半定量PCR比较正常小鼠和Tfm小鼠Leydig细胞17α－羟化酶mRNA的水平

Comparison of 17α-hydroxylase mRNA Levels in normal and Tfm Mice Leydig cells with semi-quantification PCR

本研究运用反转录合成ｃＤＮＡ和半定量ＰＣＲ的方法，测定了正常小鼠和Ｔｆｍ小鼠Ｌｅｙｄｉｇ细胞中１７α－羟化酶ｍＲＮＡ／β－肌动蛋白ｍＲＮＡ的比值，以比较二者睾丸Ｌｅｙｄｉｇ细胞中１７α－羟化酶ｍＲＮＡ的水平。结果显示：１．正常小鼠Ｌｅｙｄｉｇ细胞的１７α－羟化酶和β－肌动蛋白ｃＤＮＡ的ＰＣＲ扩增指数期均在２８个循环时完成，Ｔｆｍ小鼠的β－肌动蛋白ｃＤ－ＮＡ的ＰＣＲ扩增指数期在２８个循环完成，而１７α－羟化酶ｃＤＮＡ的ＰＣＲ扩增指数期直至３２个循环仍没完成；２．在指数期时，正常小鼠Ｌｅｇｄｉｇ细胞中１７α－羟化酶ｃＤＮＡ／β－肌动蛋白ｃＤＮＡ为４．２８±０．８８，Ｔｆｍ小鼠为０．０７９±０．０４；正常小鼠１７α－羟化酶ｍＲＮＡ的水平高于Ｔｆｍ小鼠５４．１８倍。结果进一步证实Ｔｆｍ小鼠Ｌｅｙｄｉｇ细胞１７α－羟化酶ｍＲＮＡ水平低下是导致该酶活性降低的根本原因。

In this study,the ratio of P45017a. mRNA levels in normal and Tfm mouse Leydig cells to β actin mRNA was determined using a reverse transcription and semi-quantitative polymerasechain reaction (RT-PCR). The results showed that: 1. The exponential phase of the amplification of P45017α cDNA in normal mouse Leydig cells and β-actin cDNA in both normal and Tfm mouse Leydig cells was completed in 28 cycles; but the exponential phase of the amplification of P45017a cDNA in Tfm mouse Leydig cells was not completed even in up to 32 cycles; 2. The amount of P45017α. cDNA relative to β-actin cDNA was 4. 28±0. 88 in normal mice and 0. 079±0. 04 in Tfm mice Levels of P45017α mRNA relative to β-actin in normal mice were 54. 18-fold higher than those in Tfm mice. The results indicated that the reduction of 17α-hydroxylase mRNA may decrease 17α-hydroxylase activity of Leydig cells in Tfm mice.