PCR和探针自我杂交──Northern　Blot方法对二甲基苯蒽诱导的P_（450）Cyp^（1b－1）mRNA的定量

Quantitation of P_(450) Cyp￣(1b-1) mRNA by PCR and Probe Self Hybrid──Northern Blot

通过ＰＣＲ和探针自我杂交──ＮｏｒｔｈｅｒｎＢｌｏｔ方法对０．３μＭ二甲基苯蒽（ＤＭＢＡ）诱导的２×１０６个小鼠胚胎成纤维细胞１０Ｔ１／２产生的Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ进行了定量。在这个方法中，与Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ相同的特定的单链ＣＤＮＡ探针片断通过ＰＣＲ合成。而双链探针中的一条链与单链探针和Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ的特定片断是相同的。双链探针上的另一条链（Ｈ链）则与单链探针和Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ的特定片断是互补的。用３２Ｐ－ｄＣＴＰ标记Ｈ链后，它就能够与单链探针和Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ同时进行杂交。这样，就能用已知浓度的单链探针作为标准物制做标准工作曲线和对Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ的含量进行定量。经测定，在０．３μＭＤＭＢＡ分别于１、２、３、４、６小时对２×１０６个细胞进行诱导后，２×１０６个细胞在不同时间产生的Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ分别为０．００９８、０．０１３５、０．０１７４、０．０１９０和０．０１２０μＭ。

mRNA of p450 Cyp1b-1 gene produced by 2 million 10T1/2 cells(mouse embryo fibroblastcells)under the induction of 0. 3 μM DMBA has been quantified by the method of PCR andprobe self hybrid-Northern Blot. In the method,a single-strand cDNA probe and a double-strand cDNA probe were synthesize by PCR. One strand in the double-strand cDNAprobe is the same as the single-strand cDNA probe and the specific fragment of p450 Cyp1b-1mRNA,whereas another strand(hybrid strand)is complementaty with the single-strand cDNA probe and the specific fragment of P450 Cyp1b-1 mRNA. In this way,we can use the singlestrand cDNA probe of the known concentration as the standard for making a standard curveby the self hybrid of the single-strand probe and the hybrid strand of double-strad probe.Then,by the hybrid of the hybrid strand of the double-strand probe and the specific fragmentof P450 Cyp1b-1 mRNA,we can accurately detect the concentration of the mRNA. Applying thismethod,we have quantified the concentration of P450 Cyp1b-1 .mRNA. Under the induction of0. 3 μM DMBA at 1, 2, 3,4 and 6 hours,the concentrations of P450 CyP1b-1 mRNA produced by2 million cells 10T1/2 cells are 0. 0098, 0. 0135, 0. 0174,0. 0190,0. 0120μM respectively.