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Cervical cancer is the fourth-ranked malignant tumor of female cancer in the world, and it seriously threatens women’s health. The main treatment options for patients with cervical cancer are surgery or concurrent chemoradiotherapy. With the development of medical research, researchers are committed to exploring more effective and specific treatment options in order to increase the treatment options for cervical cancer and improve the treatment effect. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a method in which the Cas9 protein uses guide RNA (gRNA) to target the target gene and achieve precise editing of the target gene. At present, CRISPR/Cas9 technology has become a promising and powerful gene editing tool, a new and effective targeted therapy that has been applied in the treatment of various tumors. The research progress of CRISPR/Cas9 technology in the treatment of cervical cancer is mainly reviewed in terms of action targets, combination therapy strategies, and related drug resistance gene screening in order to provide new strategies for the treatment of cervical cancer.
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Objective:To investigate the effects of 30% ethanol elution fraction of Artemisia absinthium extract with macroporous resin (AAEM-30%) on the dendritic cell (DC) and immunity of mice. Methods:AAEM-30% was obtained from the alcoholic extracts of A. absinthium by AB-8 macroporous resin, and its polysaccharide, flavonoid, and terpenoid contents were determined. The expressions of AAEM-30% on DC surface molecular cluster of differentiation (CD) 40, CD80 and CD86 were detected in vitro by flow cytometry, and the expressions of DC cytokines IL-6 and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). The effect of AAEM-30% on the immune function of ICR mice was measured in vivo with different doses (50 and 100 mg/kg) and different administration methods (subcutaneous injection, intraperitoneal injection, and gavage). Results:The contents of polysaccharides, flavonoids, and terpenoids in AAEM-30% were 24.30%, 22.50% and 28.19%, respectively. AAEM-30% significantly enhanced the expression of CD40, and CD86 and the secretion of IL-6 and TNF-α (all P<0.001). Compared with the control group, no statistically significant differences were found in the body mass of mice compared with the three administration methods (all P>0.05). The thymus index in the 50 and 100 mg/kg AAEM-30% intraperitoneal injection groups and the spleen index in the 50 mg/kg AAEM-30% gavage group were increased (all P<0.05). CD19 + cells increased in the 100 mg/kg AAEM-30% intraperitoneal injection group ( P<0.01) and in the 50 mg/kg AAEM-30% gavage group ( P<0.05). The CD11b + and CD11c + counts increased in the 100 mg/kg AAEM-30% gavage group ( P<0.05). The number of CD4 + and CD8 + T lymphocytes was increased by both gavage and intraperitoneal administration (all P<0.05). Conclusions:AAEM-30% can promote the maturation of DC and enhanced the immunity of mice without obvious side effects.
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Objective:To investigate the immunoregulatory effects of Glycyrrhiza uralensis ethanol extract(GUEE) on the maturation of dendritic cells (DCs) and the adjuvant effect of GUEE on OVA in na?ve BALB/c mice and an ovalbumin (OVA)-induced asthma mouse model. Methods:GUEE was prepared, and the effects of different concentrations of GUEE on the maturation of DCs and the secreted cytokines as well as the effects of GUEE on bacterial lipopolysaccharide (LPS)-induced DC maturation were examined in vitro. The effect of GUEE on the morphology of mouse bone marrow derived DCs was observed using microscopy. Molecular expression levels on the surface of DCs were detected using flow cytometry. The levels of interleukin-1β (IL-1β), IL-6, IL-12, and tumor necrosis factor-α (TNF-α) in the supernatant of DCs cultures were measured by enzyme-linked immunosorbent assay (ELISA). The maturation status of DCs was detected by flow cytometry by injecting different concentrations of GUEE into the paws of mice and isolating the draining lymph nodes 24 h later. The naive BALB/c mice were co-immunized with OVA, and the changes in regulatory T cells (Treg) were detected by flow cytometry. An OVA-protein-induced mouse asthma model was established to investigate whether GUEE as a tolerogenic adjuvant has an antigen-specific therapeutic effect on asthmatic mice. Pulmonary pathological changes were analyzed by hematoxylin-eosin staining (HE) and PAS staining. OVA-specific antibodies in serum and the frequencies of Tregs, CD4 + IFN-γ + and CD4 + IL-4 + T cells in the spleen were detected by ELISA and flow cytometry, respectively. Results:GUEE suppressed DCs maturation induced by LPS both in vitro and in vivo (all P<0.05), and reduced proinflammatory cytokine production, including IL-1β, IL-6, IL-12 and TNF-α in the absence or presence of LPS (all P<0.05). Moreover, co-immunization with OVA and GUEE increased the amount of Tregs in na?ve BALB/c mice ( P<0.05). In OVA-induced asthmatic mice, OVA and GUEE co-immunization and GUEE alone treatment substantially ameliorated the inflammation of lung tissues, decreased the levels of IgG 1 and the amount of CD4 + IL-4 + T cells, and increased the amount of Tregs (all P<0.05). Conclusions:GUEE alone or as the tolerogenic adjuvant can ameliorate allergic diseases through inhibition of DC maturation and type 2 helper T cell responses and induction of Tregs.
