基于RNA干扰技术的多巴胺D2受体低表达的高催乳素MMQ细胞模型构建  

Establishment of D2R down regulated MMQ cells model for hyperprolactinemia using RNA interference technigue

作　　者：张志洋 徐志卿[2,3] 刘丽娜[2,3] 薄奇静 王传跃[1,3] Zhang Zhiyang;Xu Zhiqing;Liu Lina;Bo Qijing;Wang Chuanyue(The National Clinical Research Center for Mental Disorders&Beijing Key Laboratory of Mental Disorders&Beijing institute for Brain Disorders Center of Schizophrenia,Beijing Anding Hospital,Capital Medical University,Beijing 100088,China;Beijing Key Laboratory of Neural Regeneration and Repair,Department of Neurobiology,Capital Medical University,Beijing 100069,China;Advanced Innovation Center for Human Brain Protection,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学附属北京安定医院国家精神心理疾病临床医学研究中心精神疾病诊断与治疗北京市重点实验室北京脑重大疾病研究院精神分裂症研究所,100088 [2]首都医科大学神经生物学系神经再生修复研究北京市重点实验室,100069 [3]首都医科大学人脑保护高精尖创新中心,北京100069

出　　处：《神经疾病与精神卫生》2022年第7期463-467,共5页Journal of Neuroscience and Mental Health

基　　金：国家自然科学基金项目(81901355);北京市自然科学基金项目(7192081)。

摘　　要：目的利用RNA干扰技术建立多巴胺D2受体(D2R)低表达的高催乳素大鼠垂体瘤MMQ细胞模型。方法设计3对可沉默D2R基因表达的小干扰RNA(siRNA)序列,转染MMQ细胞,建立包括siRNA1组、siRNA2组、siRNA3组、阴性对照(siNT)组和空白对照(CTRL)组的5组细胞模型,采用Western blot法检测各组MMQ细胞D2R蛋白表达。选择干扰高表达细胞株,加入溴隐亭1μmol/L进行干扰,采用酶联免疫吸附测定(ELISA)法、Western blot法及实时定量聚合酶链反应(qRT-PCR)法分别观察干扰后MMQ细胞催乳素的分泌、催乳素蛋白相对表达量及催乳素mRNA水平。结果5组D2R蛋白相对表达量比较,差异有统计学意义(F=19.936,P<0.01);其中siRNA1组、siRNA3组D2R蛋白相对表达量[(0.23±0.12)、(0.57±0.24)]与siNT组(0.81±0.24)、CTRL组(0.94±0.21)比较,差异均有统计学意义(均P<0.01);siRNA1组、siRNA3组对MMQ细胞的D2R蛋白表达抑制率分别为74%和35%。干扰后,siRNA组、CTRL组、siNT组MMQ细胞催乳素分泌、催乳素蛋白相对表达量、催乳素mRNA水平比较,差异均有统计学意义(F=10.898、7.485、7.898,均P<0.05);siRNA组催乳素分泌高于siNT组、CTRL组[(2.91±0.12)ng/ml比(2.14±0.15)、(2.09±0.44)ng/ml];siRNA组催乳素蛋白相对表达量高于siNT组、CTRL组[(0.99±0.67)比(0.85±0.13)、(0.82±0.12)];siRNA组催乳素mRNA水平高于siNT组、CTRL组[(1.00±0.07)比(0.69±0.09)、(0.73±0.14)],差异均有统计学意义(均P<0.05)。结论基于RNA干扰技术构建的D2R低表达的高催乳素MMQ细胞模型为目标药物治疗高催乳素血症的靶点研究提供了可靠的细胞模型。Objective To construct dopamine D2 receptor(D2R)gene low expression model in rat MMQ cells by RNA interference(RNAi)technique.Methods Three fragments of small interfering RNA(siRNA)were designed,which could induce decrease in D2R mRNA expression,then the plasmid was constructed to transfect the MMQ cells,and siRNA1,siRNA2,siRNA3,nontarget siRNA(siNT),normal control(CTRL)groups were established.The expression of D2R protein was detected by Western blot.High interference expression cell line was chosen,and then adding bromocriptine(1μmol/L).Secretion of prolactin,relative expression of prolactin and prolactin mRNA level in MMQ cells after interference were observed by ELISA,Western blot,qRT-PCR.Results The relative expression levels of D2R protein was significantly different among the 5 groups(F=19.936,P<0.001).Among them,the relative expression levels of D2R protein in MMQ cells of siRNA1 and siRNA3 groups[(0.23±0.12),(0.57±0.24)]were significantly different from those of siNT group(0.81±0.24)and CTRL group(0.94±0.21)(all P<0.05).siRNA1 and siRNA3 groups inhibited the repression of D2R protein in MMQ cells by 74%and 35%,respectively.After interference,there were statistically significant differences in secretion of prolactin,relative expression of prolactin and prolactin mRNA level of MMQ cells among the siRNA group,CTRL group and siNT group(F=10.898,7.485,7.898,all P<0.05).The secretion of prolactin of siRNA group was higher than that of siNT group and CTRL group[(2.91±0.12)ng/ml vs(2.14±0.15)ng/ml,(2.09±0.44)ng/ml].The relative expression of prolactin protein of siRNA group was higher than that of siNT group and CTRL group[(0.99±0.67)vs(0.85±0.13),(0.82±0.12)].The prolactin mRNA level of siRNA group was higher than that of siNT group and CTRL group[(1.00±0.07)vs(0.69±0.09),(0.73±0.14)].All the differences were statistically significant(all P<0.05).Conclusions The constructed hyperprolact in MMQ cell model with low D2R expression by RNAi provides a reliable cell model for the target study of target

关 键 词：高催乳素血症 RNA干扰 受体 多巴胺D2 MMQ细胞 

分 类 号：R965[医药卫生—药理学]