The Preliminary Mechanism Of Promoting Adoptive Immunotherapy By IL-12 Activating Immune Tolerance Dendritic Cells

Tumor, especially malignant tumor is a threat to human life and a disease that very difficult to be cured.Tumor prevention and treatment have become a global problem urgently to be solved, and also are a focus in of medicine study. In view of the traditional treatments such as surgery, radiotherapy and chemotherapy have some deficiencies,and tumor biological treatment including cellular immunotherapy become a hotspot in tumor therapy research. Adoptive immunotherapy is used as a potential biological treatment, much attention has been paid and it has been applied in clinical.But the study found that adoptive immunotherapy efficacy is limited by tumor immune inhibitory microenvironment. How to break the local immune inhibitory tumor microenvironment becomes the key issue in the tumor immunotherapy. Tolerogenic dendritic cells participating in tumor immune tolerance, and playing an important role in the tumor immune escape.Our previous study found that IL-12 can make the tolerogenic dendritic cells produce antitumor immune responses,but mechanism is still unclear.In this paper, we use immunosuppressive factor IL-10 and TGF-β induce tolerogenic dendritic cells in vitro and establish the tolerogenic dendritic cells model.We test the validity of the cell model by flow cytometry, qRT-PCR, Western blot, mixed lymphocyte proliferation reaction experiment. In addition, we use molecular cloning technology including the PCR, T4 connections, homologous recombination to build the oncolytic adenovirus expressing IL-12,and detect the effectiveness of adenovirus by flow cytometry and ELISA.We also detect the function of IL-12 to tolerogenic dendritic cells by flow cytometry, mixed lymphocyte proliferation reaction experiment, tumor cell killed experiment, qRT-PCR, Western blot,and definite part of the molecular mechanisms.At the same time, we also discussed the regulating effect of mi R-125 b for liver cancer stem cell self-renewal ability.Through the research of previous studies, we get liver cancer stem cells by the flow cytometry and infect liver cancer stem cells by the lentivirus of expressing miR-125 b.Through the detection of the sphere formation ability, the clone formation ability, proliferation, apoptosis and migration ability,we study the function of mi R-125 b for liver cancer stem cells.To sum up, in this paper, some conclusions are drawn as follows:1. We have confirmed this method that inducing tolerogenic dendritic cells by IL-10 and TGF-β is feasible, and the tolerogenic dendritic cells can be used for future study.2. We successfully build the oncolytic adenovirus expressing IL-12, which produce high levels of IL-12.3. We prove the IL-12 can change the biological characteristics of tolerogenic dendritic cells to make it play the immune effect, and preliminary illustrates a part of the mechanism.4. We illustrate the miR-125 b has a negative regulatory role for liver cancer stem cell self-renewal ability.This study preliminary explore the way to improve the tumor microenvironment and build the oncolytic adenovirus expressing IL-12.It also has implications of openning up new treatment strategies for the tumor microenvironment.It provides the experimental data for the subsequent improvement of tumor biological treatment and clinical application and provides the experimental basis for miR-125 b with immune cells targeting liver cancer stem cells therapy and has a certain theoretical significance and application value.