Oncolytic Virus Promotes The Proliferation Of Cervical Cancer Infiltrating Lymphocytes

Research purpose: We use gene-edited oncolytic adenoviruses 1421,1423,1416 to promote the proliferation of TIL cells in cervical cancer tissues,so that oncolytic adenoviruses can replace the cytokines IL-2,IL-7 and other functions,reducing culture costs.A large number of TIL cells are expanded by a simple method.After the expansion reaches a certain amount,the TIL cells are returned to the human body to achieve the anti-tumor effect.Materials and methods: We use a scalpel to cut the tumor tissue into a diameter of about 1-2mm,put it into a 24-well plate and add oncolytic adenovirus 1421,1423,1416 and complete medium for cultivation,and invert every day during the cultivation process.We observe the density and status of lymphocytes in the wells under the microscope;select the cells in the wells added to the virus for HE staining to distinguish the presence or absence of lymphocytes;take the cultured supernatant for ELISA experiment to detect IL-2 and IL-7 Cytokine secretion;take cells from the wells for flow cytometry at the 10 th day of culture to detect the value-added status of CD3 +,CD3 + / CD4 +,CD3 + / CD8 +,CD45 +,CD56 + cells.Results: 1.ELISA test showed that the amount of IL-2 secreted by oncolytic virus1421 reached nearly 700 pg / ml at 120 h,and almost no secretion at 24 h;while IL-7was detected in 1421 group,1423 group,1421+ In the 1423 group,1421 + CTX + MI,1423 + CTX + MI group,1421 + 1423 + CTX + MI group,the secretions at 120 hours were 1700 pg / ml,2200 pg / ml,1750 pg / ml,3000 pg / ml,At 2000 pg / ml and1500 pg / ml,IL-7 secreted the highest amount in the 1421 + CTX + MI group,and the improved combination had a certain effect on IL-7 secretion.2.We can see the presence of lymphocytes under the microscope and HE staining.In the combination of 1421,1421 + 1423,the cells proliferated obviously,but the cells cultured in all the wells began to die after the 10 th day,especially the cell death rate is higher in the combination of CTX + MI;3.Through flow cytometry detection,1421 and 1416 viruses can promote the proliferation of CD3 + and CD8 + TIL cells in cervical cancer tissues,and theproportion of CD3 + cell proliferation is higher than that of CD3 + / CD8 +proliferation,and the proportion of CD3 + cell proliferation Approximately double the proliferation of CD3 + / CD8 + cells.The proportion of CD3 + and CD3 + / CD8+ cells in well 1416 is slightly more than that in well 1421,but the difference is not obvious;1416 adenovirus can promote the proliferation of CD3 +,CD3 + / CD4 +cells in cervical cancer tissue mass,and the proportion of CD3 + proliferation it is almost 3 times the ratio of CD3 + / CD4 + proliferation;comparing the 1421 and1416 viruses,there is no obvious difference between CD45 + cells,which are both around 40%,indicating that both viruses promote the proliferation of initial T cells;In the 24-well plate of improved virus combination,CD3 + cells can be seen in the Blank group,1421 group,1423 group,1421 + 1423 combination,the proportion of CD3 + cells is increasing,and in the 1421 + 1423 combination CD3 + The highest proportion is about 6 times that of the BLank group.Conclusion: 1421,1423,and 1416 oncolytic adenoviruses can promote the secretion of IL-2 and IL-7 factors by cells in cervical cancer tissues,and then promote the proliferation of CD3 +,CD3 / CD4 +,CD3 + / CD8 + cells in cervical cancer tissues CD3 +,CD3 + / CD8 + amplification ratio is relatively high,CD3 + / CD4 +amplification ratio is relatively small;in the improved combination of virus wells,oncolytic adenovirus is more likely to promote the proliferation of CD3 + cells.The traditional method of amplifying TIL requires a large amount of IL-2.The IL-2 is expensive.This experimental method is simple and cheap,but it encounters many problems during the cultivation process.1.Because the cervical tissue is in a bacteria environment,cultivating its primary cells is easy to contaminate and is relatively difficult;2.TIL cells are likely to die when the tissue block is cultured to the left and right,probably due to the exhaustion caused by oncolytic adenovirus;3.Cervical cancer tissue block after internal radiation radiotherapy,TIL cells are more likely to die during culture,and radiation therapy may damage the immune environment of cervical tumors.