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目的建立检测人广泛型线粒体肌酸激酶(ubiquitous mitochondrialc reatine kinase,uMtCk)mRNA荧光定量的方法,并检测了结直肠腺癌病人肿瘤组织和癌旁组织中uMtCk基因表达的水平。方法基于TaqMan荧光探针技术,以GAPDH作为内参,建立实时荧光定量RT-PCR方法,并检测了30名结直肠腺癌病人肿瘤组织和癌旁组织uMtCk mRNA的表达。结果30例结直肠肿瘤患者肿瘤组织中有uMtCk mRNA的表达,范围为7.4×105~5.3×108拷贝/μgRNA,均值为(9.4±5.7)×106拷贝/μgRNA;30例癌旁组织uMtCk表达为4.5×103~6.9×105拷贝/μgRNA,均值为(8.6±3.4)×104拷贝/μgRNA。结论成功地建立了人uMtCk基因表达含量的荧光定量检测方法,检测结果可用绝对拷贝数表示,定量准确可靠。且结直肠腺癌病人肿瘤组织uMtCk mRNA的含量是升高的,提示uMtCk mRNA含量的检测有助于结直肠腺癌病人临床诊断。
OBJECTIVE: To establish a method for the quantitative detection of uMtCk mRNA in ubiquitous mitochondrial c reatine kinase (uMtCk), and to detect the expression of uMtCk gene in tumor tissues and paracancerous tissues of patients with colorectal adenocarcinoma. Methods Based on TaqMan fluorescent probe technology and real-time fluorescent quantitative RT-PCR with GAPDH as internal control, the expression of uMtCk mRNA in 30 specimens of colorectal adenocarcinoma was detected. Results The expression of uMtCk mRNA in the tumor tissues of 30 patients with colorectal cancer ranged from 7.4 × 105 to 5.3 × 108 copies / μgRNA with the mean of (9.4 ± 5.7) × 106 copies / μgRNA. The expression of uMtCk in 30 paracancerous tissues was 4.5 × 103 ~ 6.9 × 105 copies / μg RNA, mean (8.6 ± 3.4) × 104 copies / μg RNA. Conclusion The fluorescence quantitative detection method of human uMtCk gene expression was successfully established. The detection results can be expressed in absolute copy number, and the quantification is accurate and reliable. Moreover, the content of uMtCk mRNA in the tumor tissue of patients with colorectal adenocarcinoma is increased, suggesting that the detection of uMtCk mRNA content is helpful for the clinical diagnosis of patients with colorectal adenocarcinoma.