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Objective:To investigate the regulatory effects of nuclear factor-κB (NF-κB) on dendritic cell (DC) maturation and function through solute carrier family 1 member 2 (Slc1a2).Methods:Mouse bone marrow-derived DCs were transfected with Slc1a2-specific siRNA and an overexpression Slc1a2 eukaryotic expression vector. The real-time fluorescence quantitation (RT-PCR) and Western Blot methods were used to detect knockdown and overexpression efficiency. The expression of surface molecules (CD40, CD80) and major histocompatibility complex Ⅱ (MHCⅡ) of DCs was detected by flow cytometry. ELISA was used to detect the secretion of the cytokines interleukin (IL)-12, IL-6, and transforming growth factor-β (TGF-β). The effects of knockdown of Slc1a2 on DC maturation and function and the effects of overexpression of Slc1a2 on DC maturation and function were reflected by the above assay results. A mixed lymphocyte culture assay was used to investigate the effect of Slc1a2 on T cell proliferation, and an ELISA was used to detect the lavel of IL-17A. Changes in the relative fluorescence intensity of FITC in DCs were analyzed by flow cytometry to investigate the ability of Slc1a2 overexpression on antigen phagocytosis. Finally, DCs were pretreated with an NF-κB inhibitor, toluoylphenylalanine chloromethyl ketone (TPCK), and the effect of TPCK on the expression of Slc1a2 in DCs and DC maturation was examined.Results:Slc1a2 expression was found to be high in DC treated with lipopolysaccharides (LPS) ( P<0.001). The knockdown of Slc1a2 decreased DC maturation and ability to stimulate the proliferation of CD4 + T cells ( P<0.001) and inhibited IL-17 secretion ( P<0.01). Overexpression of Slc1a2 promoted DC maturation and ability to stimulate the proliferation of CD4 + T cells(all P<0.01) Pretreatment of DC with the NF-κB inhibitor TPCK inhibited the expression of Slc1a2 at mRNA and protein levels induced by LPS. Conclusions:NF-κB regulates Slc1a2 expression, which affects the maturation and function of DC.
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Fucoidan is a kind of sulfated polysaccharide with various biological activities that mainly exists in the cell walls of brown algae. It is also found in marine invertebrates such as sea cucumbers and sea urchins. Fucoidan has received a lot of attention due to its tumor-killing and immune-boosting properties. Moreover, the combination of fucoidan with chemotherapeutic drugs not only improves antitumor efficacy but also reduces the side effects of these drugs. The function of fucoidan is closely correlated with its structure, molecular weight, degree of sulfation, monosaccharide component, algae source, and time of collection. In this review, the antitumor and immunomodulatory effects of fucoidan are reviewed from the aspects of promoting cell apoptosis, inducing cell cycle arrest, inhibiting angiogenesis and cell migration, and activating immune cells, to provide theoretical guidance for the development and clinical application of fucoidan.
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Dendritic cells (DC) as professional antigen-presenting cells can activate na?ve T cells and play an important role in bridging innate immunity and acquired immunity. Therefore, the differentiation and maturation of DC and the mechanism in its function regulatory have attracted much attention. With the rapid development of high-throughput sequencing, noncoding RNAs (ncRNAs) have been gradually known. In recent years, increasing studies have reported that ncRNAs play an importantly role in regulating the differentiation and maturation of DC. In this paper, the regulatory effects of microRNAs and long ncRNAs on the differentiation, maturation and function of DC were reviewed.
