The Inhibitory Effect On The Mouseglioma’s Growth With The Overexpression Of IL-33and The Research Of Its Mechanism

Over a period of time, the central nervous system was thought to be a immune immunity organ due to the presence of the blood brain barrier, but a growing number of studies have found that the brain can occur immune response when it is under certain conditions.In pathological conditions, due to the permeability of the blood brain barrier had been changed, the central nervous system can be infiltrated with T cells, NK cells,and then occur immune response.Compared with the immune escape and tolerance of the tumor cells, the effects of the central nervous system’s immune response is negligible, therefore, how to enhance the central nervous system’s immune response to the tumor cells, especially how to directionally collect a large number of cytokine induced killer has became the research focus of many people. It has been confirmed that the activated lymphocytes can pass through the blood-brain barrier, which makes the Immune adjuvant therapy for glioma possible. With the glioma specific antigen was identificated and discovered, as well as the unceasing enhancement of the cultivation technology in vitro, glioma specific cytotoxic T lymphocyte (CTL) adoptive therapy become one of the most anticipated biological therapy research fields.Some reports showed IL-33can not only mediate TH2immune response through its specific receptor ST2, but also has direct effects on NK, DC, CTL immune cells through some related mechanism; not only the direct killing of tumor cells, but also the release of cytokines can be promoted, and a immune amplification effect will be found. IL-33plays an important role both for innate immunity and acquired immunity, IL-33has a great value in the studies about infectious, autoimmune diseases and tumor diseases.At present, a new progress has been made about the relation of IL-33and tumors outside the central nervous system. A small amount of expression of IL-33has been found in some types of cells in the central nervous system. In normal cells, IL-33exists as a nuclear factor, and will be released only from necrotic cells. How to make the central nervous system have a certain effective concentration of IL-33is the first problem to solve.The glioma cell line GL261will be transfected by the vector of plasmid and lentivirus, whether IL-33can be expressed or high stablly expressed is the key to all trials. GL261cells which can high stablly express IL-33will be injected in vivo, then be killed by immunocytes just like NK and then a large amount of IL-33will be released,which will mobilize the body’s DC, NK and CTL. IL-33has an immune amplification effect, and plays a major role in the body’s immune response of resistance of glioma. The glioma will be inhibited during its beginning and developing period. A CTL adoptive treatment also be made in this experiment, just for the further studies focus on the mechanism of the glioma growth inhibition when there is an over expression of IL-33. In order to define wether there was a common phenomenon of anti-tumor by IL-33, we had established mouse melanoma models with B16-F10cells stably transfected by IL-33, as the control group. These new findings and research will provide a new direction for the treatment of glioma, especially high grade glioma, and also provide a solid foundation for its clinical application.Chapter Ⅰ The construction of the plasmid of mouse IL-33and the preliminary study about it’s over exprssion in GL261Abstract Objective To build the plasmid of mouse IL-33and discuss the feasibility of its expression in mouse glioblastoma multiforme cell lines GL261. Methods The gene sequences of mouse IL-33was found from the genebank, artificially synthesized, and loaded in the expression vector pEZ-M03, also enzyme identificated after amplification. The correct plasmid was used to transfect GL261by using the transfection reagent Lipofectamine TM LTX. The expression of the target gene was detected by real-time PCR and RT-PCR, and the expression of the target protein was detected by ELISA. Results The eukaryotic plasmid pEZ-M03-mIL33of mouse IL-33was successfully constructed and the GL261was successfully transfected by liposome transfecion reagent. We found that the transfection efficiency was the highest48hours after transfected. A high exprssion of the target gene was detected by Real-time PCR and PCR. Also a significant over expression was found by ELISA. Conclusion The successfully constructed eukaryotic plasmid was high expressed in GL261, and this will provids a foundation for the further study about how to establish stable transfection cell lines through lentiviral vector and the next functional studies. Chapter II The establishment of the GL261cell lines with a stable expression of mouse IL-33and the functionalstudies in vitroAbstract Objective The GL261cell lines with stable expression of mouse IL-33was established by a transfection through lentiviral vector which was successully packaged with the target gene, and the fuctional study in vitro would be done, which provided a foundation for the further study in vivo.Methods The GL261cell lines were transfected by the lentiviral vector with mouse IL-33, and selected by the complete medium containing puromycin. The positive clones were amplified and its growth condition was observed. The expression of the target gene was detected by real-time PCR and RT-PCR, and the expression of the target protein was detected by ELISA and Western-blog. The invasion of the cells was detected through matrigel invasion Assay by using transwell. Results The GL261cells was successfully transfected by the lentiviral vector, and was selected by the the complete medium containing puromycin with a concentration of6ug/ml.The positive clones was amplified with a huge number. A stablly high exprssion of the target gene was detected by Real-time PCR and PCR. And a significant over expression was found by ELISA and Western-blog.The growth condition and invasion of the transfected GL261cells were not affected. Conclusion The GL261cells was successfully transfected by the lentiviral vector with the target gene.The GL261cell lines in which mouse IL-33was stable and high expressed was successfully established,and the growth condition and invasion of it were not affected,this laid a foundation for the further study in vivo. Chapter Ⅲ The study in vivo about the inhibition of the mouse glioma with the over exprssion of IL-33Object The intracranial tumor growth condition of different types of mouse which included normal and immunodeficiency was observed through the establishment of the mouse glioma models, with CTL adoptive therapy, explore IL-33inhibit the growth of glioma The immune mechanism about inhibiting effect of IL-33on the glioma’s growth was explored. Methods Under the guidance of the stereotactic instruments, the glioma modles were estabilisded by using SPF mouses of C57,Balb/c-NU and NOD/SCID.The mouses of C57were divided into two groups by wether the NK cells were closed or not.The glioma modles were established by using three types of cells included the positive and negative clones of IL-33and the control.The CTL adoptive therapy was combined with IL-33in the mouses of IL-33+/NK-.The sealing effect of NK cells in mouses was detected by flow cytometry.The condition of the intracranial tumors was dynamically observed through the7.0T magnetic resonance. The brain tissue was made into paraffin sections after had been removed.The condition of the tumor was detected by HE staining. The tissue distribution of IL-33was detected by immunohistochemi stry.The expression of CD8and CD31was observed by immunofluorescence. We also established subcutaneous melanoma models with mouse melanoma cell lines B16-F10as control group, according to the same method as the GL261group. The condition of the tumor growth was observed too. Results All sorts of mouse glioma modles were established successfully. An effective sealing effect of NK cells in mouses was detected by flow cytometry. A negative result was found in C57modles with positive clones of IL-33through HE staining or MR of the brain.The growth of the tumor was found in the rest of the modles.A large number of expression was confirmed in the brain tissue by immunohistochemistry. High expression of CD8was confirmed by immunofluorescence. All kinds of subcutaneous melanoma modles were established successfully, by comparing subcutaneous tumor growth of all groups; we confirmed that mouses of normal C57had a lower tumor growth rate when the modles were made by B16-F10cells in which IL-33was over expressed. Conclusion All kinds of mouse glioma and melanoma models were established successfully, and the inhibitory effect to the mouse glioma and melanoma growth with the over expression of IL-33was observed. In the medium of the NK cells, IL-33played direct or indirect actions on all types of immune cells and stimulated the body with the augmentation of CTL and directional infiltration, gived play to the role of immune to enlarge, resulting in a strong inhibitory effect on tumor growth.