ABSTRACT
The dynamic expression of cytokines and phenotypes during the differentiation process of dendritic cell precursors (pre-DCs) to mature dendritic cells (DCs) was investigated,and the effects of inflammatory stimulation with lipopolysaccharide (LPS) on DCs differentiation were understood.The differentiation of bone marrow cells isolated from Balb/c mice was induced to DCs in an 8-day cell culture system with RPMI-1640 complete culture medium containing 10％ FBS,20 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng/mL recombinant mouse interleukin-4 (rmIL-4).On the day 3,6 and 7 after culture,DCs were divided into non-LPS group and LPS group,given 500 ng/mL LPS for 24 h stimulation and no stimulation respectively.The expression levels of CD 1 l c+,MHC-Ⅱ +,CD40+,CD80+ and CD86+ were detected by flow cytometry,and those of IL-2,IL-4,IL-10,IL-12 p70 and IFN-γ in the supernatant by ELISA.On the day 3 and 6 after culture,the expression of IL-2,IL-4,IL-10 and IFN-γ in DCs showed no significant differences between non-LPS group and LPS group,whereas the differences were significant at day 7.The expression levels of cytokines (for IL-2,IL-4,IL-10,IFN-γ and IL-12 p70:152.86±6.91,778.33±8.35,44.55±2.54,5.8.26±1.09 and 2423.00±57.21 pg/mL respectively) in LPS group were higher than those in non-LPS group,especially IL-12 p70 increased obviously at day 7.It was concluded that during the differentiation process of pre-DCs to mature DCs,LPS stimulates DCs producing large amounts of IL-12 p70 and Thl-type cytokines as compared with Th2-type cytokines,and day 7 may be a key time-point for DCs polarization.
ABSTRACT
The effects of nanometer realgar suspension on proliferation and apoptosis of human uterine cervix cancer cell line SiHa cells and oncogenic genes HPV16E6/E7 were investigated. A "micro-jet efflux" strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations (6.25,12.5,25 and 50mg/L) for different durations (12,24,48 and 72h). The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MIT method. Special morphological changes of apoptosis were observed by transmission electron microscopy (TEM) and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry (FCM). The expression of HPV16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25-50mg/L nanometer realgar suspension for 48h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group (P<0.01), and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50mg/L for 48h.RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.