺激增加EPSP斜率。 4. TRPV4受體激動(dòng)劑4a-PDD能增加海馬CA1錐體神經(jīng)元INMDA的幅度。同樣低滲刺激也能增加海馬CA1錐體神經(jīng)元INMDA的幅度。低滲刺激增加TMDA受體活性的作用能完全被TRPV4受體阻斷劑HC-067047所阻斷,提示低滲刺激通過(guò)激活TRPV4受體能增強(qiáng)谷氨酸NMDA受體功能活性。 5.與]TRPV4受體激動(dòng)劑4α-PDD的作用相同,低滲刺激能夠增加INMDA量效曲線(xiàn)中的最大反應(yīng)能力,但對(duì)ECso值無(wú)明顯影響,并對(duì)INMDA電流的I-V曲線(xiàn)無(wú)明顯作用,提示低滲刺激激活TRPV4受體能增強(qiáng)谷氨酸INMDA受體離子通道的開(kāi)放。 6.NR2B亞基特異性阻斷劑ifenprodil能有效阻斷低滲刺激對(duì)INMDA的增強(qiáng)作用,而NR2A亞基阻斷劑NVP-AAM007對(duì)低滲刺激的作用無(wú)明顯影響。CaMKII的阻斷劑能夠阻斷低滲刺激對(duì)INMDA的增強(qiáng)作用。PKC和CKII的阻斷劑對(duì)低滲刺激的作用無(wú)影響。 7. NMDA受體阻斷劑MK801能減輕TRPV4受體過(guò)激活誘導(dǎo)海馬CA1神經(jīng)元的損傷。 結(jié)論 缺血再灌注后TRPV4受體過(guò)表達(dá)和腦水腫增強(qiáng)TRPV4受體活性能增加TRPV4受體的鈣離子內(nèi)流,以促進(jìn)谷氨酸的釋放,同時(shí)激活CaMKII通過(guò)提高NR2B亞基磷酸化水平增加NMDA受體的鈣內(nèi)流。結(jié)果提示缺血再灌注后TRPV4受體功能增加可以引起鈣超載,激活細(xì)胞凋亡信號(hào)通路,進(jìn)而造成神經(jīng)元死亡。因此,本研究提出阻斷TRPV4受體對(duì)腦缺血再灌注損傷有保護(hù)作用。
[Abstract]:With the rapid growth of the population of the world, the incidence of cerebral apoplexy (ischemic cerebrovascular disease) is increasing year by year. It has become one of the most important diseases that threaten human life. Because of the high incidence of stroke, high disability rate and high mortality, stroke has brought great mental pressure and economic burden to individuals, families and society. Although thrombolytic therapy can protect neurons by restoring cerebral oxygen supply as early as possible, reperfusion after ischemia may also aggravate the injury of brain tissue (reperfusion injury) and lead to necrosis of neurons. Therefore, it is important to study the pathophysiological mechanism of cerebral ischemia induced brain injury and find effective therapeutic targets for the improvement of the cure rate of stroke patients. It's meaning.
Cerebral injury induced by ischemia-reperfusion is mainly related to excitatory toxicity of glutamic acid, intracellular calcium (intracellular Ca2+concentration, [Ca2+]i) overload, damage of free radicals and nitric oxide, brain edema and other pathological mechanisms. It is important to explore the mechanism of neuronal damage induced by non glutamic acid dependent calcium overload.
The transient receptor potential (TRP) receptor family is an important class of non selective cation channels. The transient receptor potential vanillin receptor subfamily (TRP vanilloid4, TRPV4) is widely distributed in the central nervous system, mainly expressed as.TRPV4 receptors in the hippos, cerebral cortex, thalamus, cerebellum and other brain regions. A cation channel based on calcium ions, which is activated, can cause Ca2+ - based introversion current. In recent years, more and more studies have reported that the members of the TRP receptor family involved in the pathological process of cerebral ischemia reperfusion injury, the nelastati family type II in the.TRP family, type VII and canonical family VI have been reported to have been mediated The neuron death.TRPV4 receptor caused by cerebral ischemia has been discovered since it has been activated by a variety of stimuli, such as hypotonic, warm, mechanical, peanut four enoic acid and its metabolites. A large number of arachidic acid produced by metabolic disorders can also activate the TRPV4 receptor through its metabolite 5,6- epoxy twenty carbon three glycic acid. It is suggested that the pathological process of cerebral injury induced by ischemia-reperfusion may be related to the activation of TRPV4 receptor. Blocking the TRPV4 receptor can reduce the impairment of oxidative stress on astrocytes in the hippocampus. Injury also has protective effects on hippocampal CA1 neurons damage induced by oxygen glucose deprivation, suggesting that blocking TRPV4 receptor may alleviate ischemic brain damage.
