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目的:建立检测小鼠脂肪组织中趋化因子CCL20(CC chemokine ligand 20)mRNA的实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)的方法。方法:分离小鼠脂肪组织细胞,经PMA和离子霉素刺激4 h后收集细胞提取总RNA并逆转录为cDNA。采用qRT-PCR方法检测趋化因子CCL20 mRNA的表达水平。评价该方法的特异性,同时应用该方法检测小鼠脂肪组织中趋化因子CCL20mRNA相对表达水平。结果:建立了小鼠脂肪组织中趋化因子CCL20的SYBR Green实时荧光定量PCR检测方法,结果显示该方法的熔解曲线为单峰,同时核酸电泳显示一条特异性条带。检测结果表明高脂饮食诱导的肥胖小鼠脂肪组织中趋化因子CCL20的相对表达水平明显高于正常饮食对照组(t=3.02,P<0.05)。结论:建立了检测小鼠脂肪组织中趋化因子CCL20 mRNA的实时荧光定量PCR方法,特异性好、灵敏性强,为进一步研究小鼠CCL20的生物学功能提供了实验基础。
Objective: To establish a real-time fluorescence quantitative PCR (qRT-PCR) method for the detection of CC chemokine ligand 20 mRNA in mouse adipose tissue. METHODS: Mouse adipose tissue cells were isolated and stimulated with PMA and ionomycin for 4 h. The total RNA was collected and reverse transcribed into cDNA. QRT-PCR method was used to detect the chemokine CCL20 mRNA expression level. The specificity of the method was evaluated. At the same time, the relative expression level of chemokine CCL20 mRNA in mouse adipose tissue was detected by this method. Results: SYBR Green real-time PCR detection of chemokine CCL20 in mouse adipose tissue was established. The results showed that the melting curve of this method was single peak, and a specific band was detected by electrophoresis. The results showed that the relative expression level of chemokine CCL20 in adipose tissue of obese mice induced by high-fat diet was significantly higher than that of normal diet control group (t = 3.02, P <0.05). CONCLUSION: A real-time fluorescence quantitative PCR method for detecting chemokine CCL20 mRNA in mouse adipose tissue has been established, which has good specificity and strong sensitivity and provides an experimental basis for further study on the biological function of mouse CCL20.