摘要
目的 :建立一种客观、敏感的检测IL 2 /IL 4细胞因子基因表达变化的方法。 方法 :采用TRIzol试剂提取免疫排斥 /免疫耐受大鼠脾脏组织总RNA ,分光光度计法定量。取 2 μg总RNA ,采用RT PCR法逆转录 扩增IL 2、IL 4和内参照 β actincDNA ,扩增产物经电泳分离后 ,用凝胶图像分析仪照相和扫描分析 ,检测其相对表达量。 结果 :RT PCR产物经电泳后 ,β actin、IL 2和IL 4分别在 2 2 6bp、342bp和 332bp处出现明显条带 ,与预定条带大小相一致 ;免疫耐受组IL 4与免疫排斥组IL 2的表达有明显差异 ,在免疫耐受组中IL 4表达增高 ,而在免疫排斥组中IL 2表达增高 ,内参照 β actin扩增条带亮度在两组中相同 ,PCR产物量与扩增初始模板量之间存在良好的对应关系。 结论 :半定量RT PCR检测是一种从少量细胞中快速、简便。
Objective: To establish an objective and sensitive method for the assessment of IL 2/IL 4 gene mRNA expression in heart transplantation in rats. Methods: Total RNA was abstracted from rat spleen with TRIzol kit and quantified by Smartspec 3000 OD machine. Using 2 μg total RNA as a template, the relative content of IL 2 and IL 4 mRNA was analyzed by semi quantitative reverse transcription and polymerase chain reaction method (SqRT PCR). β actin mRNA was synthesized as the inner control. The quantity of IL 2/IL 4 mRNA expression was determined by the method of sqRT PCR. Results: The length of β actin, IL 2 and IL 4 PCR products were 226 bp,342 bp and 332 bp as designed, respectively. According to the brightness of the band, the IL 4 mRNA expression was increased in immunized tolerance group and IL 2 mRNA expression was increased in immunized rejection group, but the β actin expression was same in two groups. This showed that there was a significant difference of the mRNA expression between the IL 2 in immunized rejection group and the IL 4 in immunized tolerance group. The current experimental study indicates that there may be a correlation between the quantity of PCR products and the original template. Conclusion: A rapid, simple and reliable method is established for determination of specific mRNA expression from minute quality of cells.
出处
《新疆医科大学学报》
CAS
2003年第5期427-429,共3页
Journal of Xinjiang Medical University
基金
国家自然科学基金资助项目 (3 0 160 0 81)
新疆维吾尔自治区科技厅基金资助项目(2 0 0 2 2 110 1)
