背景:血管性痴呆的发生与脑缺血缺氧河北省有关,前期的研究表明临床效方—醒脑启智胶囊可明显改善血管性痴呆小鼠的行为学障碍,但其作用途径是否为保护缺氧细胞从而改善血管性痴呆症状。目的:采用血清药理学方法,体外培养嗜铬细胞瘤PC12细胞,用Na2S2O4产生缺氧损伤,观察醒脑启智药物血清是否对缺氧损伤的PC12细胞具有保护作用。设计:随机对照实验。单位:河北医科大学中医学院。对象:实验于2003-06/12在河北医科大学动物实验室和细胞培养实验室进行。40只SD大鼠随机分成5组,即对照血清组(n=12)和醒脑启智药物血清25.42,12.71,6.35,3.18g/kg组(n=7)。PC12细胞株购自中国医学科学院细胞中心。方法:①药物血清制备:醒脑启智药物血清25.42,12.71,6.35,3.18g/kg组按相应剂量以醒脑启智药物(枸杞子、石菖蒲、川芎、橘络等)灌胃给药,对照血清组以生理盐水10mL/kg灌胃,均为2次/d,每天灌胃间隔12h,连续给药3d,末次给药后1h麻醉状态下股动脉采血,分离血清。②PC12细胞分组培养:将体外培养的PC12细胞分为6组:对照组加入对照血清培养;模型组加对照血清培养1h后加入Na2S2O4产生缺氧损伤;醒脑启智25.42,12.71,6.35,3.18g/kg组分别加入5%不相应剂量的药物血清,培养1h后加入Na2S2O4。③观察指标:各组细胞加入Na2S2O4培养16h后,倒置相差显微镜下观察PC12细胞形态变化;计算乳酸脱氢酶释放抑制率和PC12细胞损伤的抑制率(MTT法)来评估细胞活力。主要结局观察:①各组PC12细胞的形态学观察。②各组PC12细胞的乳酸脱氢酶活性释放抑制率和PC12细胞损伤的抑制率。结果:①细胞形态:醒脑启智各剂量组PC12细胞折光性较好,细胞碎片形成减少。②乳酸脱氢酶活性释放抑制率:醒脑启智25.42,12.71,6.35,3.18g/kg组分别为81.6%,69.6%,54.4%,27.8%。③PC12细胞损伤的抑制率:醒脑启智25.42,12.71,6.35,3.18g/kg组分别为82.9%,75.6%,65.9%,53.7%。结论:醒脑启智药物血清能明显减轻PC12细胞的缺氧损伤,可增强细胞活力,降低乳酸脱氢酶活性,并呈现一定的剂量依赖关系。 BACKGROUND: The occurrence of vascular dementia is related to cerebral ischemia and hypoxia in Hebei Province. Previous studies have shown that clinical efficacy of Fangnao Qizhi Capsule can significantly improve ethological disorders in mice with vascular dementia, but whether its protective effect is protected Hypoxia cells thus improve the symptoms of vascular dementia. Objective: To use serum pharmacology method to culture PC12 cells of pheochromocytoma in vitro and produce hypoxic injury with Na2S2O4. Observe whether the serum of Xingnaoqizhi has protective effect on hypoxic PC12 cells. Design: Randomized controlled trials. Unit: School of Traditional Chinese Medicine, Hebei Medical University. Subject: The experiment was performed at the Animal Laboratory and Cell Culture Laboratory of Hebei Medical University from June 2003 to December 12th. Forty Sprague-Dawley rats were randomly divided into 5 groups: control serum group (n=12) and Xingnaoqizhi drug serum 25.42, 12.71, 6.35, 3.18 g/kg group (n=7). The PC12 cell line was purchased from the Cell Center of the Chinese Academy of Medical Sciences. Methods: 1 Preparation of drug serum: Xingnaoqizhi drug serum 25.42, 12.71, 6.35, 3.18g/kg group was given intragastrically according to the corresponding dose of Xingnaoqizhi drugs (Fructus Gardeniae, Shichangpu, Chuanxiong, Citrus, etc.) The control sera were intragastrically administered with normal saline at 10 mL/kg, both of which were given twice per day. The rats were intragastrically administrated at intervals of 12 h, administered continuously for 3 days, and blood was collected from the femoral artery under anesthesia for 1 h after the last administration to separate the serum. 2PC12 cell subculture: PC12 cells cultured in vitro were divided into 6 groups: the control group was incubated with the control serum; the model group was incubated with control serum for 1 h and Na2S2O4 was added to produce hypoxic injury; Xingnao Qizhi 25.42, 12.71, 6.35, 3.18g In the /kg group, 5% of drug serum was not added, and Na2S2O4 was added after 1 h of culture. 3 Observation indexes: After adding Na2S2O4 to each group of cells for 16h, the morphological changes of PC12 cells were observed under an inverted phase contrast microscope; the inhibition rate of lactate dehydrogenase release and the inhibition rate of PC12 cell injury (MTT method) were calculated to evaluate cell viability. The main outcomes were observed: 1 Morphological observation of PC12 cells in each group. 2 Inhibition rate of lactate dehydrogenase activity and inhibition rate of PC12 cell injury in PC12 cells. RESULTS: 1 Cell morphology: The PC12 cells in each dose group of Xingnaoqizhi had better refractive index and reduced cell debris formation. 2 The release inhibition rate of lactate dehydrogenase activity: Xingnaoqizhi 25.42, 12.71, 6.35, 3.18g/kg group were 81.6%, 69.6%, 54.4%, 27.8%. The inhibitory rate of 3PC12 cell injury was 25.9%, 12.71%, 6.35%, 3.18g/kg, respectively, in the brain-raising-intelligence group, which was 82.9%, 75.6%, 65.9%, and 53.7%, respectively. Conclusion: Xingnaoqizhi drug serum can significantly reduce the hypoxic injury of PC12 cells, enhance cell viability, and decrease the activity of lactate dehydrogenase. It shows a dose-dependent relationship.