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背景:神经生长因子和脑源性神经因子对神经细胞的存活和增殖非常重要。阿尔茨海默病患者的神经生长因子和脑源性神经因子水平均较低。目的:探讨酸性肽能否增加大鼠星形胶质细胞分泌神经生长因子和脑源性神经因子。设计:随机对照动物实验。单位:郑州大学医学院生物化学与分子生物学教研室。材料:实验于2003-09/2005-05在郑州大学生物活性肽研究所第一实验室和郑州大学基础医学院细胞培养中心完成。选取出生后2d内的SD乳鼠15只作为实验对象。方法:①将SD乳鼠在无菌条件下断头取出大脑皮质部分,进行星形胶质细胞的纯化培养,采用胶质纤维酸性蛋白免疫组化方法鉴定星形胶质细胞。②将培养的星形胶质细胞随机分为6组:空白对照组、血清对照组、阳性对照组、酸性肽37.5,75,150mg/L治疗组。空白对照组不施加任何实验因素,血清对照组加入体积分数为0.2的血清,阳性对照组加入1000U/mL干扰素,酸性肽治疗组则分别加入37.5,75,150mg/L的酸性肽。③将长满瓶底的第2代星形胶质细胞消化成单细胞悬液,以5×105mL等量接种于3块12孔培养板中。各组均于培养24,48,72h各时间点测定细胞存活率、细胞上清液中和细胞内神经生长因子及脑源性神经因子含量。主要观察指标:①不同培养时间各组细胞计数和存活率的检测结果。②酸性肽对大鼠星形胶质细胞增殖的影响。③不同培养时间各组星形胶质细胞上清液中神经生长因子及脑源性神经因子含量的变化。结果:①与空白对照组比较,酸性肽75,150mg/L治疗组细胞计数和细胞存活率在培养24,48,72h时均明显增加(P<0.05,0.01,0.001);酸性肽37.5mg/L治疗组虽有所增加但不明显。②与空白对照组比较,酸性肽37.5,75,150mg/L治疗组细胞增殖率均显著升高(0,17.5%,45.5%,72.5%,P<0.001)。③与空白对照组比较,酸性肽37.5,75,150mg/L治疗组细胞上清液中神经生长因子的吸光度值在培养24,48,72h时均明显增加(P<0.001);除了在培养24h时间点酸性肽37.5mg/L治疗组不能增加星形胶质细胞分泌脑源性神经因子外,其他各浓度酸性肽治疗组在培养24,48,72h时脑源性神经因子的吸光度值均显著提高(P<0.05,0.001)。结论:酸性肽能够不同程度地增加大鼠星形胶质细胞分泌神经生长因子和脑源性神经因子。
Background: Nerve growth factor and brain-derived neurotrophic factor are important for the survival and proliferation of nerve cells. Alzheimer’s disease patients with low levels of nerve growth factor and brain-derived neurotrophic factor. Objective: To investigate whether acidic peptide can increase the secretion of nerve growth factor and brain-derived neurotrophic factor in rat astrocytes. Design: Randomized controlled animal experiments. Unit: Department of Biochemistry and Molecular Biology, Zhengzhou University School of Medicine. MATERIALS: Experiments were performed at the First Laboratory of Bioactive Peptide Research Institute of Zhengzhou University and the Cell Culture Center of Zhengzhou University Basic Medical College from September 2003 to May 2005. Fifteen SD sucklings within 2 days after birth were selected as experimental subjects. Methods: (1) The SD rat was taken out of the cerebral cortex by decapitation under aseptic conditions. The astrocytes were purified and cultured. The astrocytes were identified by glial fibrillary acidic protein immunohistochemistry. ② The cultured astrocytes were randomly divided into 6 groups: blank control group, serum control group, positive control group, acidic peptide 37.5,75,150mg / L treatment group. In the blank control group, no experimental factors were applied. The serum control group was added 0.2 volume fraction serum, the positive control group added 1000U / mL interferon, acidic peptide treatment group were added 37.5,75,150mg / L acidic peptide. ③ overlay the bottom of the 2nd generation astrocytes digested into single cell suspension, in 5 × 105mL equal volume inoculated in three 12-well plates. The cell viability, the content of intracellular nerve growth factor and brain-derived neurotrophic factor in each group were measured at 24, 48 and 72h after culture. MAIN OUTCOME MEASURES: ① The test results of cell count and survival rate in different culture time groups. ② acid peptide on rat astrocyte proliferation. (3) The changes of nerve growth factor and brain-derived neurotrophic factor in astrocyte supernatants of different groups at different culture time. Results: ①Compared with the blank control group, the cell counts and cell viability in the 75,150 mg / L acidic peptide group increased significantly at 24, 48 and 72 hours (P <0.05,0.01,0.001); the acid peptide 37.5 mg / L Although the treatment group increased but not obvious. ② Compared with the blank control group, the cell proliferation rate of the treated group with 37.5,75,150 mg / L acidic peptide were significantly increased (0, 17.5%, 45.5%, 72.5%, P <0.001). Compared with the blank control group, the absorbance of nerve growth factor in the supernatant of the 37.5,75,150 mg / L acidic peptide group were significantly increased at 24, 48 and 72h (P <0.001) Point acid peptide 37.5mg / L treatment group can not increase the secretion of brain-derived neurotrophic astrocytes, the other concentrations of acid peptide treatment group 24, 48, 72h cultured brain-derived nerve factor absorbance values were significantly increased (P <0.05, 0.001). Conclusion: Acidic peptide can increase the secretion of NGF and BDNF in rat astrocytes to varying degrees.