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目的为了寻找一种切实可行的检测肿瘤的生物标志物及其方法。方法对100例结直肠癌患者取血清,另取献血员19例,非肿瘤患者13例为对照;并另取癌组织26份,癌旁组织22份及非恶性组织29份作对比观察,同时用3种方法进行检测。一为对P16基因甲基化分析;二为对其进行缺失分析,三为对其进行点突变的分析。结果患者血清异常甲基化为69%,缺失为4%,点突变为15%,癌组织之结果与血清相似;而癌旁组织显然较低。SSCP阳性者经序列分析证实有3例为错义突变,1例为移位,1例为同义突变;其中4例造成P16蛋白的缺失。作为肿瘤的生物标志物,3项联合应用其灵敏度为88%,特异性为96.87%,准确率为90.15%。比文献用CEA+CA19-9+CA72-4+CA2424项指标联合之灵敏度、特异性、准确率皆高,尤其是特异性。如果仅用甲基化加突变亦高于前者。结论血清DNA含量不高但能反映人体对肿瘤的负荷,本实验对DNA的质和量的要求不是太高,能检出10-3的DNA片段,可用于临床诊断及大面积普查,并可用于对出院患者的长期随访。并提示P16基因异常甲基化在结直肠癌癌变机制中起决定性作用。
The purpose is to find a feasible biomarker to detect tumor and its method. Methods 100 cases of colorectal cancer patients were taken serum, another blood donation in 19 cases, 13 cases of non-tumor patients as a control; and another take 26 cases of cancer tissue, 22 cases of non-cancerous tissues and non-malignant tissue compared for observation 29, at the same time Using three methods for testing. One is methylation analysis of P16 gene; the other is deletion analysis; the third is point mutation analysis. Results In patients with abnormal methylation of 69%, deletion of 4%, point mutation to 15%, the results of cancer tissue and serum similar; and paracancerous tissue was apparently lower. Sequence analysis showed that 3 cases were missense mutation, 1 case was shift and 1 case was synonymous mutation. Among them, 4 cases resulted in the deletion of P16 protein. As a biomarker of tumor, the sensitivity and specificity of the three combinations were 88%, 96.87% and 90.15% respectively. Than the literature with CEA + CA19-9 + CA72-4 + CA2424 indicators combined sensitivity, specificity, accuracy are high, especially the specificity. If only methylation plus mutation is also higher than the former. Conclusions Serum DNA content is not high but can reflect the human body’s tumor burden. The quality and quantity of DNA in this experiment is not too high, DNA fragments of 10-3 can be detected, which can be used in clinical diagnosis and large area census, and can be used Long-term follow-up of discharged patients. And prompted abnormal methylation of P16 gene plays a decisive role in carcinogenesis of colorectal cancer.