Effect Of Ginseng Polysacchride On Peritoneal Elicited Macrophages And Its Relationship Between Inhibitory Proliferation And Apoptosis Of Human Leukemia Cell Lines

BackgroundLeukemia is a malignant hematopoietic tumor which threatens the health of humanity. Until now, traditional chemotherapy and radiotherapy are still the main therapies in leukemic treatment. But the therapy have many side effects and ratio of relapse is very high. What's more, it greatly damages the immunity and hematopoiesis. Polysaccharides of Chinese traditional medicine can non-specific enhance immune function; improve the anti-cancer effect of chemotherapy and radiotherapy; reducing their side effects. In recent years, the immunomodulatory activity and anti-tumor effect of polysaccharides were much concerned, and become a center of Chinese medicine research.It is an well-known that Panax Ginseng is Chinese traditional important medicine on"buqi",it has been used in clinical treatment for thousands of years, Ginseng polysaccharide(GPS)is one of the main effective ingredients extracted from Ginseng. The"qixue"theory of Chinese medicine consider that the Ginseng has the effect of"buqi"promoting hematopoietiesis,and strengthening body resistance to dispel pathogenic factors. Our previous study suggested that GPS is the effective ingredients of promoting hematopoiesis, can markedly promote the proliferation and differentiation of murine multipotential hematopoietic stem cell(CFU-S),murine and human hematopoietic progenitor cells (BFU-E,CFU-GM,CFU-MK,CFU-E), and also can inhibit the proliferation of leukemia cells. GPS could inhibit the proliferation of human promyelocytic leukemia cell line HL-60, and also had a significant effect on human leukemia transplanted mice. macrophage (MФ) is an important immune cell with a variety of functions, it can regulate immune functions through phagocytize antigen and release soluble factors. peritoneal elicited macrophages(PEMФ) are represented as a substitute for macrophage. At present, molecular biological mechanism for GPS have almost no reports about the activation of PEMФ.In this study ,by using techniques of modern biology, systemLy explore the effect of GPS on PEMФand its relationship with inhibitory proliferation and apoptosis of human leukemia lines. our purpose is to clarify the modern biological mechanism for GPS"buqi and sheng xue"and inhibiting tumor, and to provide the theoretical basis and the experimental evidence for study natural inducer .Method1. isolate,purificate and analyze mouse peritoneal elicited macrophagesIn this study ,we used the techniques of anchorage-dependent culture of separated PEMФin RPMI-1640 culture media (contain 10%FBS) in vitro,and identified these cells by specifically biological marker of macrophage,such as 1. morphological observation:invert phase contrast microscopy;2. enzyme cytochemistry: acid phosphatase(ACP),a-acetic acid naphthol esterase(a-ANE);3. phagocytic experiment:phagocytosis of chicken erythrocytes and prepared Chinese ink; 4.immunocytochemistry:surface specific antigen of macrophage(CD68 stain).2. the effect of GPS on the function of PEMФCo-cultured PEMФwith GPS at different concentrations,observed expression of ACP,α-ANE,CD68 and phagocytosis of the cultured cells.3. the influence of the peritoneal macrophage cell-conditioned media (GPS-MФ-CM) on leukemic cellsCo-cultured K562,HL-60,KG1a cells with GPS-MФ-CM, The inhibitory effects of GPS-MФ-CM on K562,HL-60,KG1a cells was studied by MTT assay.The opoptotic of GPS-MФ-CM on K562,HL-60,KG1a cells was studied by TEM,FCM.4. The effect of GPS on the secrete factors of PEMФ,and the relationship with inhibitory effects and opoptotic on K562,HL-60,KG1a cells.The expression levels of Nitric oxide(NO),tumor necrosis factor alpha(TNF-a) and IL-1,IL-6,IL-12 levels of PEMФwere measured by the Griess reaction,ELISA and thymocytes proliferation test respectively.ResultOur results indicated that:morphological observation show that these anchor cells take on morphological characteristics of macrophage;enzyme cytochemistry find that ACP and a-ANE stain be strong positive;phagocytic experiment detect that the cells can phagocytose chincken erythrocytes and prepared Chinese ink,and have a high phagocytic rate.Immunocytochemistry show that cell surface antigen-CD68 express positive.So the anchor cells are purified and functional satisfactory macrophage according to morphological observation,enzyme cytochemistry,phagocytic experiment and immunocytochemistry.Our results indicated that:the synthesis and expression of ACP and a-ANE is increased,the phagocytosis of chicken erythrocytes and prepared Chinese ink is strengthened and the expression rate and index of CD68 are much stronger in PEMФinduced by GPS.According to ,the results we conclude that GPS can enhance the biological activity of PEMФ, and the final concentration of 100mg/L GPS was significance.The result of MTT assay indicated that: GPS-MФ-CM(25~200mg/L) increased inhibitory effects on K562,HL-60,KG1a cells,at 100mg/L arrived the most. The result of TEM indicated that: chromatin condensation nuclear,Fragmentation and apoptotic body appeared in these cells.And the apoptosis has enhanced by GPS(25~200mg/L) induce.Our results indicated that: NO,TNF-a,IL-6 and IL-1 has improve in the peritoneal macrophage cell-conditioned media,but the product of IL-12 hasn't increase significance.ConclusionGPS could enhancing macrophages activity significance.It could inhibit proliferation of leukemia cells and induce K562,HL-60,KG1a cells apoptosis by promoting the production of NO,TNF-a,IL-6 and IL-1.This may be one of the cellular and molecular biology mechanism of ginseng strengthening body resistance to dispel pathogenic factors.