The Basic Experiments And Clinical Application Of Isolating Circulating Tumor Cellsin Patients With Pancreatic Adenocarcinoma Using A Microfluidic Chip

Objective:1, to investigate the effect of epithelial adhesion molecular dependent method on the isolation rate of circulating tumor cells; 2, to develop a diameter-dependent microfluidic chip for the isolation of circulating tumor cells for pancreatic adenocarcinoma, as well as the effective evaluation system; 3, to investigate the value of circulating tumor cells in the diagnosis and the peri-operative evaluation of pancreatic adenocarcinoma.Materials and Methods:1, the studies on the circulating tumor cells of pancreatic adenocarcinoma patients were collected and analyzed using meta-analysis, to compare the difference between the isolation rate using epithelial adhesion molecular dependent method or independent method; 2, a microfluidic chip was developed to isolate the circulating tumor cells, and the blood of healthy volunteers for spike of cells. Immunofluorescent stain was used for the enumeration of isolated cells; 3, the blood of healthy volunteers for spike of cells were collected to isolate the spiked cells, and loop mediated isothermal amplification was used for the evaluation of amplification time of CK19, which was correlated with the number of spiked cells; 4, patients with pancreatic benign and malignant diseases were enrolled in this study, and the numbers of circulating tumor cells were analyzed using loop mediated isothermal amplification of CK19. The difference of amplification time between patients with different disease were analyzed, and receiver operating curve was used to evaluate the diagnostic effect; 5, the blood samples of patients underwent operation for pancreatic adenocarcinoma were collected pre- and postoperatively, to evaluate the difference of number of circulating tumor cells, and to analyze the correlation between the number and operative methods or nerve invasion.Results:1, sixteen studies were enrolled, and the detection rate was 29% in pancreatic adenocarcinoma patients using epithelial adhesion molecular dependent method, compared with 68%using independent methods, with a significant difference, Chi2 = 12.68, P=0.0004; 2\the microfludic chip was prepared succesfully, and Capan-1 as well as panc-1 cells were successfully isolated;2, the amplification time of CK19 using loop mediated isothermal amplification was significantly correlated with the number of spiked cells, can be used for the evaluation of circulating tumor cells; 3, there was a significant difference between the amplification time of CK19 of patients with different disease (240.11±189.70min Vs 39.82±8.06 min, t=3.165, P=0.013), which can be used in the diagnosis of pancreatic adenocarcinoma; 4, there was a significant difference between the amplification time of CK19 of patients before and after operation (39.47±6.74min Vs 51.15±11.44 min, t=-5.781, P＜0.001); 5, the amplification time of CK19 was significantly shorter in patients who can not be radically treated (31± 6.08min vs 1.06±5.698min, t=-2.784, P=0.013).Conclusions:1, Detection rate of circulating tumor cells were significantly reduced using epithelial adhesion molecular dependent methods; 2, the microfludic chip based on diameter-dependent isolation of circulating tumor cells can isolate cells without any label, and the amplification time of CK19 using loop mediated isothermal amplification can be used in the evaluation of circulating tumor cells of pancreatic adenocarcinoma; 3, there was a significant difference between the amplification time of CK19 of patients with pancreatic benign or malignant disease, which can be used in the diagnosis of the disease; there was a significant difference between the number of circulating tumor cells of patients before or after operation, and the amplification time of CK19 can be used to predict the surgical result.