【作者】 金鑫；

【导师】 李玉子； 张锦玉；

【作者基本信息】 延边大学 ， 内科学， 2021， 硕士

【摘要】 研究目的:利用急性脑片全细胞膜片钳记录、组织化学染色技术,探讨研究胰高血糖素样肽-1（GLP-1）对大鼠下丘脑室旁核（PVN）神经分泌大细胞（MNCs）活动的影响。材料与方法:选择的实验动物是出生2-3周龄的Wistar系大鼠,均为雄性,给予异氟烷进行吸入麻醉后,马上断头取出脑组织,随后利用莱卡全自动振动切片机,制备厚度为250μm的含PVN的下丘脑切片。在室温（约24-25℃）条件下,将制备后的下丘脑脑片置于持续充有混合气体（95%O₂和5%CO₂）的人工脑脊液（ACSF）中孵育60分钟以上。渗透压保持在295-300 m Os M之间,PH值为7.25-7.35。记录电极中用特制枪头灌装10μl的电极内液,电极的阻抗控制在5-7 M（?）。利用膜片钳放大器（Axopatch-700B）及Clampex数据采集软件对PVN神经分泌细胞的电活动进行记录与彩集。在电生理记录结束后,含有室旁核的脑切片于4%的多聚甲醛溶液进行固定24小时后,进行DAB染色、显微镜下拍照,并观察所记录神经分泌细胞,其位置、类型、形态学特点与投射情况。电生理实验数据分析使用Clampfit 10.4软件。各组数据的表示采用Mean±S.E.M.,数据的统计学分析采用SPSS 16.0软件,均数的比较采用配对T检验,当P<0.05时记录结果认为有统计学差异。结果:（1）在电流钳记录条件下,PVN神经分泌大细胞对去极化电流刺激敏感,并伴随明显的超极化活化的外向整流电流。（2）给予GLP-1（100 nM）可导致PVN神经分泌大细胞的自发性放电频率显著降低并伴随明显的膜电位超极化,冲洗20分钟以后恢复到给药前水平。（3）非选择性离子型谷氨酸受体拮抗剂（kynurenic acid,KYC）不能消除GLP-1所导致的部分PVN神经分泌大细胞兴奋性降低和膜超极化作用。（4）在阻断GABA_A受体的条件下,GLP-1对PVN神经分泌大细胞的自发性放电活动的抑制作用仍然存在。（5）同时给予了离子型谷氨酸受体阻断剂KYC及GABA_A受体阻断剂Picrotoxin,GLP-1仍然可以抑制PVN神经分泌大细胞兴奋性,并存在一定的浓度依存性,半数有效剂量为107nM。（6）GLP-1可显著增加PVN神经分泌大细胞的后超级化电位,而对动作电位的半宽值、上升常数、衰退常数均没有明显影响。结论:GLP-1通过活化突触后离子通道来降低PVN神经分泌大细胞的兴奋性,同时抑制其自发性放电的频率。提示GLP-1通过直接方式调节PVN神经分泌大细胞的活动。更多还原

【Abstract】 Objective:To study the effect of Glucagon-like peptide-1（GLP-1）on the activity of hypothalamic paraventricular nucleus（PVN）magnocellular endocrine cells（MNCs）,by using whole-cell patch-clamp recording technique and imunohistochemistry methods.Methods:Wistar male rats of 2-3 weeks were selected in the present study.Following the rats were anesthetized by isoflurane,the brains of rats were cut off and put into ice-cold artificial cerebral spinal fluid（ACSF）quickly.Then,the PVN included coronal slices were cut by a vibratome.The thickness of these slices was 250μm.At room temperature（about 24-25℃）,the slices of hypothalamus were incubated in ACSF filled with 95%O₂and 5%CO₂for more than 60 minutes.PH value was7.25-7.35,and osmolarity was 295-300 m Os M.The recording electrodes were filled with 10μl internal solution.Patch pipette resistances were 5-7 M（?）in the bath.The spontaneous spike firing of PVN neurons was recorded by patch clamp amplifier（Axopatch-700B）and data acquisition software.At the end of electrophysiological recording,the slices were fixed with 4%paraformaldehyde solution for 24 hours,then immunohistochemical staining and micrography were carried out to determine the type,location,morphological characteristics and projection of the recorded cells.Clampfit10.4 software was used for data analysis of electrophysiological experiments.All data were expressed by Mean±SEM.The data were analyzed by Student’s paired t-test using SPSS software.P<0.05 was considered statistically significant.Results:（1）Under current-clamp condiation,PVN Magnocellular Neurosecretory Cells were sensitive to the injection of depolarization current,and they exhibit outwardly rectification currents.（2）Application of GLP-1（100 nM）induced a significant decrease in spike firing and membrane hyperpolarization rate of PVN Magnocellular Neurosecretory Cells,which were recovery after washout for 20 min.（3）Non-selective ionic glutamate receptor antagonist（kynurenic acid,KYC）,can not block perfusion application of GLP-1-induced the frequency of spike firing decrease and membrane hyperpolarization of PVN MCNs.（4）Blocking the GABA_Areceptor,the inhibitory effect of GLP-1 on the spike firing of PVN Magnocellular Neurosecretory Cells still existed.（5）In the presence of KYC and Picrotoxin,GLP-1 still can inhibit the activity of neurons and membrane hyperpolarization.And the effect of GLP-1onMNCs was does-dependent.The half-inhibition concentration was 104 nM.（6）Application of GLP-1 induced a significant increase in AHP of PVN MNCs,but had no effect on the half-width,rise tau and decay tau.Conclusion:GLP-1 depresses the excitability of PVN magnocellular neurosecretory cells and inhibits the frequency of spontaneous discharge by activating postsynaptic ion channels,suggesting that GLP-1 directly regulates the activity of PVN magnocellular neurosecretory cells.更多还原