| Objective Glioma is the most common malignant tumor originated from glial cells in central nervous system (CNS),which including strocytoma, mesoglioma, admixed spongiocytoma and ependymoma. According to statistics, the percentage of glioma in primary intracranial tumors range from35.26percent to60.96percent. Due to the. infiltrative growth, the surgical removal of glioma were incomplete, surgical resection combined with radiotherapy and chemotherapy have same result. Mostly patients resulted in poor outcomes. The mean survival time of glioma was14.6months approximately, usually less than two years. The situation becoming today’s health menace, so looking for a new therapy become more and more important in the past20years.As the realization of central nervous system and immune characteristic began to sink in, biological immunotherapy has become the fourth therapeutic mode of glioma, which seems like the most promising options for future cancer treatment. Due to the features like safe, effective and low toxicity, DC vaccine becoming the focus of glioma immunotherapy. The dendritic cells (DC) is known as the most powerful antigen presenting cell in the body, which have dendritic structure and High expression levels of the major histocompatibility complex and costimulatory molecules as CD80, CD86. It can uptake and processing antigens, present them to T lymphocytes effectively, triggering a series of immune response. The study found that the DC vaccines made by different methods have certain anti-tumor effect.DC can be sensitization by antigen as purified protein, antigenic peptide, lysates of tumor Cells, and DNA or RNA, Because glioma has low antigenicity and different individuals heterogeneity, we not yet found specific antigen, the DC vaccine was only used in individualized treatment.so,find a antigen which expressed extensively in glioma become urgent problem to be solved. Interleukin-13receptor alpha2(IL-13Ra2) is a membrane receptor composed of380amino acid residues, belong to the erythropoietin receptor family. Its encoding gene is located on Xq24.In recent years, studies have found that IL-13Ra2have highly tissue-specific expression in malignant rumor,especially in gliomas. The higher malignancy glioma has, the stronger it express, while almost no expression in normal tissues and organs. So as a possible glioma-specific marker, IL-13Ra2may make a contribution to the diagnosis and treatment of glioma in future.research abroad have found IL-13Ra2showed the expected antigenicity and immunogenicity, that’s big potential for future cancer treatment. DC sensitized by IL-13Ra2also have certain anti-tumor effect, and this fruit would be contributed to the public of DC vaccine. Today we found there are mall deposits of cells with stem cell characteristics in glioma, have characteristics like Unlimited proliferation, self-renewal, multiple differentiation potential and tumorigenicity. This kind of cells express specific markers of stem cells.The cells are called for cancer stem cells, who have high expression of some anti-apoptotic genes and drug resistance genes.most of them stay in relative resting stage of the cell cycle,so it is’t sensitive to the chemotherapy,and difficult to perish.Ignatova found they expressing CD133and Nestin,and Singh found when mice challenged with100CD133+cells intracranial induced tumor.So, tumor stem cells are the source of tumor recurrence.As the research of tumor stem cells began to sink in, DC vaccine which used tumor stem cell as antigen have induced stronger immune response than traditional DC vaccines, proved that tumor stem cells own antigenicity duing the treatment,offer exciting new insights into immunotherapy. In this study, we detect the expression of IL-13Ra2in glioma and normal brain tissue, observe the ability to trigger immune response of DC vaccines which sensitized with IL-13Ra2, glioma cells and brain tumor stem cells, and compare with traditional DC vaccines, Discuss the importance of IL-13Ra2and brain tumor stem cell in immunotherapy.Methods The experiment was divided into four parts; Part one, Expression of IL-13Ra2in human glioma and U251cell line:Collect tissues duing operation to get paraffin blocks,resuscitate and culture U251cells to let them growing on glass coverslips The SABC immunohistochemical staining method was used to examine the protein expression of IL-13Ra2in59gliomas,5normal brain tissues and U251cells. The correlations of their expression of IL-13Ra2with malignant degrees was analyzed statistically. Part two, Culture of Glioma cell and brain tumor stem cell and preparation of cellular antigen: Collect the glioma tissues during surgery,After digested and dissoeiated, use DMEM medium containing10%Human plasma to cultured Primary glioma cells. Then cultured the cells in serum-free medium (DMEM/F12+20ng/mLbFGF,+20ng/mLEGF). cell differentiation experiment have done when spheres formed, and use flow cytometry to measured the rate of CD133+cells in the brain tumor stem cells, use DMEM medium containing10%FBS to resurrected and cultured U251cell line. Preparation of DC and DC vaccine, and Vitro cytotoxicity experiment induced to the glioma:Taken normal human peripheral blood, lymphatic separation medium, centrifuged after1640+10%autologous plasma absorb mononuclear cells cultured in37°C,5%CO2incubator for2hours, separated human peripheral blood mononeuclear cells(PBMC) from Human PeriPheral blood, then cultured the cells in1640medium containing10%for2hours.then collected and induced the non-adherent cells in1640medium containing10%Human plasma,add GM-CSF and IL-4. observed cells with light microscope, and use flow cytometry to measured their surface markers。