鸡血管活性肠肽RNA探针制备及其在鸡肠Remak神经的原位杂交反应

Preparation of chicken vasoactive intestinal peptide RNA probe and its detection in intestinal nerve of Remak by in situ hybridization

应用RT-PCR方法从鸡脑中扩增血管活性肠肽(VIP)基因片段,将其连接于pGM-T easy。分别利用pGM-T easy中T7和SP6启动子及其RNA聚合酶,以线性化的VIP/pGM-Teasy为模板转录合成正、反义DIG标记RNA探针。通过原位杂交方法将合成的探针用于探查VIP-mRNA在鸡肠Remak神经(INR)中的分布情况。结果表明,INR的神经元胞体中有VIP-mRNA的转录,且这种神经元数量众多;原位杂交阳性神经元胞体大部分是大型神经元细胞,呈圆形或椭圆形;阳性神经元胞体在INR神经节中呈层状或成群分布。INR的神经纤维呈弱阳性。在节间束也有少量的阳性细胞分布。本研究从基因水平证明INR中有VIP神经元存在。

To study the distribution of vasoactive intestinal peptide （VIP） mRNA in chicken intestinal nerve of Remak （ INR）, the sense and anti-sense digoxigenin （DIG） labeled RNA probe were prepared and utilized. The fragment of VIP gene was obtained by RT-PCR through total RNA of chicken brains. Amplified cDNA fragment was subeloned into pGM-T easy vector, and the plasmid was transformed into E. coli DFISet and chosen by ＂white-blue plaque selection＂. The recombinant plasmid was identified by EcoR I restriction enzyme digestion and sequencing, then VIP/pGM-T easy vector was linearized with the restriction enzyme of Nco Ⅰand Sal Ⅰrespectively. The sense and anti-sense DIG labeled RNA probe were producted by SP6 and T7 RNA polymerase respectively and transcription in vitro according to the protocol of ＂DIG RNA Labeling Kit （SP6/TT） ＂. Certificated by dot blot hybridization, the sense and anti-sense RNA probe were prepared successfully. The distribution of VIP-mRNA in chicken INR was examined by in situ hybridization histochemistry （ISHH）. There were many labeled neurons distributing in ganglion and nerve trunk between ganglia, most of these labeled neurons were large size neurons and had round or oval shapes. Some the fibers of INR were weakly positive. In conclusion, the sense and anti-sense DIG labeled RNA probes for ISHH of VIP were prepared successfully in this experiment, which provided an approach to study further the location of VIP-mRNA in nerve tissue ; the result of ISHH suggested the existence of VIP-mRNA in neurons of chicken INR.