Expression of90K/Mac-2Bp in Human Brain Astrocytic Tumors and90K/Mac-2Bp Pulsed DC Vaccine for Human Brain Astrocytic Tumors Immunotherapy
|School||Central South University|
|Keywords||90K/Mac-2BP astrocytomas RT-PCR Western blot Immunohistochemical90K U251 DC vaccine immunotherapy glioma MACS|
Objective To investigate the expression of90K/Mac-2BP mRNA and protein in human brain astrocytomas.Methods The expression of90K mRNA in human normal brain tissue and astrocytoma tissue was detected by RT-PCR; The expression of90K protein in human normal brain tissue and astrocytoma tissue was detected by WB and immunohistochemical staining.Results RT-PCR reveal that90K mRNA was lowly expressed in nor-mal human brain tissues(0.116±0.017).The90K mRNA expression was significantly higher in astrocytomas (0.407±0.151) than in normal human brain tissues (t=6.065P<0.05), and significantly higher in higher grade astrocytomas(WHO Ⅲ-Ⅳ)(0.516±0.128) than in lower grade(WHO I-II)(0.295±0.067)(t=8.138, P<0.05). The90K mRNA expression had no significant relationship with age, sex, and tumor size(all P>0.05).WB reveal that90K protein was lowly expressed in normal human brain tiss-ues(0.291±0.064). The90K protein expression was significantly higher in astrocytomas (1.163±0.391) than in normal human brain tissues (t=15.68, P<0.05), and significantly higher in higher grade astrocytomas (1.415±0.318) than in lower grade (0.902±0.272)(t=6.539, P<0.05). The90K protein expression which detected by WB had no significant relationship with age, sex, and tumor size(all P>0.05). Immunohistochemical stain-ing reveal that positive rate of90K protein expression was10%(1/10) in normal human brain tissues70.18%(40/57) in astrocytomas. There Was significant difference between the positive rate of90K protein expression in astrocytomas and that in normal human brain tissues(P<0.05). The po-sitive rates of90K protein in higher grade group and lower grade group were86.21%(25/29) and53.57%(15/28) respectively. The significant difference was seen between higher grade group and lower grade group (P<0.05). With the combination of histopathological data, we found that the expression level of90K protein was closely correlated the differentia-tion degrees of astrocytomas, but not with patient age, sex and tumor size (allP>0.05).Conclusion90K expressed positive in astrocytomas and weakly in normal human brain tissues. The expression of90K is higher in higher grade astrocytomas.90K may contribute to occurrence and progression of astrocytomas.90K protein may be a astrocytomas-associated antigen and target antigen in immunotherapy. Objective To investigate the effection of90K protein in DC vaccine for human brain astrocytomas Immunotherapy.Methods The expression of90K protein in U251cells was detected by WB;DC were cultured from peripheral blood monocytes; Apoptosis U251cell was obtained by heat shock; DC vaccine pulsed with Apoptosis U251cell and90K protein;Cytokines secreted by DC and T lymphocytes was detected by ELISA; CD4+T lymphocytes and CD8+T lymphocytes were purified by MACS; Induction of antigen pulsed DC-specific T lymphocytes proliferation and lethal effect of CTL for U251cell were detected by CCK-8.Four group were setted up:imDC,DC, Apoptosis U251cell-DC, Apoptosis U251cell+90K-DC.Results The high expression of90K protein in U251cell was detected by WB. Mature DC had typical dendritic structure in optical microscope and electron microscope;The high expression of immunophenotyping CD80、CD86and HLA-DR was detected by Flow cytometry assay. Apoptosis U251cell percentage was highest in44℃3h group and Apoptosis U251cell was well fused with DC. The concentrations of IL-12p70were highest in Apoptosis U251cell+90K-DC group,and the concentrations of IL-10was lower. The proliferation of T lymphocytes can be activated by Antigen pulsed DC, Apoptosis U251cell+90K-DC has the strongest ability to activate T lymphocytes proliferation.The CD4+T lymphocytes can be activated to Th1which secret IFN-yby Antigen pulsed DC; The concentrations of IFN-ysecreted by Thl were highest in Apoptosis U251cell+90K-DC coculture with CD4+T lymphocytes. The CD8+T lymphocytes can be activated to CTL which secret IFN-yby Antigen pulsed DC; The concentrations of IFN-ysecreted by CTL were highest in Apoptosis U251cell+90K-DC coculture with CD8+T lymphocytes. The CD8+T lymphocytes can be activated to CTL which kill U251cell by Antigen pulsed DC;The ability of killing U251cell by CTL were strongest in Apoptosis U251cell+90K-DC coculture with CD8+T lymphocytes.Conclusion The DC were cultured from human peripheral blood monocytes had typical dendritic structure and immunophenotyping,this method can provide source of DC reliably. The proliferation of T lymphocytes can be activated by Apoptosis U251cell+90K pulsed DC in vitro.The high purity of CD4+T lymphocytes and CD8+T lymphocytes was separated by MACS. Apoptosis U251cell+90K pulsed DC vaccine can induce antigen-specific CTL to kill U251cell,and the ability of kill U251cell was stronger than Apoptosis U251cell pulsed DC vaccine only.The appearance of90K enhance the effection of DC vaccine.The dependent pathway and non-dependent pathway of glioma related antigen-specific immune response were both participated in anti-glioma effection by Apoptosis U251cell+90K pulsed DC vaccine.