| In order to increase the survival of patients with lung cancer, it is important to find out sensitive and specific tumor markers of lung cancer. Circulating DNA is the free DNA in body fluid (eg: serum or plasma etc.) and it has been used to gene diagnosis. Loss of heterozygosity occurs commonly in human lung cancer. Our study is to investigate the loss of heterozygosity (LOH) of plasma circulating DNA of patients with lung cancer and evaluate the value of circulating DNA as lung cancer tumor marker.This study included two parts as following:1. To establish a method to extract plasma DNA.Three methods, including phenol/chloroform method, QIAamp DNA extraction kit and boiling method, were used to extract plasma DNA of 10 patients. They were sucessfully extracted 10, 10, 4 cases of circulating DNA in 10 plasma samples respectively. DNA purity was identified by the electrophoresis, and DNA concentration was deterimined by the ultravioletspectrometer. Plasma DNA concentraction with phenol/ chloroform method was 129±87.9 ng/ml, and it with QIAamp DNA extraction kit was 133±88.5 ng/ml (p>0.05). But there was no detectable DNA concentraction with boiling method by ultraviolet spectrometer. And the ratios of OD260/OD280 of the former two methods were 1.76 ± 0.12 and 1.68±0.12, respectively (p>0.05). It was satisfied to amplify all samples by using the extracted plasma DNA as PCR templet in phenol/chloroform method and QIAamp DNA extraction kit, but it only could amplify 4 cases by using the extracted plasma DNA in boiling method. In conclusion, phenol/chloroform method and QIAamp DNA extraction kit are better than boling method. Therefore, phenol/chloroform method was used to extract circulating DNA in our study. 2. To investigate the loss of heterozygosity (LOH) of plasma circulating DNA in patients with lung cancer and evaluate the diagnostic value of circulating DNA as lung cancer tumor markerSixty-nine blood samples and cancer tissues were taken from 69 patients with primary lung cancer before treatment. At the same time, blood samples were also taken in 40 patients with various pulmonary diseases other than lung cancer. Four microsatellite markers D3S1300, D3S1289, D13S171 and D17S2179E were tesed to analysis plasma DNA and cancer tissue DNA LOH in 69 cases of primary lung cancer, and inplasma DNA of 40 control subjects by PCR and silver staining. LOH ratios of D3S1300 locus, were detected in the tumor tissue and plasma DNA of patients with lung cancer were 40.6% and 29.0%, D3S1289 locus were 31.6 and 24.6%, D13S171 locus were 33.3% and 20.3%, and D17S2179E locus were 15.9% and 11.6 %, respectively. In contrast, there were only 5% D3S1300 LOH, 7.5% D3S1289 LOH, 5% D13S171 LOH and 0.0% D17S2179E LOH in plasma DNA of control group (p < 0.01, vs lung cancer group). In conclusion, there were some gene LOH in circulating DNA of plasma as same as cancer tissue DNA. Plasma circulating DNA LOH could be as a tumor marker to diagnose lung cancer. The efficiency of plasma DNA to diagnosis lung cancer was correlation with the choosen gene: D3S1300 LOH and D13S171 LOH were correlation with non-small-cell lung cancer (NSCLC), and D3S1289 LOH was correlation with small-cell lung cancer (SCLC), but D17S2179E LOH was correlation with the two ones. The diagnosis specificy and sensitivity of plasma DNA D3S1300 LOH were 95% and 29.0%, D3S1289 LOH were 92.5% and 24.6%, D13S171 LOH were 95% and 20.3%, D17S2179E LOH were 97.5% and 11.3%, and CEA were 90.0% and 29.0%, respectively. Using the combination of microsatellite markers can improve the efficiency of plasma DNA to diagnosis lung cancer: combining the two markers of D3S1300 and D3S1289, the diagnosissensitivity improved to 49.3%; and combining the two markers of D13S171 and D3S1289, it improved to 37.7%; combining the three markers of D3S1300 and D3S1289 and D17S2179E, it improved to 56.2%; combining the three markers of D13S171 and D3S1289 and D17S2179E, it improved to 46.4%; and combining the three markers of D3S1300 and...
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