Our previous study on primary sensory neurons found that the activation of the TRPV4 receptor was enhanced by regulating the voltage dependent ion channels and the function of the TRPV1 receptor on the cell membrane by affecting the downstream signal pathways (PKA, PKC, PKG, etc.). A study on the retinal deity ganglion cells found that TRPV4 receptor agonists could be used. Dose dependence increases [Ca2+]i, increases cell excitability and induces apoptosis. In addition, activation of TRPV4 receptors can increase the frequency of small excitatory postsynaptic currents between cultured hippocampal neurons, suggesting that cerebral ischemia-reperfusion may promote the release of glutamic acid by activating the TRPV4 receptor.
Therefore, the subject will first clarify whether the TRPV4 receptor is involved in the pathological process of cerebral ischemia reperfusion injury in vivo, and then focus on the protection of TRPV4 receptor on cerebral ischemia reperfusion injury and its molecular mechanism, and provide new targets and ideas for the prevention and treatment of cerebral ischemia reperfusion injury in the future.
research objective
1. whether TRPV4 receptor is involved in the injury induced by cerebral ischemia-reperfusion.
2. elucidate the molecular mechanism of blocking TRPV4 receptor in protecting cerebral ischemia-reperfusion injury.
Part 1 the role of TRPV4 receptor in cerebral ischemia-reperfusion injury
Materials and methods
1. mouse model of cerebral ischemia and reperfusion was made: the right middle cerebral artery 60min was blocked with fish line, and the middle cerebral artery occlusion (MCAO) was prepared and the mouse model was reperfused.
2. real-time quantitative RT-PCR and Western blot:MCAO were used to remove hippocampal formation at different time points after reperfusion.
MRNA and protein were extracted to detect the nRNA and protein levels of TRPV4 receptor.
3.TTC staining: MCAO reperfusion of the brain tissue of 24h and reacting in TTC solution after coronary slice, the normal brain tissue is red and the infarct tissue is white, and the volume of white brain tissue is the infarct volume after MCAO reperfusion.
4. toluidine blue staining: paraffin embedded sections of brain tissue were fixed with paraformaldehyde and stained with toluidine blue to carry out counting of hippocampal CA1 pyramidal neurons.
Result
The expression of mRNA in 2-72hTRPV4 receptor increased after 1. MCAO reperfusion, and the level of 4-48hTRPV4 receptor protein in MCAO reperfusion increased. After MCAO reperfusion, the protein level of 18hTRPV4 receptor reached the peak, then gradually decreased, and 72h recovered to normal level after MCAO reperfusion.
The 2. lateral ventricles were given TRPV4 receptor specific blocker HC-067047. Within the dose range of 0.1-30 mu M/2 L/, the volume of 24h cerebral infarction after MCAO reperfusion was reduced in a concentration dependent manner; the effective time window of HC-067047 (10 u M/2 L/ only) on the neuroprotective effect of cerebral ischemia-reperfusion injury was not specific to the TRPV4 receptor. Ruthenium red, a sex blocker, can also effectively reduce the volume of 24h infarction after MCAO reperfusion.
3. compared with control mice, intracerebroventricular injection of TRPV4 receptor agonist 4a-PDD can cause the death of hippocampal CA1 neurons.
conclusion
The expression of TRPV4 receptor in hippocampus of 4-48h increased significantly after cerebral ischemia-reperfusion. Blocking TRPV4 receptor had protective effect on cerebral ischemia-reperfusion injury.
The second part is to block the molecular mechanism of TRPV4 receptor protecting cerebral ischemia-reperfusion injury.
Materials and methods
1. the cell model of cerebral ischemia reperfusion injury is established: cerebral ischemia can cause brain edema due to energy metabolism disorder. Our previous study has proved that cell edema can activate TRPV4 receptor. In order to study the mechanism of cell edema activated by TRPV4 receptor after cerebral ischemia, the osmotic pressure of extracellular fluid is reduced in this study. To 240mOsm/kg (hypotonic stimulation) simulated brain edema cell model.
