Effect of osteoclast-derived apoptotic body microRNA-30a on osteogenic activity / 解剖学报

Objective To explore that whether apoptotic bodies released by osteoclasts mediate osteogenic activity. Methods The osteoclasts were induced from mouse (n = 10) bone marrow monocytes in vitro, and were identified by tartrate resistant acid phosphatase (TRAP) staining, F-actin, and DAPI double labeling immunofluorescence. The Co- culture system of osteoclasts and mouse osteoblasts MC-3T3E1 was established. The apoptosis of osteoclasts was analyzed by DNA fragment ELISA. Immunoblotting of apoptotic body markers was investigated. Real-time PCR analysis of bone formation markers was tested. MiRNA expression profiling of apoptotic body was identisfied. Results Alendronate (ALN) 100 μmol/L induced osteoclast apoptosis and caused apoptotic body release from osteoclasts. The expression of C3b and annexin V protein was enhanced by ALN; the expression of C3b in osteoclasts was negatively correlated with the activity of osteoblasts; the microarray screening of apoptotic body showed that miR-30a was correlated with bone formation markers and serum alkaline phosphatase (ALP). Conclusion Osteoclast-derived apoptotic body miR-30a can inhibit the activity of osteoblasts. Apoptotic body may participate in the dialogue between osteoclasts and osteoblasts.

Cytotoxicities of Tumor-specific T Lymphocytes Primed by Glioma Apoptotic Body-or Glioma Cell Lysate-pulsed Dendritic Cells

OBJECTIVE: The choice of tumor antigen for dendritic cell(DC)-loading has still been an unresolved problem in the DC-based vaccine strategies against malignant gliomas that has not been found well-characterized tumor specific antigens. In this study, we compare tumor-specific T cell response induced by glioma apoptotic body(GAB)-pulsed DCs to response induced by glioma cell lysate-pulsed ones quantitatively. METHODS: DCs generated in the presence of granulocyte macrophage-colony stimulating factor and interleukin(IL)-4 from peripheral blood mononuclear cells(PBMCs) of HLA-A2 positive healthy donors were cultured. Each GABs and glioma cell lysate generated from HLA-A2 positive T98G glioblastoma cells were co-incubated with DCs. CD8+ T lymphocytes isolated from PBMCs of same donors were cultured in media containing IL-2 and either stimulated by GAB- or lysate-pulsed DCs three times at a weekly interval. The interferon(IFN)-gamma concentrations of each cell culture supernate were measured by enzyme immunoassay technique. Cytolytic activity of the generated cytotoxic CD8+ T cells either stimulated with GAB- or lysate-pulsed DCs was determined by a standard 4-h 51Cr-release assay. RESULTS: IFN-gamma production and cytolytic activity of effector T cells stimulated by GAB-pulsed DCs were significantly higher than those of T cells stimulated by lysate-pulsed ones. CONCLUSION: These results indicate the choice of antigen is a critical determinant in the induction of antitumor immunity against malignant glioma. Antigen preparations from GABs represent a promising alternative to glioma cell lysate in DC-based glioma vaccine strategies.

Induction of Cellular Immune Response by Dendritic Cells Pulsed with Glioma Apoptotic Bodies

OBJECTIVE: The aim of this study is to verify the hypothesis that human dendritic cells(DCs) can process antigens from glioma cell apoptotic bodies and induce antigen-specific effector T cells. METHODS: DCs generated in the presence of granulocyte macrophage-colony stimulating factor(GM-CSF) and interleukin-4(IL-4) from peripheral blood mononuclear cells(PBMCs) of healthy donors with human leucocyte antigen(HLA) A*0201 were cultured for 7 days. Glioma apoptotic bodies(GABs) from T98G glioblastoma cells following 18 hour-actinomycin D treatment were co-incubated with DCs for 3 days. CD8 T cells isolated from peripheral blood of same donors were cultured in media containing IL-2 and were stimulated by GAB-pulsed DCs three times at a weekly interval. The interferon-gamma(IFN-gamma), a cytokine related to cytotoxicity, concentrations of cell culture supernates were measured by enzyme immunoassay technique. RESULTS: Induced DCs had DC's own phenotypic characteristics such as highly expressed major histocompatibility complex(MHC) class II, CD1a and CD86 molecules. They also had high endocytotic activity. Preteatment of T98G glioma cells with actinomycin D resulted in 53% of cells undergoing apoptosis. IFN-gamma production of effector T cells stimulated by GAB-pulsed DCs was significantly higher than that of T cells stimulated by non-pulsed DCs. CONCLUSION: Naive CD8 T cells can be activated by human GAB-pulsed DCs to become antigen-specific effector T cells. Using GABs as a antigen source may be a novel approach in future DC-based immunotherapeutic trials for malignant glioma.

Studies on the Radiation Induced Apoptosis by Morphological and Biochemical Analysis in A431 Cells / 대한핵의학회잡지

PURPOSE: We performed this study to evaluate the process of radiation induced apoptosis in A431 skin epithelial cancer cell line. MATERIALS AND METHODS: Low to high dose radiation (0, 2, 5, 10, 25 Gy) was given to A431 cells by Cs-137 cell irradiator. Apoptosis was evaluated by cell morphology, dye exclusion test, and DNA laddering. RESULTS: Cell viability decreased as the radiation dose increased. Number of apoptotic bodies increased as radiation dose increased. It increased most significantly at 12 hours after irradiation. Lactate dehydrogenase activity in culture medium increased according to radiation dose and time after irradiation. CONCLUSION:: Radiation-induced apoptosis which was the main course of cell death in A431 cells could be analyzed quantitatively by counting apoptotic bodies under microscope. Apoptosis increased as radiation dose increased.