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皮质脊髓投射神经细胞(cortico-spinal projection neurons,CSPN)对于大脑的功能非常重要,但目前还缺乏研究小鼠发育期CSPN基因表达谱的有效方法。将出生后2 d(P2)的小鼠在脊髓交叉处注射逆行标记的神经示踪剂,并在P8时进行激光显微切割。提取捕获细胞的RNA并线性放大,而后利用定量PCR进行皮层标记基因的验证,线性放大后的RNA与大脑皮层组织的RNA相比,第五皮层标记基因Fezf2(Fez family zinc fi nger 2)表达明显升高,第四、第六皮层标记基因Rorβ(RAR related orphan receptorβ)、Foxp2(forkhead box P2)的表达相对较低。之后,进行小鼠基因芯片表达谱检测,通过基因功能富集度分析发现,在CSPN中大多数富表达的基因都与G蛋白偶联受体信号通路以及运动行为有关。而且,染色质的修饰和编排相关基因在CSPN里也有富集表达。通过定量PCR进一步验证了芯片中CSPN的富集基因,结果发现,Meg3(maternally expressed gene 3)等7个基因在大脑第五皮层表达丰富。该研究将在体神经细胞荧光标记、激光捕获显微切割、RNA线性放大、基因芯片及定量PCR等多种技术结合起来,提供了一种研究发育期小鼠CSPN基因表达谱的方法。
Cortical-spinal projection neurons (CSPNs) are very important for the function of the brain. However, there is a lack of effective methods to study the expression profiles of CSPN genes during mouse development. Mice born 2 days after birth (P2) were injected retrogradely labeled neurotracer at the spinal cord intersections and laser microdissected at P8. The RNA of the trapping cells was extracted and amplified linearly. Then, the cortex marker gene was verified by quantitative PCR. The expression of Fezf2 (Fez family 2 gene) was significantly increased in the linear amplified RNA compared with the RNA in the cerebral cortex The expression of RARβ or Foxp2 (forkhead box P2) in the fourth and sixth cortex was relatively low. Afterwards, the gene expression profile of mouse microarray was detected. According to the enrichment of gene function, most of the genes expressed in CSPN were related to G protein-coupled receptor signaling pathway and motor activity. Moreover, chromatin modification and orchestration related genes are also enriched in CSPN expression. Quantitative PCR further validated the chip CSPN enrichment genes and found that Meg3 (maternally expressed gene 3) and other seven genes in the fifth cortex expression is rich. In this study, a combination of techniques such as fluorescent labeling of human neural cells, laser capture microdissection, RNA linear amplification, microarray and quantitative PCR, provides a method for studying the expression profile of mouse CSPN in developmental stages.