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Phagosomes undergo the fusion of early endosomes, late endosomes and lysosomes, and then degrade antigens and kill pathogenic microorganisms, which is called phagosome maturation. The functional differences of phagosomes after maturation in different immune cells lead to distinct characteristics on their processing of phagocytic antigens. It has been found that the antigen degradation ability of immune cells is opposite to their antigen presentation capacity through comparing the functional differences of phagosomes from neutrophils, macrophages and dendritic cells after antigens uptake. Among them, dendritic cells have the strongest antigen presentation capacity, followed by macrophages and neutrophils. The in-depth study of immune cell phagosome maturation and antigen presentation will provide new strategies for vaccine development and immunotherapy.
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Objective:To investigate the effect of silica gel column separation component of Artemisia asiatica (AEM-SC) on the maturation and immune function of mouse dendritic cells (DCs). Methods:Artemisia asiatica components were prepared by macroporous resin eluted with 70% ethanol, and then isolated by silica gel column to obtain AEM-SC. The contents of polysaccharides, flavonoids and triterpenes were quantified. Flow cytometry was used to detect the expression level of DCs surface molecules and antigen phagocytosis ability and to stimulate the proliferation of allogeneic T cells. ELISA method was used to detect the effect of DCs on cytokine secretion. Results:The contents of polysaccharides, flavonoids and triterpenes in AEM-SC were 10.12%, 5.7% and 3.62%, respectively. Functional tests showed that AEM-SC significantly reduced the expression levels of LPS-induced DCs surface molecules CD40, CD86 and MHC-II, reduced the expression levels of inflammatory cytokines IL-12p40, TNF-α and IL-6 (all P<0.05), improve the ability of phygocytosis ( P<0.01), and reduce the ability of DCs to stimulate the proliferation of CD4 +T and CD8 +T lymphocytes in the spleen of mice (all P<0.001). In the inflammatory mouse model experiment, AEM-SC significantly reduced the expression levels of DCs surface molecules CD40, CD86, CD80 (all P<0.001), and the expression levels of inflammatory cytokines TNF-α, IL-12p40 in serum (all P<0.01). Conclusions:AEM-SC can inhibit the maturation of DCs-induced LPS both in vitro and in vivo, indicating that AEM-SC has the immunosuppressive effect.
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Objective To investigate the effects of ethanol extracts of Lepidium meyenii Walp (LMEE) from two different areas in Xinjiang on the maturation of mouse macrophages (RAW264. 7 cells) and dendritic cells (DCs). Methods Ethanol extracts of LMEE from Tashikuergan County (Ta xian) and A La gou of Xinjiang were prepared and named as LMEE-T and LMEE-A, respectively. RAW264. 7 cells and bone marrow-derived DCs from C57BL/6 mice were treated with different concentrations of LMEE-T/A. The viability of RAW264. 7 cells was analyzed by MTT assay. Expression of costimulatory molecules and MHCⅠ on the surface of RAW264. 7 cells and DCs was detected by flow cytometry. Secretion of cytokines and the release of nitrogen oxide (NO) were measured by ELISA and Griess method, respectively. Results LMEE-T/A had no significant influence on the viability of RAW264. 7 cells when the concentration was lower than 1 mg/ml. Treating RAW264. 7 cells with LMEE-T/A promoted surface molecule expression, cytokine secretion and NO release through TLR4 signaling pathway in a dose-dependent manner. Moreover, LMEE-T was more potent than LMEE-A. LMEE-T/A at the concentration of 0. 4 mg/ml promoted the expression of DC surface molecules and the secretion of cytokines. Infrared and ultraviolet spectra showed that LMEE-A and LMEE-T contained polysaccharides, macaenes, macamides and flavanols. Compared with LMEE-A, LMEE-T contained more benzene ring compounds but less polysaccharides. Conclusion Both LMEE-T and LMEE-A could activate RAW264. 7 cells and promote the maturation of DCs. The differences between their effects might be related to the differences in their contents.
