| Lung cancer is one of the most frequent malignant tumors in humans and the prognosis is poor. In order to improve the clinical approach to lung cancer patients, several serum tumor markers such as CYFRA2 1-1 CEA NSE have been studied. The sensitivity of each of these markers is not high enough, the study about combination ofCYFRA2I-1. CEA and NSE is few. Distant metastases are a key factor in planning the treament of lung cancer, so recent interest has been focused on the detection of circulating cancer cells in the peripheral blood of lung cancer patients. Now a technique named reverse -transcriptase polymerase chain reaction (RT-PCR) has been used to detect the hematogenous micrometastases. The sensitivity and specificity of this techinique are higher than immunoctology. Cytokeratin 19 (CKI9) is used as a target gene. Immunologic studies have been showed CK19 mRNA expression in a significant 5 number of lung cancer specimens, but low-level expression of CKI9 mRNA in mormal peripheral blood mononucleated cells (PBMCS) is still debated. Based on these data, we analyzed expression of CK19 mRNA in PBMCS from 67 lung cancer patients and 30 benign lung diseases (BLD) .The rate of CK 19 mRNA positivity was significant higher in lung cancer patients than in BLD controls (P < 0.05). Combination of CYFRA2I-1 and CEA or NSE can improve the sensitivity. 1 Patients and Methods 1.1 Patients 1.1.1 The study population consisted of 67 histologically documoned lung cancers. Staging procedures were based on TNM stage in 1997 of UICC, 23 stage I + II, 27 stage III, 17 stage IV. Histopathologic examination showed 25 squamous cell cancinomas, 28 adenocarcinomas, 14 small-celIl lung cancers (SCLC). 1.1.2 The control population consisted of 30 BLD, it contained pneumonias, chronic bronchitis, lung abscess, pulmonary tuberculosis, lung benign tumor. 1.2 Sample Collection and RNA Preparation We used Vacutainer collection tubes to collect 7ml blood, 2m1 blood in the first tube was used to detect CYFRA21-1 CEA and NSE, 5m1 blood in the second tube containing heparin was used to detect CKI 9 mRNA. then used Trizol Kit to extract total mRNA from PBMCS and solved it in I Opi DEPC water. 1.3 RT-PCR 1.3.1 RT 6 2OiiI reaction mix contained oligo dT primer, 10mM deoxynucleotide trisphophate (dNTP) , 0.1 M dithiothreitol (DTT) , RNAs in, MMLV-RT, 5 x MMLV-RT buffer, DEPC water. Negative control contained DEPC water instead of RNA. 1.3.2 PCR PCR reactions were performed in 25.tl final volume that contained cDNA, lOx PCR buffer, 25mM Mgcl2, 10mM dNTP, Taq polymerase, primers for CK19 and ~ -actin . PCR conditions were consisted of one amplification round of 40 cycles at 950C for 1 minute, 54擟 for 1 minute, 72扖 for 90 seconds and 720C for 1 Ominutes . Negative control contained DEPC water instead of cDNA. 1.3.3 After the PCR was completed, 1 Oi.tl of PCR product were analyzed on agarose gel . CK19 produt was 460 bp size and ~ -actin produt was 370 bp size. 1.4 CYFRA2 1-1 and NS...
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