【摘要】 目的 采用化学遗传学技术选择性激活雌鼠下丘脑弓状核（arcuate nucleus, ARC） Kisspeptin神经元以研究其在骨代谢方面的作用。方法 采用双侧卵巢切除（ovariectomy, OVX）构建绝经后骨质疏松小鼠模型，将8～10周龄杂合型Kiss1-CreGFP雌鼠，按手术类型（OVX/SHAM）、病毒类型[人毒蕈碱型乙酰胆碱受体M3亚型（Gq蛋白偶联型）（human muscarinic acetylcholine receptor M3 subtype(Gq-coupled), hM3D(Gq)/空载]、药物类型[（氯氮平-N-氧化物（clozapine-N-oxide, CNO）/生理盐水（normal saline, NS）]三因素随机分为6组：Cre-OVX-hM3-CNO组、 Cre-OVX-hM3-NS组、 Cre-SHAM-hM3-CNO组、 Cre-SHAM-hM3-NS组、 Cre-OVX-空载-CNO组、 Cre-OVX-空载-NS组，另设2组野生型对照：Control-OVX、 Control-SHAM。通过脑立体定位技术对小鼠双侧下丘脑注射腺相关病毒（adeno-associated virus, AAV）-EF1α-DIO-hM3D(Gq)-mCherry，使小鼠表达hM3D(Gq)，在饮用水中加入CNO，通过CNO和hM3D(Gq)受体的结合以激活Kisspeptin神经元。造模完成后，取小鼠全脑，进行冰冻切片和免疫荧光，以评估病毒的感染和表达情况；对小鼠股骨使用micro-CT扫描并进行三维图像重建和数据分析，同时进行骨组织切片和苏木素伊红（hematoxylin-eosin, HE）病理染色，以评估下丘脑ARC部位Kisspeptin神经元的激活对雌鼠骨量和骨髓腔脂肪细胞的影响。结果 与Control-SHAM组小鼠相比，Control-OVX组雌鼠骨量减少，骨体积分数（bone volume/total volume, BV/TV）降低（P<0.01）、骨小梁数量（trabecular number, Tb. N）减少（P<0.001），骨小梁分离度（trabecular separation, Tb. Sp）升高（P<0.01）；股骨远端骨小梁和骨髓细胞分布稀疏，骨髓腔中脂肪细胞数量增多。在Kiss1-CreGFP小鼠双侧下丘脑注射AAV-EF1α-DIO-hM3D(Gq)-mCherry, 3周后病毒成功感染并表达相应受体。Cre-OVX-hM3-CNO组骨量减少，骨髓腔中脂肪细胞增多，BV/TV降低（P<0.01）、 Tb. N减少（P<0.01）, Tb. Sp升高（P<0.05）。结论 雌鼠下丘脑ARC部位的Kisspeptin神经元对骨骼稳态起负性调控作用。更多还原

【Abstract】 Objective To explore the influence of Kisspeptin neurons on bone metabolism by selectively activating them in the arcuate nucleus(ARC) of mice using chemical genetic techniques.Methods A bilateral ovariectomy method was used to construct a postmenopausal osteoporosis model in female mice. Kiss1-CreGFP female mice aged 8-10weeks were randomly divided into six groups based on three factors: surgery type(OVX/SHAM), virus type(human muscarinic acetylcholine receptor M3 subtype( Gq-coupled) [ hM3D( Gq) ] /empty vector), and drug type( clozapine-N-oxide [ CNO ] /normal saline [ NS ]): Cre-OVX-hM3-CNO group, Cre-OVX-hM3-NS group,Cre-SHAM-hM3-CNO group, Cre-SHAM-hM3-NS group, Cre-OVX-empty vector-CNO group, Cre-OVX-empty vectorNS group. Two additional wild-type control groups, Control-OVX group and Control-SHAM group were included. The expression of hM3D(Gq) receptor was induced by bilateral injection of adeno-associated virus(AAV)-EF1α-DIO-hM3D(Gq)-mCherry into the hypothalamus of female mice by stereotactic technique, and clozapine-N-oxide(CNO) was administered daily in drinking water to activate Kisspeptin neurons through the binding of CNO and hM3D(Gq) receptors.After the model was established, the whole brain of the mice was taken for frozen section and immunofluorescence to evaluate the virus infection and expression. Mouse femur were scanned by micro-CT, three-dimensional image reconstruction and data analysis were performed. Bone tissue sections and hematoxylin-eosin(HE) staining were performed to evaluate the effect of Kisspeptin activation on bone mass and bone marrow cavity adipocytes in female mice.Results Compared with the female mice in the Control-SHAM group, the bone volume of female mice in the Control-OVX group was reduced,bone volume/tissue volume(BV/TV) was reduced(P< 0. 01), bone trabecular number(Tb. N) was reduced(P<0. 001), and trabecular separation(Tb. Sp) was increased(P<0. 01); femur distal trabecular and bone marrow cells were sparsely distributed, and the number of adipocytes in the bone marrow cavity was increased. Injected AAV-EF1α-DIO-hM3D(Gq)-mCherry virus into the hypothalamus of Kiss1-CreGFP female mice, and the virus successfully infected and expressed the corresponding receptor after 3 weeks. In the Cre-OVX-hM3-CNO group, selective activation of Kisspeptin neurons induced significant bone mass reduction and increased adipocytes in the bone marrow cavity, decreased BV/TV(P<0. 01), decreased Tb. N(P<0. 01), and increased Tb. Sp(P<0. 05) in OVX female mice.Conclusion Kisspeptin neurons in the hypothalamic arcuate nucleus of female mice negatively regulate skeletal homeostasis.更多还原