CAJ | 학술논문

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6 h，LPS组细胞以含终浓度为100 ng／mL LPS的无血清胶质细胞培养液孵育6 h，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0．5 h，然后加入LPS（终浓度为100 ng／mL）继续孵育6 h。 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0．5 h，再加入终浓度为1μmol／L的NPY孵育0．5 h，最后加入终浓度100 ng／mL的LPS继续孵育6 h。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20 min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。
목적：탐토신경태Y（ NPY）이중추신경계통소효질세포위파점대대서전간발작적영향。방법배양화순화원대SD대서피질소효질세포，면역세포화학염색，감정소효질세포순도급관찰세포형태。장소효질세포분위대조조、LPS조、NPY＋LPS조화BIBP3226＋NPY＋LPS조。대조조세포이무혈청효질세포배양액부육6 h，LPS조세포이함종농도위100 ng／mL LPS적무혈청효질세포배양액부육6 h，NPY＋LPS조세포시선이함NPY （종농도위1μmol／L）적무혈청효질세포배양액부육0．5 h，연후가입LPS（종농도위100 ng／mL）계속부육6 h。 IBP3226＋NPY＋LPS조세포선용함NPY Y1수체조단제BIBP3226（종농도위1μmol ／L）적무혈청효질세포배양액부육0．5 h，재가입종농도위1μmol／L적NPY부육0．5 h，최후가입종농도100 ng／mL적LPS계속부육6 h。20지SD대서수궤분위대조조、LPS조、NPY＋LPS조、BIBP3226＋NPY＋LPS조，매조5지。장각조소효질세포조건배양액리심후주입상응각조대서적측뇌실，관찰각조대서적행위학표현。근거Diehl분급표준대대서전간발작정도진행평고。결과20 min내，LPS조화BIBP3226＋NPY＋LPS조5지대서균출현중도전간발작，NPY＋LPS조유4지출현경도전간발작，대조조미견전간발작。 LPS조발작정도현저중우대조조， NPY＋LPS조발작정도현저경우 LPS 조， BIBP3226＋NPY ＋LPS 조발작정도현저중우 NPY ＋LPS 조， LPS 조화BIBP3226＋NPY＋LPS조발작정도비교차이균무통계학의의。결론격활후적소효질세포가이도치대서전간발작，저충작용시통과소효질세포여신경원지간적비직접접촉도경실현적，가능여소효질세포분비도세포외적생물활성물질유관。 NPY가이통과작용우소효질세포상NPY Y1수체，억제대서적전간발작。
Objective It is to explore the influence of NPY on the epileptic seizure of rats by acting on microglia.Methods The primary cortex microglia of rats was cultured and purified, morphology of microglia were observed through immunocyto-chemistry staining, Primary cerebral cortical microglia of rats was divided into control group, LPS group, NPY+LPS group, NPY group and BIBP3226+NPY+LPS group.Microglia cells in control group were incubated with serum-free medium for 6 h;microglia cells in LPS group were incubated with serum-free medium including LPS( final concentration 100 ng/mL) for 6 h;microglia cells in NPY+LPS group were incubated with serum-free medium including NPY( final concentration 1μmol/L) for 0.5 h firstly, then continued the incubation for 6 h after adding LPS (final concentration 100 ng/mL);microglia cells in NPY group were incubated in serum-free medium including NPY ( final concentration 1 μmol/L ) for 6 h; microglia cells in BIBP3226+NPY+LPS group were incubated in serum-free medium including BIBP3226 ( final concentration 1 μmol/L ) which was NPY Y1 receptor blocking reagent for 0.5 h, then we incubated them for 0.5 h after adding NPY (final concentra-tion 1μmol/L) ,at last the cells were incubated for 6 h after adding LPS with the final concentration 100 ng/mL.20 adult SD rats were divided into control group,LPS group,NPY+LPS group and BIBP3226+NPY+LPS group,every group included five rats.The levels of epileptic seizure of rats in each group were observed after microglia conditioned mediums were injected re-spectively into ventricle of the adult rats.Behaviors of the rats in every group were observed, and epilepsy degree was evalua-ted based on Diehl’ s method.Results In 20 minutes, all the 5 rats in LSP group and BIBP3226+NPY+LPS group appeared severe epileptic seizure.There were 4 rats in NPY+LPS group appeared mild epileptic seizures in the same time.There was no rat appeared epileptic seizure in control group.The degrees of epileptic seizure of LPS group were significantly higher than that of the control group.the degrees of NPY+LPS group were significantly lower than that of LPS group.The degrees of BIBP3226+NPY+LPS group were significantly higher than that of NPY+LPS group.There was no difference in the degrees between LPS group and BIBP3226+NPY+LPS group.Conclusion LPS could stimulate microglia cells to cause epileptic sei-zure of rats.NPY can restrain epileptic seizure of rats through acting on NPYY1 receptor on microglia.This may be related to something with biological activity.