Study On Purification Of NK Cells And The Relationship Between The Expression Of MICA/B On Tumor Cell Lines With Their Sensitivity To Cytotoxicity Of NK Cells

Objective1. To establish an effective and economical method to obtain NK cells of high purity at low cost;2. To provide experimental data for NK cell clinical application by detecting cytotoxicity of NK cells;3. To analyze the relationship between the expression of MHC class I chain-related gene A/B (MICA/B) on different human tumor cells with their sensitivity to NK cytotoxicity.Methods1. The purification methods of human NK cellsNK cells were purified from the peripheral blood by complement lysis, T cell panning, Percoll discontinuous density gradient centrifugation, and RosetteSep(?) procedure. Compared these methods and modified the methods. The proportion of NK cells (CD3-CD56+) was determined by flow cytometry.2. The cytotoxicity of human NK cellsThe cytotoxicity of NK cells at different effect-to-target ratios (E:T) against the leukemic cell line K562, breast carcinoma cell line Bcap37, renal cell carcinoma cell line 769-P,lung carcinoma cell line A549 were measured by CCK-8 assay.3.The expression of MICA/B in tumor cell linesThe expression of MICA/B in K562, Bcap37,769-P and A549 cells was measured by flow cytometry. Results1. The purification methods of human NK cellsThe purity of NK cells obtained by T cell panning, complement lysis, Percoll discontinuous density gradient centrifugation were (30.90±2.35)%,(42.59±4.01) %,(60.02±3.25)% respectively, and obtained by RosetteSep(?) directed method and modified method were (95.39±3.87)%,(96.52±2.42)%, no difference between these two methods(P＞0.05).2. The cytotoxicity of human NK cellsThe cytotoxicity of NK cells against the same tumor cell line was significance at different effect-to-target ratios (P＜0.01); The cytotoxicity of NK cells against K562,Bcap39 and 769-P was much higher than that of A549 at same effect-to-target ratio (P＜0.01),no difference between K562 and Bcap37(P＞0.05).3. The expression of MICA/B in tumor cell linesThe expression of MICA/MICB on K562,Bcap37,769-P,A549 measured by flow cytometry were (60.35±0.50)%,(90.72±0.64)%,(55.59±0.55)%,(3.84±0.10) % respectively.4. The relationship between the expression of MICA/B and the cytotoxiciy of NK cellsThe expression of MICA/B on NK cells was correlated to the cytotoxicity of NK cells at effect-to-target ratios of 5:1,10:1 and 20:1 respectively(P＜0.05),the correlation enhanced with the ratio get higher for the same tumor cell lines.Conclusion1. Comparing with complement lysis, T cell panning, Percoll discontinuous density gradient centrifugation, The modified RosetteSep(?) method is time-saving and effective to obtain NK cells of high purity at low cost.2. The expression of MICA/B on tumor cell lines was correlated with NK cell-mediated cytolysis. The expression of MICA/B on tumor cell surface affect the cytotoxicity of NK cells.