| Objective In order to evaluate the clinical significance of plasma circulating cell free DNA size distribution state and DNA integrity in the diagnosis and treatment of NSCLC, and investigate the application value of serum tumor marker and its corresponding cell free DNA the diagnosis and treatment with NSCLC.Methods First Part Construction the standard curve of β-actin standard plasmid, set up a method for the detection of SYBR Green real time fluorescence quantitative PCR. Design 100 bp, 183 bp and 278 bp specific primers of the amplification product length to the β-actin reference gene. Collected the peripheral blood of 104 cases of NSCLC, 30 cases of benign lung disease patients, 30 cases of healthy control group, 37 cases NSCLC patients followed up for postoperative 1 day and postoperative 3 weeks, 19 patients after postoperative chemotherapy, detected the content of three free fragments of plasm circulating cell free DNA, then calculated of DNA integrity, at the same time, combined with clinical pathological data of the patients, to evaluate the value of clinical application of cell free DNA and integrity in the diagnosis and treatment of NSCLC.Second Part Design the specific primers of CEA, KRT19 target gene. Collected the peripheral blood of 95 patients with NSCLC, 30 cases of benign lung disease patients, 30 cases of healthy control group, 37 cases NSCLC patients followed up for postoperative 3 weeks, 19 cases of postoperative chemotherapy patients.Detected the content of their serum CEA and CYFRA21-1, and its corresponding cell free DNA(CEA-cf DNA KRT19-cf DNA) in their plasma, besides, the content of hs-CRP also were detected. Then, combined with clinical and pathological data of the patients, analyzed its potential application value in the diagnosis and treatment of NSCLC.Results First Part(1) The standard curve of β-actin standard plasmid were successfully constructed, set up a method for the detection of SYBR Green real-time fluorescence quantitative PCR.(2) The content of 100 bp, 183 bp, 278 bp β-actin gene in NSCLC group were significantly higher than those in benign disease group and healthy control group(P < 0.001), and three fragments of benign lung disease group were higher than those in healthy control group(P < 0.01). The content of three different fragments of β-actin gene in peripheral blood of NSCLC were different, the gene content gradually reduced with the increase of DNA fragment length. The small fragment of cell free DNA in the group of one day after operation was increased than preoperative group(P < 0.05); The content of cell free DNA of the patients followed-up 3 weekends after operation decreased significantly compared with the preoperative group(P < 0.001), but its content in the chemotherapy group were raised than that after operation 3 weekends(P < 0.01).(3) The DNA integrity of NSCLC was significantly higher than that in benign lung disease group and healthy control group(P < 0.01), the DNA integrity of the patients followed-up 3 weekends after operation were reduced compared with the preoperative group(P < 0.05).(4) The content of plasma cell free DNA was related with the clinical stage, metastasis and tumor size of NSCLC patients; There were no significant correlation between DNA integrity and the age, gender, smoking status, clinical stage, pathological type, degree of tumor cell differentiation, metastasis, and tumor size of NSCLC patients.(5) The area of ROC curve for the content of three fragment β-actin gene and DNA integrity for the diagnosis of NSCLC were 0.831, 0.814, 0.785, and 0.702.Second Part(1) The levels of plasma CEA-cf DNA, KRT19-cf DNA of NSCLC were significantly higher than those in benign disease group and healthy control group(P < 0.001). The content of postoperative CEA-cf DNA and KRT19-cf DNA decreased significantly compared with the preoperative(P < 0.05); however, the content of CEA-cf DNA after chemotherapy were increased compared with postoperative group(P < 0.05).(2) The serum levels of CEA, CYFRA21-1 of NSCLC were significantly higher than those in benign disease group and healthy control group(P < 0.05), the content of CEA in benign disease group were significantly higher than those in healthy control group(P < 0.01); The content of hs-CRP had no significant difference between NSCLC and pulmonary benign disease group, but they both higher than that in healthy control group(P < 0.01). In the group of operation, CEA and CYFRA21-1 were significantly decreased(P < 0.05). There were no difference between the chemotherapy group and the patients after operation; the content of hs-CRP had no significant difference before and after surgery and chemotherapy.(3) The content of CEA-cf DNA was related to the size of NSCLC, and the KRT19-cf DNA to tumors stage, metastasis tumor size. While, the CEA content was related to the type of tumor pathology, the CYFRA21-1 levels to tumor differentiation, transfer and tumor size of patients, and hs-CRP content to the degree of tumor differentiation, clinical stage, metastasis and tumor size.(4) The area of ROC curve of CEA-cf DNA, KRT19-cf DNA, serum CEA, CYFRA21-1 and hs-CRP in the diagnosis for NSCLC were 0.911, 0.803, 0.839, 0.759,and 0.622. There was a significant correlation between the serum levels of CEA, CYFRA21-1 and their corresponding cell free DNA(R = 0.324, 0.401, P < 0.01).Conclusion1. There is a highly discrimination of plasma cell free DNA and DNA integrity in NSCLC, its have a certain clinical application value of the early diagnosis and postoperative followed-up with NSCLC.2. There is a highly application value of serum tumor markers and their corresponding plasma cell free DNA in the early diagnosis and treatment of NSCLC.3. The plasma cell free DNA content and serum markers have a certain reference value of patients with NSCLC staging, differentiation, tumor burden, and metastasis clinical condition.4. Detection of peripheral blood cell free DNA has broad prospects in clinical application for its advantages of convenient and simple operation, small and easy to get and carry out large-scale. |
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