2. field potentials recording: the hippocampal coronary slices were prepared to detect the excitatory postsynaptic potential (excitatory post-synaptic potentiation, EPSP) and double pulse facilitation (paired-pulse facilitation, PPF) of the hippocampal Schaffer branch -CA1 synapses.
3. whole cell patch clamp recording: preparation of hippocampal coronary slices to detect the micro excitatory postsynaptic current (miniature excitatory postsynaptic current, mEPSC) of hippocampal CA1 pyramidal neurons, NMDA receptor mediated current (NMDA-indcued current, INMDA) and high voltage activated calcium current (high voltage-gate) in the hippocampal synaptic pyramidal neurons (high voltage-gate). D calcium current, Ica).
4. toluidine blue staining: same as the first part.
Result
The results of 1. field potential recording showed that the perfusion of TRPV4 receptor excited Qi 4 alpha -PDD in hippocampal slices could cause the increase of the slope of -CA1 synapse EPSP in the lateral branch of the hippocampal Schaffer and the ratio of PPF, suggesting that the activation of TRPV4 receptor can increase the release of presynaptic neurotransmitters. When the osmotic pressure of the fine extracellular perfusion fluid of the hippocampus slices to 240mOsm/kg (low permeability) Stimulated), the slope of -CA1 synapse EPSP in the lateral branch of the hippocampal Schaffer increased significantly, while the ratio of PPF to the.TRPV4 receptor blocker (HC-067047) could block the release of the hypotonic neurotransmitter.
2. whole cell patch clamp recording results showed that low permeability increased the frequency and amplitude of mEPSC in hippocampal slice CA1 pyramidal cells, suggesting that hypotonic stimulation not only increased the release of presynaptic neurotransmitters, but also increased the activity of glutamate receptors in the postsynaptic membrane.
3. hypotonic stimulation has no obvious effect on the amplitude of voltage gated calcium current (Ica), current voltage curve, activation curve, and inactivation curve of hippocampal CA1 presynaptic pyramidal neurons. Giving N or P/Q type voltage gated calcium channel blockers can not affect low permeability stimulation to increase EPSP skew.
4. TRPV4 receptor agonist 4a-PDD can increase the amplitude of INMDA in hippocampal CA1 pyramidal neurons. The same hypotonic stimulation also increases the amplitude of INMDA in hippocampal CA1 pyramidal neurons. The effect of hypotonic stimulation on the activity of TMDA receptor can be completely blocked by the TRPV4 receptor blocker HC-067047, suggesting that hypotonic stimulation can enhance glutamate by activating TRPV4 receptor. The functional activity of NMDA receptor.
5. the action of]TRPV4 receptor agonist 4 alpha -PDD is the same. Hypotonic stimulation can increase the maximum reaction ability in the INMDA dose effect curve, but it has no obvious effect on the ECso value, and has no obvious effect on the I-V curve of INMDA current, suggesting that the activation of the TRPV4 receptor by the hypotonic stimulation can enhance the opening of the glutamate INMDA receptor ion channel.
6.NR2B subdivision antagonist ifenprodil can effectively block the enhancement of INMDA by low permeability stimulation, while NR2A subunit blocker NVP-AAM007 has no significant effect on the effect of low permeability stimulation..CaMKII blockers can inhibit the enhancement of low permeability stimulation to INMDA, and.PKC and CKII have no effect on the effect of low permeability stimulation.
7. NMDA receptor blocker MK801 can alleviate the damage of CA1 neurons induced by TRPV4 receptor over activation.
conclusion
The overexpression of TRPV4 receptor and the enhancement of TRPV4 receptor activity after ischemia reperfusion can increase the calcium influx of TRPV4 receptor, promote the release of glutamate, and activate CaMKII by increasing the level of NR2B subunit phosphorylation to increase the calcium influx of the NMDA receptor. The results suggest that the increase of TRPV4 receptor function after ischemia-reperfusion can cause calcium overload. Activation of apoptotic signaling pathway, which leads to neuronal death, therefore suggests blocking TRPV4 receptors to protect against cerebral ischemia-reperfusion injury.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R743

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 高艷琴,高慧,周正怡,陸世鐸,孫鳳艷;大鼠局灶性腦缺血再灌注損傷后紋狀體和海馬區(qū)瞬時(shí)受體電位通道蛋白4的表達(dá)增加[J];生理學(xué)報(bào);2004年02期

，

本文編號(hào)：1875842