鸡CDC25B-mRNA探针制备及其在鸡十二指肠黏膜的原位杂交反应

Preparation of Chicken CDC25B-mRNA Probe and Its Detection in Duodenal Mucosa by in situ Hybridization

应用RT-PCR方法从鸡胚中扩增CDC25B基因片段,将其连接于pGM-Teasy。分别利用pGM-Teasy中T7和SP6启动子及其RNA聚合酶,以线性化的CDC25B/pGM-Teasy为模板转录合成正、反义DIG标记RNA探针。以合成的地高辛(DIG)标记的CDC25B-mRNA为探针,进一步应用原位杂交组织化学方法(in situhybridiza-tion histochemistry,ISHH)探查CDC25B-mRNA在鸡十二指肠黏膜的分布。结果表明,成年鸡十二指肠肠腺上皮细胞中有丰富的CDC25B-mRNA的转录,其中CDC25B-mRNA探针杂交阳性细胞在肠腺基底部和小肠绒毛中部分别占上皮细胞总数的81.60%±9.63%和36.21%±8.81%。原位杂交阳性产物大部分分布于十二指肠肠腺上皮"干细胞部"和"增生部"细胞的胞质和胞核,肠腺基底部杂交信号由下至上逐渐减弱,至小肠绒毛下部消失,转为阴性。在黏膜肌层以及肌肉层杂交反应呈阴性。本研究从基因水平证明了鸡十二指肠黏膜肠腺上皮"干细胞部"和"增生部"细胞中有CDC25B-mRNA的存在,表明该区域有活跃的增殖过程,以补充绒毛上端死亡脱落的细胞。

In order to investigate the distribution of CDC25B-mRNA in the duodenal mucosa of chicken, the sense and anti-sense digoxigenin （DIG） labeled RNA probe were prepared and utilized. The fragment of CDC25B gene was obtained by RT-PCR through total RNA of chicken embyros. Amplified cDNA fragment was subcloned into pGM-T easy vector, and the plasmid was transformed into E. coli DH5α and screened by ＂white-blue plaque selection＂. The recombinant plasmid was identified by EcoR Ⅰ restriction enzyme digestion and sequencing, then CDC25B/ pGM-T easy vector was linearized with the restriction enzyme of Nco Ⅰ and Spe Ⅰ respectively. The sense and anti-sense DIG labeled RNA probe were produced by SP6 and T7 RNA polymerase respectively and transcription in vitro according to the protocol of ＂DIG RNA Labeling Kit （SP6 /T7）＂. The sense and anti-sense RNA probes were prepared successfully. The distribution of CDC25B-mRNA in the duodenal mucosa of chicken was examined by in situ hybridization histochemistry（ISHH）. There were many labeled cells distributing in the duodenal mucosa of adult chicken. Of these labeled cells, 81.60%±9.63% and 36.21%±8.81% CDC25B-mRNA positive cells were distributed in the basilar part and middle portion of duodenal mucosa of adult chickens respectively. Most of these labeled cells were positive in the regions of the stem cell and proliferation. The positive signals were both in the cytoplasm and cell nucleus. The signals of ISHH were decreased from basilar part to upper in the crypt of lieberkuhn and disappeared in the inferior of villi of small intestine. The labeled cells were both negative in the lamina muscularis mucosae and muscular layer. In conclusion, the sense and anti-sense DIG labeled RNA probes for ISHH of CDC25B were prepared successfully in this experiment, which provided an approach to study further the location of CDC25B-mRNA in chicken. The results of ISHH confirmed the existence of CDC25B-mRNA and athletic proliferation activities in the duodenal mucosa of adult chicken.