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Objective To investigate the effects of Glycyrrhiza uralensis Fisch L. crude polysac-charides (GUCP) as an adjuvant on the immunity of mice and the immune responses induced by human pap-illoma virus ( HPV)-DNA vaccine. Methods ICR mice were injected with different concentrations of GUCP by different ways to detect the influences of GUCP on body weight, organ indexes and the numbers of immune cells in spleen. C57BL/ 6 mice were co-immunized with HPV-DNA vaccine and GUCP to detect the adjuvant efficacy on antigen-specific cellular and humoral immune responses. Results GUCP in all injected groups had no side effect on mouse body weight and liver, heart, lung and kidney indexes, but intraperitone-al injection of GUCP significantly increased spleen and thymus indexes and the numbers of B cells, CD4+ T cells, macrophages and dendritic cells in spleen. Subcutaneous injection of GUCP significantly increased the numbers of B cells and macrophages in spleen and intragastric administration significantly increased the num-bers of CD4+ and CD8+ T cells in spleen. Furthermore, GUCP as an adjuvant enhanced the antigen-specific CD4+ and CD8+ T cell responses and the levels of IgG, IgG1 and IgG2a induced by HPV-DNA vaccine at a certain degree. Conclusion GUCP enhanced the immunity of mice and the antigen-specific cellular and hu-moral immune responses induced by HPV-DNA vaccine. These results suggested that GUCP might be used as an adjuvant for DNA vaccine.
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Objective To investigate the effects of Artemisia absinthium L. ( A. absinthium L. ) extracts on the maturation and function of dendritic cells (DCs). Methods A. absinthium water (AAW) and ethanol ( AAE) extracts were prepared. DCs were separated into several groups and treated with differ-ent concentrations of AAW (containing 5, 50 and 150μg/ml of polysaccharide) and AAE (containing 0. 6, 3 and 6 μg/ml of flavonoid) , respectively. Cell viability, antigen phagocytosis and maturation of DCs were detected by flow cytometry. Levels of cytokines were analyzed by ELISA. Western blot assay was performed to analyze the activation of key molecules in NF-κB, mitogen-activated protein kinase ( MAPK) and janus kinase/signal transducer and activator of transcription ( JAK/STAT) signaling pathways. Results AAW promoted the maturation of DCs, significantly decreased antigen phagocytosis and increased cytokine produc-tion (IL-12p40 and TNF-α). AAE significantly enhanced the expression of co-stimulatory molecules on the surface of DCs and decreased antigen phagocytosis, but had no significant effect on cytokine production. Mo-reover, AAE significantly inhibited the LPS-induced expression of TNF-α, IL-12p40 and IL-6. Further anal-ysis revealed that AAW and AAE could activate the phosphorylation of p38, extra-cellular signal regulated kinase (ERK), IKKα/β, NF-κBp65 and JAK2. Besides, AAE could activate the phosphorylation of c-Jun N-terminal kinase ( JNK ) and inhibit the LPS-induced phosphorylation of inhibitor of NF-κB ( IκB-α) , IKKα/β, NF-κBp65, p38, ERK and JAK2. Conclusion AAW could enhance immunity and AAE could inhibit inflammation.
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Objective To investigate the effects of water extracts of Lepidium meyenii walp (LMWE) collected from two different places in Xinjiang on the maturation and function of dendritic cells (DCs) and to evaluate their roles in immunoregulation. Methods Water extracts of LMWE growing in Tashikuergan County (Ta xian) and A La gou of Xinjiang were prepared and named as LMWE-T and LMWE-A,respectively. Bone marrow-derived DCs and splenocytes isolated from C57BL/6 mice were treated with different concentrations of polysaccharide extracts from LMWE-T/A. Effects of LMWE-T/A on the per-centage and apoptosis of DC,expression of co-stimulatory molecules and secretion of cytokines were detected by flow cytometry and ELISA. MTT assay was used to analyze the proliferation of splenocytes. Changes in the functions of DC were evaluated by mixed lymphocyte reaction(MLR). Results LMWE-T/A had no in-fluence on the percentage and viability of DC. Expression of CD40 and CD86,and secretion of TNF-α,IL-12p40 and IFN-γ were significantly increased by LMWE-T/A treatment in a dose-dependent manner. LMWE-T/A treatment enhanced the functions of DCs and also dose-dependently promoted the proliferation of splenocytes. LMWE-A was more effective than LMWE-T in promoting CD86 expression,IFN-γ secretion and splenocyte proliferation. Pretreatment with TAK-242,an inhibitor of Toll-like receptor 4(TLR4),could sig-nificantly inhibit the regulatory effects of LMWE-T/A on CD40 expression and the secretion of TNF-α and IL-12p40. Conclusion This study shows that LMWE could promote the maturation of DC through TLR4 signaling pathway,enhance the functions of DC without side effects on DC survival,and increase the prolif-eration of splenocytes,indicating that LMWE has a potential immunopotentiating effect. LMWE-A has better effects than LMWE-T on immune enhancement.
