小鼠脑干3β羟基类固醇脱氢酶1免疫阳性神经元信号检测方法的优化

Optimization of signal detection method for 3βhydroxysteroid dehydrogenase 1 immunoreactive neurons in mouse brain stem

类固醇激素合成关键调节酶-3β羟基类固醇脱氢酶1(3β-HSD1)是类固醇激素合成途径中的关键调节酶,其活性的改变可影响雄激素(睾酮)的分泌水平。检测3β-HSD1在脑组织的分布对研究脑干调节类固醇激素的分泌具有重要意义。但常规检测手段难以检测出3β-HSD1阳性信号的分布。为优化检测小鼠3β-HSD1在脑神经元中分布的方法,本研究分别采用不同固定液(4%多聚甲醛、20%甲醇+0.2%丙酮、4%多聚甲醛+10 mmol/L柠檬酸钠)预先灌注小鼠,取其脑组织制作冰冻切片,经优化的封闭前预处理后,经免疫组化方法检测3β-HSD1在小鼠脑组织中的分布以及不同固定液对其在小鼠脑干中的阳性信号检测结果的影响。同时采用western blot验证该免疫组化试验结果。结果显示,3β-HSD1主要在小鼠后脑脑干内分泌类固醇激素的主要核团,即在脑干中的缝苍白核(RPa)、巨细胞网状核(GRN)及臂旁核(PB)中表达。4%多聚甲醛固定组小鼠在上述核团内均未观察到3β-HSD1阳性荧光信号;20%甲醇+0.2%丙酮固定组小鼠在上述核团内虽可见3β-HSD1阳性信号分布,但荧光信号较模糊;4%多聚甲醛+10 mmol/L柠檬酸铵固定组小鼠在其脑干上述核团内获得染色结果清晰,神经元形态饱满的3β-HSD1阳性荧光信号。Western blot结果也证实了3β-HSD1蛋白在小鼠脑干内上述3个神经核团内的特异表达。综上所述,采用免疫组化试验检测3β-HSD1在小鼠脑内的定位时,经4%多聚甲醛+10 mmol/L柠檬酸钠固定液组的小鼠脑组织切片可在小鼠脑干RPa、GRN及PB核团内获得饱满的3β-HSD1阳性荧光信号。本实验方法的优化将为进一步研究脑内3β-HSD1影响类固醇激素合成过程的研究提供技术手段。

3β-hydroxysteroid dehydrogenase-1(3β-HSD1),the key regulator of steroid synthesis,is the key regulator of steroid hormone synthesis pathway,which can ultimately affect the secretion level of androgen(testosterone).However,the distribution of 3β-HSD1 in the brain has not been reported,which limits the understanding of energy metabolism,reproduction,endocrine and other functions in the central nervous system.In order to optimize the method of detecting the distribution of brain stem 3β-HSD1 in mice brain neurons,different fixation methods such as 4% paraformaldehyde,20%methanol+0.2% acetone,or 4% paraformaldehyde+10 mmol/L sodium citrate,were used to preperfuse the mice,and frozen sections were then prepared,optimized and pretreated before sealing[1% TritonX-100+0.4% SDS perforation,10 mmol/L citric acid].The distribution of 3β-HSD1 in the brain tissue of mice and the influence of different fixations on the neuron positive signal were detected by immunohistochemical method.Meanwhile,western blot was used to verify the results of the immunohistochemical test.The results showed that 3β-HSD1 was mainly expressed in the major nuclei of steroid hormones in the posterior brain stem of mice,such as RPA,GRN and PB.No 3β-HSD1 positive fluorescence signal was observed in 4% paraformaldehyde fixed mice;3β-HSD1 positive signal distribution was observed in 20% methanol+0.2% acetone fixed mice,but the fluorescence signal was vague;The mice in 4% paraformaldehyde+10 mmol/L sodium citrate were able to obtain 3β-HSD1 positive fluorescent signals with clear staining results and full neuron shape in the above nuclei in the brain stem.Western blot also confirmed the specific expression of 3β-HSD1 protein in the mouse brain stem.In conclusion,when using immunohistochemistry to detect the localization of 3β-HSD1 in the mouse brain,a full 3β-HSD1 positive fluorescence signal can be obtained in the RPA,GRN and PB nuclei of the brain stem of mice by the 4% paraformaldehyde+10 mmol/L sodium citrate fixation.The optimization of this method will provide a technical means for further study of the distribution of 3β-HSD1 in mice brain and the effect of 3β-HSD1 on the synthesis of steroids.