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ABSTRACT: This study aimed to examine the expression of immune suppression factors and the mechanisms of antitumor effects of cord blood dendritic cells (DCs) stimulated by soluble cluster of differentiation 40 ligand (sCD40L) and cytokines in vitro in ovarian cancer patients.
The expression levels of interleukin (IL)-10 and transforming growth factor (TGF)-β messenger RNA in peripheral blood were detected by reverse transcription polymerase chain reaction; expression levels of CD80 and CD86 in DCs stimulated by sCD40L were detected using flow cytometry and confocal laser scanning microscopy.
Expression levels of IL-10 and TGF-β genes in the peripheral blood of ovarian cancer patients were significantly increased compared with patients with benign ovarian tumors (P < 0.05). The expression levels of CD80 and CD86 in DCs cultured in the granulocyte-macrophage colony-stimulating factor + IL-4 + stem cell factor + Flt-3 ligand + sCD40L group were significantly increased compared with those in the control group, as assessed by flow cytometry and confocal laser scanning microscopy (P < 0.05).
A variety of cytokines in combination with sCD40L can promote the proliferation of cord blood-derived DCs and induce their maturation as well as stimulating a specific antitumor response.
OncoTargets and Therapy 01/2013; 6:503-15. · 1.26 Impact Factor
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ABSTRACT: To investigate the immune mechanisms for Periplocin from Cortex Periplocae (CPP) in tumor-bearing mice.
H(22) tumor-bearing model BALB/c mice were applied to evaluated in vivo immunoregulatory effect of CPP. The influence of different dose CPP (0.25, 0.50 and 1.00 mg/kg) on immune organs in tumor-bearing mice were observed. T cell subsets of mice spleen were detected by flow cytometry. MTT assay was used to determine the influence of CPP on lymphocyte proliferation of mice spleen stimulated by ConA. The levels of TNF-alpha, IL-2 and IL-12 in serum from mice were detected by means of ELISA.
Thymus index and spleen index of H(22) tumor-bearing model control mice became less than that of normal mice (P<0.05). Compared to both model and normal control groups, thymus index and spleen index of H(22) tumor-bearing mice treated with CPP increased obviously (P<0.05). CPP had no influence on the number of CD8(+) T cells, but up-regulated markedly the number of CD3(+), CD4(+) T cells and the ratio of CD4(+)/CD8(+) in tumor-bearing mice. In CPP-treated mice, the percentage of CD3(+), CD4(+) T cells were not different from normal mice (P<0.05), the ratio of CD4(+)/CD8(+) was higher than that of normal mice (P<0.05). CPP enhanced obviously lymphocyte proliferation of mice spleen induced by ConA, the SI scores were even higher than that of normal mice. The levels of TNF-alpha, IL-2 and IL-12 in serum from CPP-treated mice, increased significantly compared to model control group (P<0.05) in a dose-dependent manner, were similar to or higher than that of normal mice.
CPP protected immune organs of tumor-bearing mice, increased obviously the percentage of CD4(+) and CD4(+)/CD8(+) among the T cell line, and enhanced lymphocyte proliferation of mice spleen significantly, stimulated the production of TNF-alpha, IL-2 and IL-12. The results suggested that CPP possessed potent immunoregulatory effect.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 10/2009; 25(10):887-90.
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ABSTRACT: To examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murine ovarian cancer OVHM cells (CD40L-OVHM) injected into the spleen on liver metastasis in mice.
OVHM cells were inoculated into the spleen of 6 to 8 week-old female B6C3F1 (C57BL/6N x C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVHM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-gamma, TNF-alpha, IL-12, IL-4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded.
The mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rate in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-gamma, TNF-alpha and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (p < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05).
The data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Th1 cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.
Zhonghua zhong liu za zhi [Chinese journal of oncology] 07/2009; 31(7):505-9.
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ABSTRACT: Celecoxib can inhibit cell proliferation, regulate cell cycle and induce apoptosis, but the underlying mechanisms are still unclear. This study was to investigate the association between the NF-kappaB (kappaB) pathway and the apoptosis of breast cancer cell line MDA-MB-231 induced by celecoxib.
The expression of cyclo-oxygenase-2 (COX-2) mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Cell proliferation and cell cycle were detected by MTT and flow cytometry (FCM), respectively. The protein expressions of caspase-3 and p65 in MDA-MB-231 cells were detected by western blot.
After incubation with different concentrations of celecoxib for 48 h, COX-2 mRNA expression in MDA-MB-231 cells was decreased in a dose-dependent manner compared with untreated cells (P<0.05). Proliferation of MDA-MB-231 cells was reduced drastically in a dose-and time-dependent manner after celecoxib treatment (P<0.05). Combination of prostaglandin E2 (PGE2) and celecoxib exerted similar inhibition effect to celecoxib alone on cell growth (P>0.05). High-dose celecoxib induced an increase in the percentage of G0/G1 phase cells accompanied by the change in DNA ploidy. The cellular caspase-3 level was enhanced whereas the p65 level was decreased in celecoxib-treated MDA-MB-231 cells after 24 h in comparison to those in the control cells.
