改进竞争性RT-PCR法测定缺碘大鼠颅骨中BMP-2基因表达变化

Expression of BMP-2 Gene in Calvaria of Iodine -deficient Rat Detected by a Modified Competitive RT-PCR Assay

为更加精确地测定碘缺乏状态下大鼠颅骨组织中骨形态发生蛋白 2 (BMP 2 )mRNA表达水平 ,改进竞争性RT PCR方法 ,探讨碘缺乏对骨骼发育影响的分子机制 .将构建的DNA竞争模板克隆入pSportⅠ质粒中 ,利用pSportⅠ质粒中所含有的SP6RNA聚合酶启动子序列 ,将DNA竞争模板在体外转录成为RNA .以此RNA作为竞争性RT PCR中的竞争模板 ,与所提取的总RNA一同进行RT PCR ,消除逆转录效率对定量结果的影响 .定量检测碘缺乏和正常大鼠颅骨总RNA中BMP 2mRNA的水平 .碘缺乏大鼠 (1d～ 84d)颅骨中BMP 2表达含量明显降低 .1d时降低 6 4 3倍 ,以后差距逐渐缩小 ,至 84d相差 1 5 5倍 .结果提示 ,碘缺乏所致的骨骼发育不良与BMP 2表达不足有关 ,结果首次揭示微量元素碘和甲状腺激素与BMP

To study the molecular mechanism of bone development retardation caused by iodine deficiency and measure more accurately the bone morphogenetic protein 2(BMP 2) mRNA level in rat calvaria,a modified competitive RT PCR method was established. The DNA competitor was cloned into pSportⅠ plasmid and was transcripted into RNA by using SP6 RNA polymerase in vitro. The RNA was purified and quantified by measuring the absorbance at 260 nm. This RNA competitor along with the total RNA isolated from rat calvaria were reverse transcripted and amplified by PCR in an Eppendorf tube. The modified method might be more accurate by eliminating the influence of the efficiency of reverse transcription to the data of BMP 2 mRNA level. BMP 2 mRNA levels in iodine deficient and normal rat calvaria were determined by this competitive RT PCR method respectively.BMP 2 mRNA expression in iodine deficient rats was lower than that in normal rats (1～ 84 days). It was 6 43 times decreased on day 1 and it was 1 55 times decreased on day 84. These results suggested that the retardation of bone development caused by iodine deficiency might be related to the low expression level of BMP 2 gene. It is the first report to elucidate that the microelement iodine and thyroid hormones are involved in BMP 2 expression regulation.