| Objectives:To establish a new grate micro-nano chip to capture circulating tumor cells in patients with non-small cell lung cancer,so as to help lung cancer diagnosis,disease evaluation,monitoring efficacy and prognosis.Methods:(1)To establish a set of independent laboratory CTCs separation system based on grid type micro membrane chip assembly CTCs separation device(3-D capture grid sandwich structure),composed of the 3 main aspects of micro injection pump and peristaltic pump;(2)Develop lung cancer cell line A549,and explore the cell operating conditions optimization of CTCs separation system,surfactant including size,fluid velocity of the grating chip aperture and anti cell adhesion ratio;(3)The number of different(50~250)simulated lung cancer patients with peripheral blood samples of lung cancer A549 cells with 2ml in peripheral blood of normal human,with the CTCs A549 in the separation system of the blood cell filtering,sorting out,and then the A549 lung cancer cells with FITC Ep labeled CAM antibody separation and enrichment reaction was performed after immunofluorescence staining under the microscope to determine the count.The sensitivity and recovery of the A549 cells in the whole blood were detected by the detection platform.(4)Verify the feasibility of the CTCs detection method in the peripheral blood of the patients with lung cancer.Results:The 5 m to 8 m aperture gate type micro nano chip Hang Lung cancer cell capture rate in solution had significant differences on A549 cells(P<0.05),with the chip size,the capture rate decreased gradually,selectivity of 5 m and 6 m chip is relatively good and there was statistical significance(P<0.05).A549 cell suspension in different flow conditions capture rate difference was statistically significant,the correlation coefficient was 0.973(P < 0.05),the regression equation was Y=-88.6X+108.7(bilateral P<0.01),and to filter out A549 cell liquid and transfer liquid in the diameter of the differences were statistically significant(P<0.05),with the increase of flow rate,chip gate type microporous membrane 6 m pore diameter of A549 cells capture rate,selectivity and filtration time were gradually decreased.The proportion of surfactant in PBS buffer is greater than 1:20,there was a significant difference between the effects of surfactants on A549 cell diameter(P<0.05),the proportion of A549 cells and the diameter of the linear correlation,the correlation coefficient was 0.918(P<0.05),the linear regression equation was Y=1.862X+16.274;at the same time,PBS buffer agent proportion of surface activity and centrifugal after breaking the cell number of A549 linear correlation,the correlation coefficient was 0.912(P<0.05),the linear regression equation was Y=-2266.42X+91.887,the surfactant ratio is low,the more the number of A549 cells after centrifugation is not broken.The ratio of flow velocity and surfactant can affect the residual accumulation of cell surface cells on the whole blood sample.In this experiment,when the gate type micro nano chip aperture is 6 m,micro injection pump flow rate of 0.15ml/min(peristaltic pump flow rate of 0.3ml/min),PBS surface active agent in the washing liquid ratio of 0.2:20 filtering separation,A549 cell capture rate and good selectivity,the consumption of more short,influence the cell morphology and the integrity of the small chip filter residue accumulation of blood cells at least,facilitate identification and observation of the next step in the recognition of tumor cells.In 2 healthy volunteers with different number of ML in peripheral blood(50,100,150,200,250)after A549 cells were separated and enriched,the recovery rate of A549 cells in 80% ~ 90% between the A549 cells into the chi square test number and A549 cell recovery rate,the difference was statistically significant(bilateral P<0.05),and the linear correlation,the correlation coefficient was 0.983(P < 0.05),the linear regression equation was Y=0.046X+79.3,the more the number of A549 cells in blood samples was mixed,the more the number of detected cells.The peripheral blood of the patients with lung cancer can be detected by CTCs capture method in this experiment,and the lung cancer cells can be isolated.Conclusion:In this study,we established a new grate micro nano chip to detect CTCs.It was sorted according to the size and deformability of CTCs,and was identified by fluorescent antibody immunoreaction.CTCs can be preliminarily captured in peripheral blood of clinical patients with non-small cell lung cancer. |
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