Celecoxib could inhibit MDA-MB-231 cell proliferation and promote cell apoptosis by down-regulating the NF-kappaB signaling pathway.
Ai zheng = Aizheng = Chinese journal of cancer 06/2009; 28(6):569-74.
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ABSTRACT: To examine whether the enhanced expression of CD40L cDNA on murine ovarian cancer (OVHM) cells could induce the secretion of Th1 cytokines from dendritic cells (DC).
OVHM cells were transfected with the full-length mouse CD40L cDNA by lipofectamine 2000 and then G418 resistant cells as positive cells were selected. They were examined for their expression of CD40L with flow cytometry. Bone marrow cells were firstly depleted of erythrocytes, macrophages, T and B cells with PE-conjugated magnetic beads, and then cultured in 10% FCS RPMI 1640 medium supplemented with recombinant mouse GM-CSF and IL-4 for 10 days. PKH67-labeled tumor cells were cultured with DC, and then the stained cells were analyzed for the expression of MHC-I, MHC-II, CD80, CD86, CCR7 in DC with flow cytometry. The expression of p40, p19, p35, p28, EBI3 subunits, IL-18, IFN-gamma, Mig gene in cocultured DC-tumor cells were detected by RT-PCR.
The CD40L cDNA was successfully transfected into OVHM cells. Bone marrow-derived DCs, when cultured with CD40L/OVHM, formed clusters with the tumors and showed an upregulated expression of MHC- I, MHC-II, CD80, CD86, CCR7. Expression of the IL-12, IL-23, IL-27, IL-18, interferon-gamma (IFN-gamma) and Mig (monokine induced by IFN-gamma) genes was induced in the DCs that were cultured with CD40L/OVHM but not with OVHM cells.
These data directly showed that the expression of CD40L on ovarian cancer cells facilitates the interaction between DCs and tumors, enhances the maturation of DCs, induces secretion of Th1 cytokines, especially for IL-12, IL-23 and IL-27, which maybe one of the possible antitumor mechanism for CD40L-transfected ovarian cancer cell line.
Zhonghua zhong liu za zhi [Chinese journal of oncology] 04/2008; 30(3):174-8.
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ABSTRACT: To investigate the influence of different stimulators on cytokines secretion of tumor-draining lymph node (TDLN) cells and study antitumor effects and mechanism of TDLN cells.
IL-12 p70, IFN-gamma, TNF-alpha and NO which were secreted by TDLN cells induced by different stimulators (IL-2 group, IL-2+autologous tumor antigen group, IL-2+GM-CSF+IL-4+autologous tumor antigen group and IL-2+GM-CSF+IL-4+LPS group) were detected by ELISA and Griess after TDLN cells were stimulated for 7, 14 and 21 d.
The rate of CD83(+) cells of TDLN cells induced by IL-2+GM-CSF+IL-4+autologous tumor antigen and IL-2+GM-CSF+IL-4+LPS was significantly increased. The TDLN cells in the two groups secreted much more IL-12 p70, IFN-gamma and TNF-alpha than IL-2 group and IL-2+autologous tumor antigen group. The TDLN cells stimulated by IL-2+GM-CSF+IL-4+LPS secreted significantly more NO than the other three groups. TDLN cells in each group secreted IL-12 p70, IFN-gamma and NO at the highest level after TDLN cells were stimulated for 14 d.
Different stimulators can make TDLN cells produce different number of CD83(+) cells (DC), which gives TDLN cells different antitumor activity.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 10/2006; 22(5):609-12.
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ABSTRACT: Accumulative evidences have indicated the importance of CD40-CD40L receptor-ligand pair in the initiation and regulation of human immune response. Whether triggering CD40-CD40L signaling pathway could induce antitumor effects in ovarian cancer cells is still unclear. This study was to investigate the biological and antitumor effects of CD40L on ovarian cancer cell line OVHM (CD40-positive).
CD40L cDNA was transfected into OVHM cells by lipofectamine 2000. Immunofluorescent technique and flow cytometry were used to evaluate the expression of CD40, CD40L, MHC-I, MHC-II, CD80, CD86. Cell proliferation was detected by MTT assay. CD40L/OVHM and OVHM cells were inoculated hypodermically into C57BL/6N x C3H/He F1 (B6C3F1) mice and BALB/c nude mice simultaneously. Tumor volume was measured. The production of IFN-gamma secreted by the spleen cells from CD40L/OVHM and OVHM cell-inoculated mice co-cultured with 20 Gy-irridiated OVHM cells were measured with enzyme-linked immunosorbent assay (ELISA).
