The Tumor Targeting Of CIK Cell

Objective:Separation and preparation of cytokine-induced killer cells using human peripheral blood; Human breast cancer cells MDA-MB-435Vaccinated BALB/cNude nude mice to establish animal models, then we analyze its tumor targeting chemokine characteristics of CIK cells cultured in vitro after reinfusion nude mice.Method:1. Separation and preparation of CIK cells from human peripheral blood by Ficoll density gradient centrifugation.2. Detection and identification of CIK cells of the immune phenotype by double fluorescent staining and flow cytometry.3. Use the MTT destruction experiment and Transwell invasion chamber experiments to observed CIK cells in vitro killing effect on human breast cancer cell line MDA-MB-435and the role of tumors trend migration.4. Establish the xenografts in nude mice model,inject the fluorescent dye CIK cells labeled Dil to the nude mice through the tail vein. Dynamic observing the trend migration to tumor of CIK cell by xenogen in vivo imaging system.5. Put the nude mice to death at6h、24h、48h、7day、14day,then stripping the lungs、liver、stomach、kidney、spleen and tumor tissue to prepared as paraffin sections, HE staining,light microscope to observe the performance of different pathological,and observed fluorescence expression in different tissues under fluorescence microscope. TUNEL apoptosis assay tumor cell apoptosis rate.Results:1. The human peripheral blood mononuclear cells were cultured by different cytokines in vitro. Induced differentiation of CIK cells grew rapidly at3days and grew up to the peak at14days. The proliferation multiples of CIK cells was reached more than50times at14days. Double staining results showed that the CD3+CD56+T cells’percentage were reached above50, and the flow cytometry results showed CD3+CD56+T cells’percentage(63.34%) increased more than50times compared with the beginning (1.10%). 2. The MTT assay showed that at an effector-target cell ratio of10:1、20:1、40:1after48hours, CIK cells showed higher cytotoxic effect for MDA-MB-435cells with the results are (32.20±2.15)%、(51.16±2.64)%and (72.16%±4.11)%, respectively. The killing cytotoxic were (10.08±2.23)%、(16.13±3.12)%and (21.34±2.75)%in PBMC,respectively. The killing rate of CIK cells against MDA-MB-435cells is higer than PBMC.3. Transwell invasion chamber experimental results show that,chemotactic migration of CIK cells to MDA-MB-435cells increased with the increasing of Concentration of the tumor cell supematant,and with a statistically significant compared with the control group (P<0.01)4. After tail vein injected MDA-MB-435cells7days, nude mice xenograft model were successful established. Then injected CIK cells labeled Dil, small animal in vivo imaging instrument detection resulted that,red fluorescence signal can be observed in the lung and lower extremity aorta in the first6h;24h, the signal can be observed in lung、gastrointestinal region and tumor tissue surrounding, and the signal intensity was significantly enhanced than in6h, the signal is the most widely distributed in The gastrointestinal region; After48h,the signal also can be observed in Lung、 gastrointestinal region and tumor tissue surrounding, and the signal intensity remained stable; At7days, red light signal is mainly concentrated in the tumor and its surrounding, but the signal intensity is significantly reduced compared to the previous; At14days, the luminous signal intensity was significantly reduced in the tumor tissue surrounding.5. HE staining of tumor tissue paraffin resulted that lymphocytes can only be found in the tumor tissue and it distributed like nest around the rumor cells under light microscope. Part of the region in tumor tissue appears apoptosis necrosis with inflammatory cell infiltration.48h,lymphocyte distributed the broadest area of tumor tissue and a maximum of apoptotic necrosis. No obvious pathological changes in the rest of the organization, or only a small amount of bleeding. Immunohistochemical results showed that lymphocyte surface markers in tumor tissue were CD3+、CD56+、 CD45RO+, all the CIK cell phenotype. Red fluorescence signal can be seen in the the lymphocyte distribution area in the tumor tissue under the fluorescence microscope, which was the CIK cells labeled DiI. And in48h, the fluorescence signal was most widely distributed in tumor tissue with the highest intensity.6.The TUNEL apoptosis experimental results show that CIK cells have a higher rate of apoptosis in the tumor CIK group compared to non-injected control group, with a statistically significant (P＜0.01).Conclusion:The separation and preparation of CIK cells in our laboratory having anti-chemotactic migration ability of tumor cells in vitro. And showing tumor targeting chemotaxis in vivo xenograft tumor model. And having a certain amount of time distribution characteristics. In addition to the normal organs and the organization in the body do not have significant side effects. Therefore, CIK cells in tumor targeted therapy as a new effective and safe cell carrier will play a more significant tumor killing effect.