過度表達紅血球生成素的 NIH/3T3 纖維母細胞株於大鼠中風模型之神經元再生研究

Abstract

缺血性中風是最常見的中風，會造成腦部缺血以致功能損傷；臨床上治療缺血性中風的方式仍然只能使用抗血栓藥物，使血栓溶解將血管疏通，目前已有許多創新的實驗研究利用細胞治療及滋養因子來改善中風治療的結果卻無法恢復對腦組織的傷害。相關實驗已證實紅血球生成素 (erythropoietin, EPO)除了可以增加血球前驅細胞的增生及分化以外，越來越多的研究亦已經證實EPO對缺血性中風可以有良好的神經保護的效果及預後。然而，由於目前使用紅血球生成素治療缺血性中風的研究多使用短時間、高劑量的靜脈給予，雖能改善病患的運動能力，但可能會增加血容比及提高全身性血栓的風險。因此，本實驗將過量表現紅血球生成素的纖維母細胞( EPO-3T3-EGFP)注射到大鼠受損區域後，觀察此細胞治療是否能夠持續性的釋出紅血球生成素；以達到神經保護及再生的效果。 我們使用中大腦動脈阻塞的大鼠來模擬臨床上常見的缺血性中風。中風後隔天使用核磁共振造影來觀察損傷的區塊以及大小。核磁造影之後使用立體定位將1×106的EPO-3T3-EGFP打入損傷區。為了解紅血球生成素對功能修復的情形，分別在手術前及術後第一到第十四天使用神經傷害評分表來觀察大鼠在平衡、動作以及感覺上的功能損傷評估，且手術後第十四天，再進行一次核磁共振造影來觀察原損傷區的恢復情形。此外，動物在第7及15天進行犧牲並灌流，並將鼠腦進行冷凍包埋，切片及組織染色分析；以及依照實驗批次於術後第3、7、14、21及28日犧牲動物，抽取大腦內蛋白質進行分析。 結果顯示，我們發現使用過量表達紅血球生成素的纖維母細胞或未表達紅血球生成素的纖維母細胞(3T3-EGFP)的組別，在缺血性中風的急性期和慢性期都可以有良好的功能性回復。並在核磁共振造影上，使用EPO-3T3-EGFP細胞的治療可以減少62%的受損區域。同時間在EPO-3T3-EGFP以及3T3-EGFP的處理組別，在免疫組織染色上，發現在腦室旁區 (SVZ) 的細胞增生比率有明顯的增加，同時也發現注射EPO-3T3-EGFP細胞於該腦區的神經前驅細胞的活化比率明顯增加。酵素免疫分析法的結果顯示，打入EPO-3T3-EGFP細胞後第三天可以在損傷區域大量產生紅血球生成素，同時在損傷側腦源性神經營養因子(brain-derived neurotrophic factor, BDNF) 的含量有顯著性的增加，因此根據以上的觀察，過度表達紅血球生成素的細胞，確實可以促進缺血性中風損傷區域提供穩定的EPO釋放，具有神經修復與再生能力，並增進大腦功能性的恢復。

Ischemic stroke is the most common subtype among all kinds of stroke in the world. Treatment of ischemic stroke is restricted to acute thrombolytic drugs, which dissolve thrombi and emboli to re-canalize blood vessels. Due to lack of efficient treatment for stroke, cell treatments and trophic factor administration have been used as a novel experimental approach. Erythropoietin (EPO) has been shown to stimulate proliferation and differentiation of erythroid progenitor and mediate the neuro-protection. There is increasing number of studies to investigate the effective neuroprotection of EPO against ischemic stroke, but application of excessive EPO could contribute to systematically high hematocrit and elevation of thrombotic risk in animal. In order to avoid the side effect of high dosage application, the cell treatment of implanting the over-expressing EPO cell line maybe another approach to treat ischemic stroke and have the effect of neuroprotection and neurogenesis. In this study, we produced ischemic stroke in adult rats by the approach of middle cerebral artery occlusion (MCAO) and examined the infarct zone with MRI. Total of 106 CFU EPO-overexpressing NIH/3T3 (EPO-3T3-EGFP) cells were directly injected to the infarct zone. The brain function was assessed via modified Neurological Severity Score (mNSS). On day 14 after stroke induction, the infarct volume was measured again by MRI and the animal was euthanized for the study of angiogenesis and neurogenesis. The animal were sacrificed on day 7 and 15 for immunohistochemistry analysis. Rats were also sacrificed on day 3, 7, 14, 21 and 28 for protein extraction and analysis. From our research, the result of neurological assessment suggested that both 3T3-EGFP-treated and EPO-3T3-EGFP-treated groups showed significantly improvement of functional ability in both acute and chronic phase of ischemic stroke. In MRI examination, the recovery rate of the EPO-3T3-EGFP treated group could up to 62% and significantly higher than 3T3-treated group and MCAO control group. In immunohistochemistry, significant increase of cell proliferation in subventricular zone (SVZ) was demonstrated in EPO-3T3-EGFP treated group and 3T3-EGFP group, and percentage of DCX immunoreactive cells in SVZ of infarct side in the EPO-3T3-EGFP group was significantly increased, which may indicate implantation of EPO-3T3-EGFP cells could further enhance the repairing process of infarct site in our MCAO animal model. In ELISA analysis, we found that the concentration of EPO in infarct striatum was detected in high level on day 3, and EPO-3T3-EGFP cells could induce the expression of BDNF in the infarct area and contralateral area. In summary, our data suggest that the EPO-overexpressing NIH/3T3 cells treatment could provide stable release of EPO in the infarct area, facilitate neurogenesis and neuroprotection ability and may contribute to the functional recovery of brain.