两种冻存方法在肿瘤微转移检测中的运用

Two methods of freezing storage for detecting tumor micrometastases

目的 建立科学的外周血有核细胞的冻存方法 ,为肿瘤的微转移逆转录PCR检测打好基础。方法 分离15例CEA分泌性肿瘤患者外周血有核细胞 ,分别用两种方法冻存 ,设为两组。对细胞进行总RNA的抽提 ,测定RNAOD值并行 1%琼脂糖凝胶电泳 ,进一步逆转录PCR反应、聚丙烯酰胺凝胶电泳分析最终产物。结果 经两种方法冻存的细胞 ,其总RNAOD值差异无显著性 ,电泳均见竹节样带。两组逆转录PCR结果一致 ,阳性率均为 66.7%。结论 在肿瘤的微转移逆转录PCR检测中 ,抽提组织或细胞总RNA是重要的环节 ,而对于后者来说 ,原始标本的冻存是很关键的。该实验中所采用的两种冻存方法都切实可行。

Objective To establish scientific methods of freezing storage of nucleated cells in peripheral blood for detecting tumor micrometastases with RT-PCR (reverse transcription-polymerase chain reaction) . Methods \ First, nucleated cells in peripheral blood from 15 patients with CEA-producing tumors were seperated. Secondly, two methods of freezing storage were used in two groups respectively. After total RNA extraction, OD value detection, agarose gels electrophoresis, RT-PCR and polyacrylamide gel electrophoresis were performed. Results\ There was no significant difference between the two groups of RNA OD values. After running on agarose gels, intact 28S and 18S RNAs of total RNA were clearly seen. The RT-PCR results of the two groups were consistent. The CEA mRNA positive rate was 66.7%. Conclusion \ Isolating total RNA from nucleated cells or tissues is an important step in detecting tumor micrometastases with RT-PCR. Furthermore, how to freeze the original samples seems crucial. The above-mentioned two methods are both practical.