化学遗传学技术调控GLP-1神经元兴奋性及其对食欲的影响  

Chemical genetics technology regulates the activity of GLP-1 neurons and its effect on appetite

作　　者：杨亚南 邵雨薇 田峻[2] 赵娟 祝叶 舒晴 Yang Yanan;Shao Yuwei;Tian Jun;Zhao Juan;Zhu Ye;Shu Qing(Department of Traditional Chinese Medicine,CR&WISCO General Hospital,Wuhan 430080,China;Department of Rehabilitation,Zhongnan Hospital of Wuhan University,Wuhan 430071,China;Wuhan Sports University,Wuhan 430079,China)

机构地区：[1]华润武钢总医院中医科,武汉430080 [2]武汉大学中南医院康复医学科,430071 [3]武汉体育学院研究生院,430079

出　　处：《中华内分泌代谢杂志》2022年第4期322-329,共8页Chinese Journal of Endocrinology and Metabolism

基　　金：国家自然科学基金(81804180);湖北省卫健委中医药科研面上项目(ZY2021M031);湖北省卫生健康委联合基金青年人才项目(湖北省卫生健康科研基金资助)(WJ2019H163)。

摘　　要：目的通过化学遗传学技术构建胰升糖素样肽1(GLP-1)神经元可控性模型大鼠,并观察GLP-1神经元兴奋性的变化对食欲的调控作用。方法将15只大鼠分为绿色荧光蛋白(GFP)组、HM3D组和HM4D组,每组5只。分别在3组大鼠的孤束核区域注射不同组合的腺相关病毒(rAAV),GFP组在孤束核区域注射rAAV-GLP-1-cre和rAAV-GFP-dio;HM3D组在孤束核区域注射rAAV-GLP-1-cre和rAAV-HM3D-mCherry-dio;HM4D组在孤束核区域注射rAAV-GLP-1-cre和rAAV-HM4D-mCherry-dio。通过观察腹腔注射不同剂量N-氧化氯氮平(CNO)后大鼠的摄食行为和体重变化来筛选其最佳剂量。通过与腹腔注射生理盐水进行比较来确认GLP-1神经元的可调控性。观察大鼠处死前30 min注射CNO后大鼠孤束核区域GLP-1神经元的激活数量及下丘脑POMC神经元的表达。结果各组大鼠孤束核区域内GLP-1神经元均成功被标记,CNO注射剂量为1mg/kg时,HM3D组大鼠摄食减少(P=0.021),而HM4D组大鼠摄食增加(P=0.002)。而注射剂量为0.5 mg/kg和3 mg/kg时均未出现此效应。免疫荧光结果显示,HM3D组孤束核中GLP-1神经元的兴奋性高于GFP组(P=0.022),GFP组高于HM4D组(P=0.049)。腹腔注射CNO后HM3D组大鼠孤束核区域内的GLP-1神经元及下丘脑的POMC神经元表达也高于HM4D组(P=0.003)。结论通过化学遗传学技术在大鼠孤束核内注射不同组合的rAAV能够成功建立GLP-1神经元可控性模型大鼠。1 mg/kg的CNO剂量能够有效激活或抑制该神经元,从而产生调控食欲的效应。Objective To conduct a glucagon like peptide-1(GLP-1)controllability model rat by chemical genetics,and observe the impact of GLP-1 neuron excitability on appetite.Methods Fifteen rats were evenly divided into Green fluorescent protein(GFP)group,HM3D group,and HM4D group.Various combinations of adeno-associated virus(rAAV)were injected into the nucleus tractus solitarius(NTS).rAAV-GLP-1-cre and rAAV-GFP-dio were administered in rats of GFP group.The rats of HM3D group were injected with rAAV-GLP-1-cre and rAAV-HM3D-mCherry-dio while rAAV-GLP-1-cre and rAAV-HM4D-mCherry-dio were injected in rats of HM4D group.The optimal dose of clozapine N-oxide(CNO)was selected based on feeding behavior and body weight changes of rats after intraperitoneal injection of different doses of CNO.The controllability of GLP-1 neurons was confirmed by comparing with intraperitoneal injection of saline.The number of activated GLP-1 neurons in the NTS area and the expression of POMC neurons in the hypothalamus were detected 30 minutes after CNO injection.Results GLP-1 neurons in the NTS area of rats were successfully labeled.The rat of HM3D group revealed a decrease in food intake(P=0.021)while the rat of HM4D group showed an increase(P=0.002),when given 1 mg/kg of CNO,no changes at the dose of 0.5 mg/kg and 3.0 mg/kg.Immunofluorescence showed that the activity of GLP-1 neurons in NTS of GFP group was lower than that of HM3D group(P=0.022),and higher compared with that of the HM4D group(P=0.049).The expression of GLP-1 neurons in NTS and POMC neurons in the hypothalamus of the HM3D group after intraperitoneal injection of CNO was also higher than that in the HM4D group(P=0.003).Conclusion Using chemical genetics technology,GLP-1 controllability model rat could be successfully established via injecting varying combinations of rAAV into the NTS area of rat.Injection of 1 mg/kg CNO can effectively activate or inhibit the neuron to regulate appetite.

关 键 词：化学遗传学 神经环路 胰升糖素样肽1 食欲 

分 类 号：R338[医药卫生—人体生理学]