【摘要】：目的： 研究細(xì)胞外尼克酰胺磷酸核糖轉(zhuǎn)移酶(eNAMPT)對(duì)腦缺血損傷的影響,并探尋其作用機(jī)制。 方法： 大鼠側(cè)腦室注射野生型NAMPT蛋白和喪失酶活性的突變NAMPT蛋白H247A,通過(guò)大腦中動(dòng)脈栓塞(MCAO)復(fù)灌誘導(dǎo)大鼠局灶性腦缺血,以缺糖缺氧(OGD)誘導(dǎo)原代混合神經(jīng)細(xì)胞和原代神經(jīng)元損傷,Western檢測(cè)NAMPT表達(dá),免疫組化觀察NAMPT分布變化,TTC染色計(jì)算梗死損傷體積,MTT與LDH釋放檢測(cè)細(xì)胞活性,PI、DAPI染色檢測(cè)細(xì)胞壞死,ELISA檢測(cè)TNF-α釋放。 結(jié)果： MCAO再灌注后24小時(shí),缺血中心區(qū)eNAMPT水平上升,mRNA水平下降,缺血中心區(qū)、周邊區(qū)和假手術(shù)組腦內(nèi),NAMPT均主要分布在神經(jīng)元。重組NAMPT蛋白腦室內(nèi)注射能夠加重MCAO引起的腦缺血性損傷。在原代培養(yǎng)神經(jīng)元,重組NAMPT蛋白對(duì)正常和OGD誘導(dǎo)的神經(jīng)元損傷均無(wú)明顯影響。但在原代培養(yǎng)神經(jīng)細(xì)胞,重組NAMPT蛋白能濃度依賴的加重OGD誘導(dǎo)的神經(jīng)元,同時(shí)可以濃度和時(shí)間依賴的引起TNF-a釋放,TNF-a中和抗體能夠減去OGD誘導(dǎo)、NAMPT蛋白加重的神經(jīng)元損傷。NAMPT突變蛋白H247A的作用野生型NAMPT相似。 結(jié)論： 腦缺血后,血NAMPT進(jìn)入腦內(nèi),使eNAMPT水平增加,eNAMPT能夠加重大鼠腦缺血性損傷,這種損傷是通過(guò)作用于膠質(zhì)細(xì)胞而釋放TNF-a發(fā)生,并且與其催化作用無(wú)關(guān)。
[Abstract]:Aim: to study the effect of extracellular nicotinamide phosphate ribosyltransferase (eNAMPT) on cerebral ischemia injury and explore its mechanism. Methods: a mutant NAMPT protein H247A was injected into the lateral ventricle of rats with wild-type NAMPT protein and lost enzyme activity. The focal cerebral ischemia was induced by middle cerebral artery embolization with (MCAO) reperfused. Primary mixed neurons and primary neurons were induced by glucose deficiency and hypoxia (OGD). NAMPT expression was detected by Western, NAMPT distribution was observed by immunohistochemistry, infarct volume was calculated by TTC staining, cell activity was measured by MTT and LDH release, and PI, was detected by PI,. Cell necrosis was detected by DAPI staining and TNF- 偽 release was detected by ELISA. Results: 24 hours after reperfusion of MCAO, the level of eNAMPT increased and the level of mRNA decreased in the ischemic center, and NAMPT was mainly distributed in the neurons in the ischemic center, peripheral area and sham operation group. Intracerebroventricular injection of recombinant NAMPT protein can aggravate ischemic brain damage induced by MCAO. In primary cultured neurons, the recombinant NAMPT protein had no significant effect on normal and OGD-induced neuronal damage. However, in primary culture, the recombinant NAMPT protein can aggravate the OGD-induced neurons in a concentration-dependent manner and induce the release of TNF-a in a concentration-and time-dependent manner. The neutralizing antibody to TNF-a can subtract the OGD-induced neurons. The NAMPT-induced neuronal damage was similar to that of wild-type NAMPT induced by Napt mutant H247A. Conclusion: after cerebral ischemia, blood NAMPT enters the brain and increases the level of eNAMPT. ENAMPT can aggravate the cerebral ischemic injury in rats, which is caused by the release of TNF-a through the action of glial cells and has nothing to do with its catalytic effect.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743

【共引文獻(xiàn)】

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