Mixed mature DC with IL-13Ra2protein,tumor cell antigen and a tumor stem cell antigen, then cultured them for24hours to get DC vaccines. Isolated T cells from human peripheral blood mononeuclear cells (PBMC),then cultured T lymphocytes and DC in the RPMI1640containing10%Human plasma, compared anti-tumor effect with different DC vaccines.Statistical analysis:Results are expressed as mean±SD. The t test was used for comparisons of two groups,oneway ANOVA was used for Comparisons of multiple groups. Difference is considered significant at P<0.05Results There was no expression of IL-13Ra2in all cases of normal brain tissues. However,33of the59glioma tissues showed positive expression of IL-13Ra2(55.9%), There were specially significant difference between them(P<0.01).duing the glioma group, the expression of IL-13Ra2was significantly different between low grade glioma (I-II grade) and high grade glioma (Ⅲ桰Vgrade)(P<0.01)。 U251cell line also showed positive expression of IL-13Ra2too. We successfully achieved enough U251cells and glioma cells by culturing them in serum containing medium, the cells seen under the microscope have shapes as spindle style, star style and polygonal style. The cell have translucent endochytema, and grown in the monolayer and adherence way on the bottle. It grows rapidly, passage in every week. After4th generation,we found the polygonal shape became the most shape in the cells. Eulturing glioma cells in serum-free medium we found BTSC grown as The cloning balls of cells,4th generation protuberance more visible, accounted for most of the polygonal shape. Serum-free stem cell cultured culture medium more than1week grown in suspension, formed the the suspensions tumor clone ball, the refractive better, flow cytometry cell markers CD133positive for expression rates of the different patients. flow cytometry found many CD133+cells in the brain tumor stem cells, Expression rate of low grade gliomas became higher with culture time increased, when cells was inoculated into serum containing medium, many cells move out of clone ball,The characterization of them was same as primary glioma cells。DCs could be isolated from PBMC by GM-CSF and IL-4. under the microscope we can seen the typical dendritic structure on DC’s membrane surface.Flow cytometry detected the PBMCs surface marker, CD14, have low expression on DCs,while the expression rates of DCS characteristic surface markers higher than before,(CD8387.1%, CDla81%, CD8684.2%、 HLA-DR83%、CD8078%), we get large number of purity mature DC from PBMC.The Vitro Cells Cytotoxic experiment results show that, Compared with pure DC group, DC vaccines loaded by IL-13Ra2, glioma cell antigen and brain tumor stem cell antigen could significantly stimulated the proliferation of T lymphocytes, and abolish glioma cells Effectively. Compared to other groups, DC vaccines loaded by brain tumor stem cell antigen have the strongest effect to activated CTL (P<0.05), while IL-13Ra2group and tumor cell antigen group have no significant difference in the effect (P>0.05). IL-13Ra2group have weakly effect to induced CTL for glioma stem cell, when compare it with the pure DC group,we got no statistical significance from statistical analysis (P>0.05). The stimulation index of used brain tumor stem cell antigen was higher than others in the same stimulator to responder ratio, and when the ratio was1:10,the stimulation index was the highest one.Conclusion:IL-13Ra2protein do not express in normal brain tissue,but widely expressed in glioma and U251cell line.The Expression of IL-13Ra2is associated with the malignant degrees of glioma, so it is an ideal immunotherapy target; We can get enough brain tumor stem cells in the way of culture Glioma cell and U251cell line in serum-free medium; Sour ce of of human peripheral blood mononuclear cells from in vitro GM-CSF and IL-4induce--d cultured available under plenty of mature DC dendritic cells provide a source of cells for tumor immunotherapy in DC vaccines. Compared to IL-13Ra2group and glioma cell antig-en, DC vaccines loaded by brain tumor stem cell antigen have the strongest effect to active-ted CTL which directed against glioma cell and brain tumor stem cell, besides, DC va-ccine loaded by IL-13Ra2and glioma cell antigen also have obviously effect to kill glio-mas cells,so they also have has potential value in clinical therapy. |
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