gudca对胆红素所致sd大鼠脑干神经元突触小体ca2过载拮抗作用-antagonism of gud ca on bilirubin-induced synaptosome ca2 overload in brain stem neurons of sd rats.docxVIP

GUDCA 对胆红素所致 SD 大鼠脑干神经元突触小体 Ca2+过载的拮抗作用中文摘要GUDCA 对胆红素所致 SD 大鼠脑干神经元突触小体 Ca2+过载的拮抗作用 中文摘要目的探讨胆红素对 SD 大鼠脑干神经元突触小体游离钙离子浓度的影响，以及甘 氨酸熊脱氧胆酸（GUDCA）的拮抗作用。方法采用出生后 7-14 天的 SD 大鼠 40 只（雌雄不拘），随机分成三大组，分别为 对照组（A 组），胆红素组（B 组，胆红素浓度分别为 0.1μmol/L、1μmol/L、10μmol/L）， 以及 GUDCA 干预组（C 组胆红素浓度同 B 组，GUDCA50μmol/L）。SD 大鼠称重并 麻醉（乙酰丙嗪，肌注，4.5mg/kg）、迅速断头、取脑干，用蔗糖密度梯度离心法（0.32 mol/L、0.8 mol/L、1.2mol/L）提纯脑干突触小体；以不含 Ca2+、Mg2+、酚酞的 HBSS 分别洗涤、离心三次后，用钙离子荧光探针 Oregon green 488 BAPTA-1/AM10μmol/L 染色，室温下孵育 40min，再以 HBSS 分别洗涤、离心三次，再次离心，加适量 HBSS 获得富有活性的突触小体悬浮液（BCA 法控制蛋白浓度）。采用光镜及透射电镜观察 突触小体活性，并以共聚焦激光扫描显微镜观察不同条件下突触小体的形态，以及突 触小体[Ca2+]i 荧光强度的实时变化。结果通过上述方法可以获得活性良好的 SD 大鼠脑干神经元突触小体；对照组突 触小体[Ca2+]i 的荧光强度稍微增加；在 0.1μmol/L、1μmol/L、10μmol/L 的胆红素的 作用下，胞内钙的荧光强度迅速升高，并随浓度增加而增加；在胆红素和 GUDCA 的 共同作用下，胞内钙荧光强度显著降低。结论胆红素能使突触小体内的钙超载，并与浓度呈正相关；GUDCA 能够显著抑 制胆红素所致的突触小体内的钙超载。GUDCA 是有效的、潜在的治疗新生儿胆红素 脑病的治疗药物。关键词胆红素；突触小体；内钙；GUDCA ； OG-BAPTA ；共聚焦激光扫描显 微镜作者：李丹萍 指导老师：时海波英文摘要GUDCA 对胆红素所致 SD 大鼠脑干神经元突触小体 Ca2+过载的拮抗作用GUDCA inhibits bilirubin-induced calcium overload in synaptosomes isolated from brainstem neurons in the SD ratsAbstractPurpose To observe real-time changes of intracellular calcium arose from bilirubin and GUDCA in synaptosome from brainstem in the SD rat.Methods Fourty P7-14 SD rats (male and female)were used in this study.They were randomly assigned to two groups. A group was bilirubin group and B group was bulirubin plus GUDCA group. Twenty rats were in each group，five rats were in every different concentration. Rats were decapitated after anesthetized with intramuscular injection of acepromazine(4.5 mg/kg of body weight).Then ， the brainstem were dissected.The synaptosomes were purified by sucrose density gradient centrifugation. After loading OG-BAPTA in synaptosomes from brainstem（45min，room temperature）， two dimensional image of intracellular calcium and analysis of fluorescence intensity were achieved by Confocal laser scanning microscopy(CLSM)．ResultsCompared with blank group， an immediate peak of fluorescent intensityof [Ca2+]i wa