地高辛标记大鼠下丘脑生长激素释放因子cRNA探针的制备及应用

PURIFICATION AND ITS APPLICATION OF DIGOXINGENIN-LABELED rhGHRF cRNA PROBE

目的:制备地高辛标记大鼠下丘脑释放因子cRNA探针.方法:大鼠下丘脑生长激素释放因子的重组质粒cDNA PGEM4经转化扩增后,用碱性裂解法获取质粒cDNR并纯化.用限制性内切酶ECORI酶切,以线性cDNA为模板.在T_7和Sp_6RNA聚合酶作用下.采用体外转录法分别合成地高辛素标记的大鼠下丘脑生长激素释放因子cRNA和RNA探针.实验采用原位杂交技术.以cDNA为探针,检测大鼠下丘脑中生长激素释放因子mRNA的分布.检测Dig-cRNA探针的灵敏度.结果:生长激素释放因子神经元呈圆形或多边形.主要集中于下丘脑弓状核团和腹内侧核团的周缘,此外.在下丘脑视前区、视交叉上核区域及视上核的结节部.还可见散在的神经元分布.结论;在对GHRF的进一步研究中,Dig-rhCHRFcRNA探针必将作为一种有力的工具而发挥重要的作用.

After conversion and amplification, recombinant rhGHRF DNA/PGEM4 was purified by al-kali denaturation and phenol-chloroform-isoamyalcohol extraction. Upon rhGHRFDNA template restricted with EcoRI, antisense and sense rhGHRF-cRNAs were labeled by digoxigenin and transcripted in vitro with T7 and SP6 RNA polymerase. The distribution of rhGHRF mRNA was showed in the rat hypothalamus within situhybridization. The results suggested rhGHRF neurons were primarily centered in the arcuate nucleus and surrounded the lateralparvicellular part of hypothalamus. In addition, a few were scattered in the suprachiasmatie nucleus, the preopticarea and the tuberal part of the supuraoptic nucleus. Almost all these cells were polygonal or round in shape. It was noted that the temperature of hybridization should be raised to 56 C.