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目的探讨神经肽Y(NPY)以中枢神经系统小胶质细胞为靶点对大鼠癫痫发作的影响。方法培养和纯化原代SD大鼠皮质小胶质细胞,免疫细胞化学染色,鉴定小胶质细胞纯度及观察细胞形态。将小胶质细胞分为对照组、LPS组、NPY+LPS组和BIBP3226+NPY+LPS组。对照组细胞以无血清胶质细胞培养液孵育6h,LPS组细胞以含终浓度为100ng/mLLPS的无血清胶质细胞培养液孵育6h,NPY+LPS组细胞是先以含NPY(终浓度为1μmol/L)的无血清胶质细胞培养液孵育0.5h,然后加入LPS(终浓度为100ng/mL)继续孵育6h。IBP3226+NPY+LPS组细胞先用含NPY Y1受体阻断剂BIBP3226(终浓度为1μmol/L)的无血清胶质细胞培养液孵育0.5h,再加入终浓度为1μmol/L的NPY孵育0.5h,最后加入终浓度100ng/mL的LPS继续孵育6h。20只SD大鼠随机分为对照组、LPS组、NPY+LPS组、BIBP3226+NPY+LPS组,每组5只。将各组小胶质细胞条件培养液离心后注入相应各组大鼠的侧脑室,观察各组大鼠的行为学表现。根据Diehl分级标准对大鼠癫痫发作程度进行评估。结果20min内,LPS组和BIBP3226+NPY+LPS组5只大鼠均出现重度癫痫发作,NPY+LPS组有4只出现轻度癫痫发作,对照组未见癫痫发作。LPS组发作程度显著重于对照组,NPY+LPS组发作程度显著轻于LPS组,BIBP3226+NPY+LPS组发作程度显著重于NPY+LPS组,LPS组和BIBP3226+NPY+LPS组发作程度比较差异均无统计学意义。结论激活后的小胶质细胞可以导致大鼠癫痫发作,这种作用是通过小胶质细胞与神经元之间的非直接接触途径实现的,可能与小胶质细胞分泌到细胞外的生物活性物质有关。NPY可以通过作用于小胶质细胞上NPY Y1受体,抑制大鼠的癫痫发作。
Objective To investigate the effect of neuropeptide Y (NPY) on the central nervous system microglia as the target of seizure in rats. Methods Cultured and purified primary SD rat cortical microglia, immunocytochemical staining, identification of microglia purity and observation of cell morphology. Microglia were divided into control group, LPS group, NPY + LPS group and BIBP3226 + NPY + LPS group. The cells in control group were incubated with serum-free glial cell culture medium for 6h. The cells in LPS group were incubated with serum-free glial cells containing 100ng / mLLPS for 6h. NPY + LPS group cells were treated with NPY (final concentration 1μmol / L) serum-free glial cell culture medium 0.5h, then added LPS (final concentration of 100ng / mL) and incubated for 6h. The cells in IBP3226 + NPY + LPS group were incubated with serum-free glial cell culture medium containing NPY Y1 receptor blocker BIBP3226 (final concentration of 1μmol / L) for 0.5h and then incubated with NPY at a final concentration of 1μmol / L for 0.5h h, and finally adding LPS at a final concentration of 100ng / mL for 6h. Twenty SD rats were randomly divided into control group, LPS group, NPY + LPS group and BIBP3226 + NPY + LPS group, with 5 rats in each group. Each group of microglia conditioned medium was centrifuged and injected into the lateral ventricle of corresponding rats to observe the behavior of each group. The degree of seizure in rats was assessed according to the Diehl scale. Results Within 20 minutes, 5 rats in LPS group, BIBP3226 + NPY + LPS group had severe seizure, 4 in NPY + LPS group had mild seizure, and no seizure in control group. The degree of seizure in LPS group was significantly higher than that in control group. The degree of seizure in NPY + LPS group was significantly lower than that in LPS group. The degree of seizure in BIBP3226 + NPY + LPS group was significantly higher than that in NPY + LPS group and seizure degree in LPS group and BIBP3226 + NPY + LPS group The difference was not statistically significant. Conclusion The activated microglia can induce epileptic seizures in rats. This effect is mediated through the indirect contact between microglia and neurons, which may be related to the extracellular bioactivity of microglia secreted Material related. NPY can inhibit seizures in rats by acting on NPY Y1 receptors on microglia.