Effects Of Ganoderma Lucidum Polysaccharide On The Expression Of Co-Stimulating Molecules And MHC Molecules On Tumor Antigen-Induced Macrophage

ObjectiveCancer is a malignant disease caused by the proliferation and invasion of some cells.Cancer is the first cause of premature death in China.Melanoma from melanocytes is the most deadly cutaneous malignancy,and osteosarcoma from osteoblasts is the most common primary malignancy of bone tissue.For malignant tumors,although the traditional treatment methods such as surgery,radiotherapy and chemotherapy progress quickly,the effect is still not satisfactory.In recent years,with the development of immunotherapy and traditional Chinese medicine,it has played an increasingly important role in the treatment of cancer.For centuries,Ganoderma lucidum polysaccharide has been used as a medicine and health product to treat various diseases,improve health and prolong life.Polysaccharides and triterpenes are regarded as the main components of Ganoderma lucidum polysaccharide,which have various biological activities including immune regulation and anti-tumor.The core mechanism of anti-tumor effect of Ganoderma lucidum polysaccharide is to enhance host immune function.The immune system plays an important role of anti-tumor.Successful clearance of cancer depends heavily on proper activation of antigen-presenting cells(APCs).APCs include dendritic cells(DCs),macrophages and B cells,which present the peptides loaded on MHC-I/MHC-Ⅱ to T lymphocytes,while APCs cells express CD80 and CD86 to provide co-stimulating signals to T cells to awaken antigen-specific adaptive immunity.Therefore,APCs and their surface molecules such as CD80,CD86,MHC-I and MHC-Ⅱ are critical for immune surveillance of cancer.Immunotherapy and traditional treatment methods such as radiotherapy and chemotherapy are required to improve the function of APCs to enhance the therapeutic effect.In this study,mouse peritoneal macrophages were prepared by intraperitoneal injection of thioglycolate,and the macrophage surface marker CD68 was detected by flow cytometry.Melanoma or osteosarcoma antigens were applied to macrophages which were pretreated with LPS or Ganoderma polysaccharides.Flow cytometry was used to detect the expression of costimulating molecules CD80 and CD86,MHC-I(H-2Kb/H-2Db or H-2Kd/H-2Dd)and MHC-Ⅱ(I-A/I-E).To observe whether tumor antigen have the ability to promote the expression of macrophage co-stimulatory molecules and MHC molecules and whether Ganoderma lucidum polysaccharide have the ability to promoting the effect of tumor antigen as above.Methods1.Induction of mouse peritoneal macrophages by thioglycolate medium and identificationC57BL/6 or BALB/c mice were injected into the abdomen with thioglycolate medium to extract mouse peritoneal macrophages,and the expression of CD68 was detected by cytometer to identify macrophages.2.Effect of tumor antigen on the expression of macrophage co-stimulating molecules and MHC moleculesMacrophages were divided into different concentrations of B16F10/ K7M2 tumor antigen groups(0μg/ml group,25 μg/ml group,50μg/ml group,100μg/ml group).Each group was treated with 300ng/ml LPS for 12 h,and then treated with different concentrations of tumor antigen for 24 h after removal of LPS.Flow cytometry was used to detect the expressions of CD80,CD86,MHCI(H-2Kb/H-2Db or H-2Kd/H-2Dd)and MHC-Ⅱ(I-A/I-E)on the surface of macrophages in each group.3.Effect of Ganoderma lucidum polysaccharide on expression of costimulating molecules and MHC molecules on tumor antigen-induced macrophagesMacrophages were divided into different concentrations of Ganoderma lucidum polysaccharide groups(0μg/ml group,0.2μg/ml group,0.8μg/ml group,3.2μg/ml group,12.8μg/ml group).B16F10/ K7M2 tumor antigen of 50μg/ml was added to each group for 24 h after Ganoderma lucidum polysaccharide removal,Flow cytometry was used to detect the expressions of CD80,CD86,MHC-I(H-2Kb/H-2Db or H-2Kd/H-2Dd)and MHC-Ⅱ(I-A/I-E)on the surface of macrophages.Results1.Mouse peritoneal macrophages were induced by Thioglycolate Medium and identified.1)Thioglycolate Medium induced macrophages were adherent to the wall,showing round or spindle shape.2)CD68 was highly expressed in macrophages induced by thioglycolate medium by flow cytometry.2.Effect of tumor antigen on the expression of macrophage co-stimulatory molecules and MHC molecules1)B16F10/K7M2 antigen promoted the expression of CD80 and CD86 on the surface of LPS-pretreated C57BL/6 or BALB/c macrophages,and was dose-dependent with tumor antigen in a certain range.2)Almost all macrophages of C57BL/6 mouse expressed MHC-I(H-2Kb/H-2Db).Almost all macrophages of BALB/c mouse expressed MHC-I(H-2Kd/H-2Dd).3)B16F10/K7M2 antigen induced the expression of MHC-Ⅱ(I-A/I-E)on macrophages of LPS-pretreated C57BL/6 or BALB/c mouse,and was dosedependent with tumor antigen in a certain range.3.Effects of Ganoderma lucidum polysaccharide on expression of costimulatory molecules and MHC molecules on tumor antigen-induced macrophage1)Ganoderma lucidum polysaccharide can promote B16F10/K7M2 antigen to induce surface co-stimulatory molecule CD86 on macrophage of C57BL/6 or BALB/c mouse,which is dose-dependent with Ganoderma lucidum polysaccharide in a certain range.There was no significant change in the expression of the co-stimulatory CD80.2)Almost all macrophages of C57BL/6 mouse expressed MHC-I(H-2Kb/H-2Db).MHC-I(H-2k D/H-2Dd)was expressed on almost all macrophages of BALB/c mouse3)Ganoderma lucidum polysaccharide promoted the expression of MHC-Ⅱ(IA/I-E)on macrophages of C57BL/6 or BALB/c induced by B16F10/K7M2 antigen.and was dose-dependent with Ganoderma lucidum polysaccharide in a certain range.ConclusionsIntraperitoneal injection of Thioglycolate Medium can induce mononuclear-macrophages in mouse to move to the abdominal cavity and obtain a large number of macrophages,which is a feasible method to prepare mouse macrophages for invitro study.Mouse melanoma and osteosarcoma antigens can promote the expression of co-stimulatory molecules CD80,CD86 and MHC-Ⅱ(I-A/I-E)on mouse peritoneal macrophages induced by Thioglycolate Medium,which were benefit for promoting antigen presentation function of tumor antigen on macrophage.Ganoderma lucidum polysaccharide have the ability to promote the effect of improvement expression of CD86 and MHC-Ⅱ(I-A/I-E)of mouse melanoma and osteosarcoma cell antigens,and have the ability to promote antigen presentation function of tumor antigen on macrophage.