制备人Ⅱ型多巴胺受体cRNA探针检测帕金森病大鼠模型纹状体内Ⅱ型多巴胺受体基因表达的变化  

Preparation of the cRNA probe of human ⅡDopamine D_2-receptor to detect the change of expression of Dopamine D_2-receptor geneⅡin corpus striatum in rat model with Parkinson disease

作　　者：赵焕英[1] 李良[1] 刘延英[1] 刘静[1] 孙晓红[1] 杨慧[1] 

机构地区：[1]首都医科大学北京神经科学研究所,北京市神经再生修复重点实验室,北京市100054

出　　处：《中国临床康复》2005年第25期25-27,i0001,共4页Chinese Journal of Clinical Rehabilitation

基　　金：北京市教委科技发展规划重点项目(KZ200310025009);北京市自然基金项目(7022005);国家科技高技术研究发展计划(八六三计划)(2003AA326010)~~

摘　　要：目的:获得单链、杂交结果稳定的人Ⅱ型多巴胺受体基因正义和反义RNA探针,检测人Ⅱ型多巴胺受体在帕金森病大鼠模型中表达的变化。方法:实验于2002-10/2003-06在首都医科大学神经科学研究所内完成。选出2,6,12周帕金森病大鼠模型60只,每组各20只,用来做原位杂交的组织处理;将已制备的pGEM-T-Easy-D2R质粒载体分别经ClaI、XbaⅠ限制性酶切反应得到线形质粒,再分别以T7和SP6聚合酶制备了人Ⅱ型多巴胺受体基因的反义和正义探针,通过斑点杂交检测探针的浓度;将反义和正义探针分别作等梯度稀释,分别与组织切片杂交,强度洗脱后,观察使对照正义链探针没有杂交信号,而反义探针杂交后也无本底的最适宜浓度,从而降低内缘性其他多巴胺受体亚型的干扰作用;用制备的最适宜浓度探针分别杂交帕金森病大鼠模型2,6,12周的组织切片,杂交后冲洗,免疫检测及呈色,观察帕金森病大鼠模型纹状体中Ⅱ型多巴胺受体表达。结果:①实验标记的人Ⅱ型多巴胺受体探针属cRNA探针,其特点为单链探针,杂交过程不需变性,可合成最可靠的反义链探针,杂交结果稳定,体外转录合成的cRNA探针长度也比较一致,其浓度分别为150,120mg/L。②将反义和正义探针分别等梯度稀释,分别与组织切片杂交,强度洗脱后,结果证明25mg/L浓度最适宜,对照正义链探针没有杂交信号,而反义探针杂交后也无本底。③原位杂交证实,反义探针能够与帕金森病大鼠模型纹状体中Ⅱ型多巴胺受体mRNA杂交。用反义探针标记的细胞中可见蓝色颗粒沉积,左右半球纹状体中标记细胞数在2周时没有变化,帕金森病大鼠损伤侧和健侧Ⅱ型多巴胺受体mRNA表达没有明显变化;6周时,损伤侧较健侧表达明显上调,12周时,损伤侧与健侧表达都下降且没有明显差别。结论:实验获得了人Ⅱ型多巴胺受体基因反义和正义cRNA探针,并得出无�AIM：To obtain the sense and anti-sense RNA probe with stable monochain and hybridization result of Dopamine D2-receptor （D2R） gene, and detect the changes of expression of D2-receptor in Parkinson （PD） model of rats. METHODS： The experiment was done from October 2002 to June 2003 in Beijing Institute for Neuroscience of Capital University of Medical Sciences. Sixty rat models with Paralysis disease at 2, 6, 12 weeks with 20 rats in each group were selected to be used for the tissue disposal of hybridization in situ; The prepared pGEM-T-Easy-D2R plasmid cartier was gained the line-form plasmid through limited ClaI ,Xba I cutting enzyme reaction, respectively, and using T7 and SP6 polymerase to make sense and anti-sense probe of Dopamine D2-receptor gene, and the concentration of probe was detected by dot blot hybridization; The same gradient dilution was done on sense and anti-sense probe, respectively, and crossed with tissue section. After the elution of intensity, there was no hybridization signal of control sense chain probe observed, while there was no the most suitable concentration after anti-sense hybridization, in order to decrease the effect of intervention of other infra-border Dopamine receptor isoforms; The prepared most suitable concentration probe was crossed with tissue section of rat models with Parkinson＇s disease at 2, 6, 12 weeks, and after hybridization they were douched, immunity detection and appeared color to observe the expression of Dopamirie D2-receptor in corpus striatum of rat models with Parkinson＇s disease. RESULTS： ① Dopamine D2-receptor probe labeled in this experiment belonged to cRNA probe and its characteristics was monochain probe and no need to degenerate during the process of hybridization and could be used as the most reliable anti-sense chain probe with a stable hybridization result. The cRNA probe length of composite transcription in vitro was more or less coincidence, and its concentration was 150,120 mg/L, respectively. ② The anti-sense and sense pr

关 键 词：帕金森病 受体 多巴胺/遗传学 原位杂交 基因 RNA 信使/生物合成 

分 类 号：R742[医药卫生—神经病学与精神病学]