CRF单克隆抗体和多克隆抗体制备及其在睡眠剥夺模型大鼠脑内的变化

目的:制备并鉴定促肾上腺皮质激素释放因子(CRF)的单克隆抗体(mAb)和多克隆抗体(pAb),并研究其在睡眠剥夺模型大鼠脑内的变化。方法:用CRF分别与牛血清白蛋白(BSA)和牛甲状腺球蛋白(BTG)交联,制成免疫原和检测用的包被抗原。将BSACRF分别免疫新西兰大白兔和雌性BALB/c小鼠,制备其mAb和pAb,经ELISA和免疫细胞化学染色等方法进行鉴定,并用所获抗体观察CRF在正常成年大鼠睡眠剥夺48h后脑内的变化。结果:所获得的CRFpAb和9株mAb具有较高的效价、特异性及亲和力。其中,pAb和2株mAb(1D10,2F4)可用于免疫组织化学染色。睡眠剥夺48h后下丘脑室旁核、杏仁核中央亚核、终纹状核卵圆亚核等核团高表达CRF样阳性产物,而对照组相关核团内则极少或未出现。结论:①成功地获得可用于免疫组化染色的CRFmAb和pAb。②提示CRF在中枢神经系统对睡眠剥夺造成的应激反应的调节过程中具有重要的意义。下丘脑室旁核、杏仁中央核和终纹床核是脑内相关调节环路的一部分。

Preparation of polyclonal and monoclonal antibodies against CRF and change of CRF in the sleep-deprived rat brain

AIM: To produce polyclonal and monoclonal antibodies against corticotropin-releasing factor (CRF) and to study the changes of CRF in sleep-deprived rat brain with the antibodies acquired. METHODS: Commercial CRF was linked to bovine throglobulin (BTG) and bovine serum albumin (BSA) respectively to produce immunogen and embedding antigen. New Zealand rabbits and BALB/c mice were immunized with the BTG-CRF immunogen to produce polyclonal and monoclonal antibodies respectively. The acquired antibodies were appraised with ELISA and immnohistochemical staining. The characterized antibodies were used to observe the changes of CRF in the rat brain 48 h after sleep deprivation. RESULTS: CRF polyclonal antibody and 9 clones of monoclonal antibodies were obtained with high titer, affinity and specificity. Among them, the polyclonal antibody and 2 monoclonal antibodies (1D10, 2F4) were excellent in immunocytochemical staining. The CRF-like immunoreactive substances were found more strongly expressed in the neurons of paraverticular hypothalamic nucleus (PVN), central subnucleus of amygdala (CeA), oval subnucleus of bed nucleus of stria terminalis (BNSTov), and nucleus of solitary tract (NTS) in sleep-deprived rat brain. While they were much weaker even absent in the control. CONCLUSION: The polyclonal and monoclonal antibodies against CRF were successfully produced for immunocytochemical studies. The results indicate that CRF may play an important role in stress-responsive modulation during sleep deprivation. PVN, CeA and BNSTov are integral part of brain circuit related to the stress modulation.