CAJ | 학술논문

目的：探讨重组人粒细胞集落刺激因子（ rhG-CSF）对缺血缺氧神经元巨自噬水平的影响及意义。方法：采取氧糖剥夺法制作细胞缺氧模型，Western blot法检测0、100、200μg/L rhG-CSF干预下细胞巨自噬标志微管相关蛋白轻链3膜型和胞质型比值（LC3Ⅱ/LC3Ⅰ）的变化，单丹磺酰戊二胺（MDC）法荧光观察巨自噬空泡变化，MTT法检测细胞活力。应用改良线栓法建立大鼠局灶性脑缺血模型，rhG-CSF干预前及干预7 d后进行改良的神经功能缺损评分评定，Western blot法检测干预7 d后大鼠皮层神经元LC3Ⅱ/LC3Ⅰ，免疫组化法检测LC3蛋白的表达。结果：随着rhG-CSF给药剂量的增加，细胞缺氧模型 LC3Ⅱ/LC3Ⅰ和巨自噬空泡数量逐渐增加（ F＝288．686，154．698，P＜0．001），细胞活力也相应增强（F＝74．456 P＜0．001）。动物实验中，干预7 d后，rhG-CSF组大鼠缺血神经元LC3Ⅱ/LC3Ⅰ（F＝573．085，P＜0．001）及LC3阳性细胞数（F＝88．945，P＜0．001）分别较假手术组及模型组上调，大鼠的神经功能缺损评分较干预前明显降低（F组间＝20．403，F时间＝19．193，F交互＝33．415，P＜0．001）。结论：局灶性脑缺血后rhG-CSF可能通过上调巨自噬水平参与神经元的保护。
목적：탐토중조인립세포집락자격인자（ rhG-CSF）대결혈결양신경원거자서수평적영향급의의。방법：채취양당박탈법제작세포결양모형，Western blot법검측0、100、200μg/L rhG-CSF간예하세포거자서표지미관상관단백경련3막형화포질형비치（LC3Ⅱ/LC3Ⅰ）적변화，단단광선무이알（MDC）법형광관찰거자서공포변화，MTT법검측세포활력。응용개량선전법건립대서국조성뇌결혈모형，rhG-CSF간예전급간예7 d후진행개량적신경공능결손평분평정，Western blot법검측간예7 d후대서피층신경원LC3Ⅱ/LC3Ⅰ，면역조화법검측LC3단백적표체。결과：수착rhG-CSF급약제량적증가，세포결양모형 LC3Ⅱ/LC3Ⅰ화거자서공포수량축점증가（ F＝288．686，154．698，P＜0．001），세포활력야상응증강（F＝74．456 P＜0．001）。동물실험중，간예7 d후，rhG-CSF조대서결혈신경원LC3Ⅱ/LC3Ⅰ（F＝573．085，P＜0．001）급LC3양성세포수（F＝88．945，P＜0．001）분별교가수술조급모형조상조，대서적신경공능결손평분교간예전명현강저（F조간＝20．403，F시간＝19．193，F교호＝33．415，P＜0．001）。결론：국조성뇌결혈후rhG-CSF가능통과상조거자서수평삼여신경원적보호。
Aim:To explore the effect and significance of rhG-CSF on neuronal macroautophagy in hypoxia ischemia rats.Methods:Cell hypoxia model was established by taking oxygen-glucose deprivation.After dealing cells with 0,100, 200 μg/L rhG-CSF, the marker of macrophage , LC3Ⅱ/LC 3Ⅰ was measured by Western blot , macroautophagy vesicle change was analyzed by monodansylcadaverine ( MDC) , and MTT was used to investigate the cell viability .Focal cerebral ischemia rat model was established by improved suture method .The modified neurological severity score (mNSS)was adopt-ed to evaluate neural function recovery before and after the intervention of rhG-CSF,LC3Ⅱ/LC3 Ⅰ of cortical neurons in rats was observed by Western blot , and LC3 protein expression was detected by immunohistochemistry .Results:With the increase of the dosage of rhG-CSF, the ratio of LC3Ⅱ/LC3Ⅰ and the number of macroautophagy vesicles gradually in-creased (F=288.686,154.698,P<0.001),and the cell viability also gradually increased (F=74.456, P<0.001). Compared with sham operation group and model group , LC3Ⅱ/LC3Ⅰand LC3-positive cells (F=573.085 and 88.945, P<0.001) raised in rhG-CSF group.After treatment, the mNSS of rats in rhG-CSF group was lower ( Fgroup =20.403, Ftime =19.193,Fmutual =33.415,P<0.001).Conclusion: rhG-CSF may participate in the neural protection after focal cerebral ischemia by raising macroautophagy activity .