细胞外尼克酰胺磷酸核糖转移酶通过非酶途径加重大鼠脑缺血损伤

发布时间：2019-03-22 17:59

【摘要】：目的：研究细胞外尼克酰胺磷酸核糖转移酶(eNAMPT)对脑缺血损伤的影响,并探寻其作用机制。方法：大鼠侧脑室注射野生型NAMPT蛋白和丧失酶活性的突变NAMPT蛋白H247A,通过大脑中动脉栓塞(MCAO)复灌诱导大鼠局灶性脑缺血,以缺糖缺氧(OGD)诱导原代混合神经细胞和原代神经元损伤,Western检测NAMPT表达,免疫组化观察NAMPT分布变化,TTC染色计算梗死损伤体积,MTT与LDH释放检测细胞活性,PI、DAPI染色检测细胞坏死,ELISA检测TNF-α释放。结果： MCAO再灌注后24小时,缺血中心区eNAMPT水平上升,mRNA水平下降,缺血中心区、周边区和假手术组脑内,NAMPT均主要分布在神经元。重组NAMPT蛋白脑室内注射能够加重MCAO引起的脑缺血性损伤。在原代培养神经元,重组NAMPT蛋白对正常和OGD诱导的神经元损伤均无明显影响。但在原代培养神经细胞,重组NAMPT蛋白能浓度依赖的加重OGD诱导的神经元,同时可以浓度和时间依赖的引起TNF-a释放,TNF-a中和抗体能够减去OGD诱导、NAMPT蛋白加重的神经元损伤。NAMPT突变蛋白H247A的作用野生型NAMPT相似。结论：脑缺血后,血NAMPT进入脑内,使eNAMPT水平增加,eNAMPT能够加重大鼠脑缺血性损伤,这种损伤是通过作用于胶质细胞而释放TNF-a发生,并且与其催化作用无关。
[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- 伪 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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743

【共引文献】