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Objective To investigate the effects of crude extract of Capparis spinosa L. fruit alka-loids (CSFA) on the maturation of murine bone marrow-derived dendritic cells (DCs). Methods CSFA was prepared and the contents were determined by high performance liquid chromatography. DCs were trea-ted with different doses (1, 2, 3 mg/ml) of CSFA. The viability of DCs, the expression of surface mole-cules and the ability of phagocytosis were detected by flow cytometry. The secretion of cytokines was meas-ured by ELISA. Western blot assay was performed to analyze the activation of key molecules in mitogen-acti-vated protein kinases ( MAPK) and nuclear factor-kappa B ( NF-κB) signaling pathways. Results The re-sults showed that CSFA alone had no significant influence on the expression of surface molecules and cyto-kines in DCs. However, it significantly decreased the expression of CD40, CD80, CD86 and MHC Ⅱ as well as the secretion of IL-12p40 and TNF-αthat were induced by lipopolysaccharides (LPS), but increased IL-10 secretion and the ability of phagocytosis after treating DCs with both CSFA and LPS. Further, the phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and the nuclear translocation of NF-κBp65 induced by LPS were inhibited by CSFA. Conclusion CSFA could inhibit the maturation of DCs and the secretion of pro-inflammatory cytokines induced by LPS while in-creasing the secretion of the anti-inflammatory cytokine IL-10 and the ability of phagocytosis, which might in-volve MAPK and NF-κB signaling pathways. This study suggests that CSFA could be used as a potential im-munosuppressant.
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Objective To investigate the effects of water extract of Glycyrrhiza uralensis Fisch.(GUWE) on the activation of RAW264.7 cells and the possible mechanism.Methods RAW264.7 cells were treated with GUWE containing different concentrations of polysaccharide (10, 50, 100, 500 μg/ml).Viability of these cells was analyzed by MTT assay.Phagocytic activity and surface molecules expressed on these cells were detected by flow cytometry.Levels of cytokines were analyzed by ELISA.Western blot assay was performed to analyze the activation of key molecules in TLR4 signaling pathway.Results GUWE at the concentration of 500 μg/ml significantly decreased the viability of RAW264.7 cells, but significantly increased the viability of RAW264.7 cells at concentrations of 50 μg/ml and 100 μg/ml.GUWE significantly enhanced the phagocytic activity of RAW264.7 cells as well as the expression of cytokines and costimulatory molecules in a dose-dependent manner.Further analysis indicated that the activation of RAW264.7 cells induced by GUWE was suppressed by TLR4 inhibitor.Moreover, GUWE enhanced the phosphorylation of NF-kB p65 and TLR4 downstream mitogen-activated protein kinases (MAPKs) including p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK).Conclusion This study indicates that GUWE promotes the activation of RAW264.7 cells through TLR4 signaling pathway.
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Many overwintering organisms produce antifreeze proteins (AFPs) that can be adsorbed onto the surface of ice crystals and modify their growth. These proteins show great diversity in structures, and they have been found in a variety of organisms. AFPs from insects have higher thermal hysteresis activity than other organisms. Recent studies revealed the structures of AFPs and put forward different ice-binding models. No mechanism, however, can apply to all antifreeze proteins and the molecular interaction between AFPs and ice are not accurately resolved. AFPs can be applied extensively to agriculture, aquaculture and low temperature storage of organs, tissues, as well as cells. To confer transgenic plant cold resistance application of AFPs is essential, while the expression and regulation of antifreeze gene need to be elucidated.
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The gene nhaA encodes functional protein that may play an important role in salt tolerance of Escherichia coli In order to study bacteria salt tolerance, a pair of primers were designed according to public nhaA sequence and was used to amplify 1 1kb DNA fragment with PCR The nhaA gene from E coli DH5?was cloned into a T vector and sequence analyses reveal that the cloned fragment contains entire nhaA gene coding region To apply the method of homology analysis,the result shows that many kinds of bacterium have nhaA gene, such as E coli K12, E coli O157:H7, Salmonella typhimurium and Salmonella enterica , et al This analysis suggests that nhaA gene lie generally in bacterial; and it has intimate relation with salt tolerance of E coli that may be of great importance in improvement of the salt tolerance of plant