大鼠脊髓Ⅰ型白细胞介素1受体mRNA定量检测方法的建立  

Foundation of quantitative detection of type Ⅰ interleukin 1 receptor mRNA in rat spinal cord

作　　者：王小飞[1] 王惠民[1] 陈建[2] 孙承龙[1] 王跃国[1] 

机构地区：[1]南通大学附属医院基因诊断实验室,江苏省南通市226001 [2]南通大学附属医院神经外科,江苏省南通市226001

出　　处：《中国临床康复》2005年第41期25-28,共4页Chinese Journal of Clinical Rehabilitation

摘　　要：目的:建立一种方便可靠的方法定量检测大鼠脊髓Ⅰ型白细胞介素1 受体mRNA。方法:实验于2004-09／2005-01在南通大学基因诊断实验室和上海第二医科大学神经生物学实验室完成。雌性SD大鼠15只。随机分为5组, 正常对照组3只,脊髓损伤4 h组3只,脊髓损伤4 h假手术组3只,脊髓损伤24 h组3只,脊髓损伤24 h假手术组3只。利用LightcCycler荧光定量聚合酶链反应仪和荧光染料SYBR green Ⅰ对聚合酶链反应条件进行优化后,对用重物坠落诱导的脊髓损伤模型大鼠的脊髓组织中Ⅰ型白细胞介素1受体mRNA进行实时荧光聚合酶链反应,通过标准曲线法进行绝对定量分析或通过比较Ct(△△Ct)法进行相对定量分析。结果:15只大鼠均进入结果分析。①聚合酶链反应特异性:熔点曲线分析和琼脂糖凝胶电泳表明每个聚合酶链反应都获得了特异性的扩增产物。②聚合酶链反应扩增线形与效率:该方法能够检测到最低100个拷贝的模板,对含有1 000或更多拷贝模板的常规检测可获得较好的定量数据。Ⅰ型白细胞介素1受体和次黄嘌呤磷酸核糖基转移酶的扩增效率分别是0．962,0．973。③定量计算公式的有效性:以cDNA稀释倍数对数值为X轴,以相应目的基因和看家基因的ΔCt为Y轴得到直线的斜率为0．084,可以应用公式2-ΔΔQ。④初步应用:脊髓损伤4 h,24 h后其Ⅰ型白细胞介素1受体mRNA表达水平分别有2．6倍(P<0．05)和 7．8倍的增加(P<0．01)。结论:应用SYBRgreen Ⅰ实时荧光定量方法具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等优点,非常适合于对表达低水平Ⅰ型白细胞介素1受体mRNA的组织进行定量分析。AIM：To provide a reliable and flexible method to quantitate type Ⅰ interleukin-1 receptor mRNA of rat spinal cord. METHODS：The experiment was conducted at the Gene Diagonosis Laboratory, Nantong University and Neurobiology Laboratory, Shanghai Second Medical University from September 2004 to January 2005. Fifteen female SD rats were assigned randomly into 5 groups：3 rats in normal control group,3 rats in spinal injury for 4 hours group,3 rats in spinal injury for 4 hours sham operation group,3 rats in spinal injury for 24 hours group and 3 rats in spinal injury for 24 hours sham operation group.After optimization of polymerase chain reaction （PCR）parameter,real-time quantitative PCR on the typeⅠ interleukin-1 receptor mRNA in spinal cord of rats with spinal injury induced by heavy matter falling was performed with a LightCycler rapid thermal cycler and fluorescence dye SYBR green Ⅰ.Absolute quantification analysis was done with standard curve method or relative quantification analysis was performed with comparative threshold cycle（△△Ct ）method. RESULTS：Fifteen rats were all involved in the result analysis.① Specificity of PCR：Melting curve analysis and agarose gel electrophoresis confirmed that every PCR gained the specificity of the amplification product.② Shape and efficiency of amplification of PCR： The technique was able to detect as few as 100 eontrol template copies,with quantitative data available routinely for 1 000 or more copies. The efficiencies of amplification of type Ⅰ interleukin 1 receptor and hyposanthine phosphoribosyhransferase were 0.962 and 0.973,respectively.③Effectiveness of quantitative formula： Taking logarithm value of cDNA dilution power as X-axis, △Ct of relative objective gene and house-keeping gene as Y-axis, the slope of straight line was 0.084 with the 2^-△△Cr formula.④ Primary application： Expression of type 1 interleukin 1 receptor mRNA after spinal injury for 4 hours or 24 hours increased by 2.6 times （P 〈0.05）and 7.8 time

关 键 词：脊髓 受体 白细胞介素1 逆转录聚合酶链反应 大鼠 

分 类 号：R651.21[医药卫生—外科学]