Expression of CD40L in OVHM/CD40L cells was significantly higher than that in OVHM cells (P<0.05). Moreover, the expression of co-stimulatory molecules CD80 and CD86 of CD40L/OVHM was increased (P<0.05). The proliferation rate of parental and CD40L-expressed OVHM cells were not different. Although, delayed tumor growth was observed when CD40L-expressing OVHM cells and OVHM cells were injected simultaneously and contralaterally, the tumor growth of OVHM and CD40L/OVHM in BALB/c nude mice was not different. The amounts of IFN-gamma secreted by the co-culture of irradiated OVHM cells with spleen cells from mice injected with CD40L/OVHM were much higher than those from OVHM/parental cell inoculated and naive mice.
The forced expression of CD40L cDNA in OVHM cells can increase the immunogenicity, and thus develop antitumor effects against OVHM-incubated mice. In vivo immunization of immunocompetent mice with CD40L/OVHM improves the generation of cytotoxic lymphocytes against parent ovarian cancer cells.
Ai zheng = Aizheng = Chinese journal of cancer 09/2006; 25(9):1102-7.
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ABSTRACT: There are no specific methods for early diagnosis of ovarian cancer, recurrence prevention and drug-resistance. The experimental mouse model of ovarian cancer could help to reveal the biological and genetic features of ovarian cancer, and provide rational basis for further intervention strategy. This study was to establish a model of ovarian cancer in mice and homologous cell line, and analyze its biological characteristics.
Ovarian cancer was developed in 8-week-old female F1 (C57BL/6N x C3H/He) mice by a single whole-body neutron irradiation of 2.7 Gy from a (252)Cf source. A metastatic cell line was established through serial subcutaneous transplantation of the primary tumor for 11 generations, and then tumor cells were transferred to in vitro cultivation. These cells were cloned for more than 6 months. The biological characteristics of the tumors and the homologous cell line were determined by cellular and molecular biological techniques.
The grafted tumors in mice were successively passaged for 11 generations with a successful inoculation rate of 96% during 23 months. A tumor cell line OV99 isolated from the grafted tumors was established after 6 months and grew steadily. Morphologic characters and ultrastructures of OV99 cells were accorded with those of ovarian cancer epithelia. The chromosomal analysis of OV99 cells revealed aneuploid pattern of 76 chromosomes. Flow cytometry (FCM) and reverse transcription-polymerase chain reaction (RT-PCR) showed same features between OV99 cells and positive control ovarian cancer cell line OVHM, including distribution of cell cycle, rapid growth rate and the expression of P21, P185, P53, proliferating nuclear cell antigen (PCNA) and Cyclin D proteins, and MAGE-1 and MAGE-3 mRNA.
Establishment of the ovarian carcinoma animal model in mice and OV99, a cell line owns biologic characteristics of ovarian cancer cells, provides experimental materials for further investigation of ovarian carcinoma.
Ai zheng = Aizheng = Chinese journal of cancer 07/2006; 25(6):701-7.
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ABSTRACT: To investigate the effect of lupane acetate of cortex periplocae (CPLA) on the differentiation, maturation and immune activity of human peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs) in vitro.
PBMC isolated from human peripheral blood was cultured with GM-CSF, IL-4 for 5 d and stimulated with TNF-alpha (as positive control) or CPLA to induce DCs. The morphological characteristics of DC were observed under inverted microscope and transmisson electron microscope. The expressions of CD1a, CD83, CD80 and CD86 were analyzed by flow cytometry. IL-12, IFN-gamma production in the culture supernatant of DCs was detected by ELISA. MTT method was used to determine the proliferation of T cells stimulated by DCs.
After 10-days culture with cytokines and CPLA, PBMC developed into mature DCs with typical morphological characteristics and high expressions of CD1a, CD83, CD80 and CD86 on the cellular surface (P<0.05). CPLA enhanced IL-12 and IFN-gamma production by DCs (P<0.05). CPLA-treated DCs markedly stimulated proliferation of T cells (P<0.05).
CPLA may induce the differentiation and maturation of DC, up-regulate cytokines production and increase the immune activity of DC.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 01/2006; 22(1):26-8, 32.
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ABSTRACT: Based on the ideology of "using the monitor data to reflect the production safety and using the risk analysis and safety evaluation methods to guide safety management to realize the objection of mine coal safety state warning and assistant decision-making support system", with Bofang coal mine of Shanxi Orchids Corporation's practical experience in safety state management, we set up a warning and assistant decision-making support system on the theme of "mine safety". The establishment of the system can make coal mine safety management staffs at all levels control themine safety situation, improve the reaction speed of all the possible risk, deal with the significant security risks in time, prevent the occurrence of disasters and reduce the incidence of major accidents and injury rate effectively, so as to open up a new way for coal mine production safety.
Intelligent Information Technology Applications, 2007 Workshop on. 3:830-834.
