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Objective To detect the change of exoression level of plasma microRNA‐21(miR‐21) and TGF‐β1 in cardiac remode‐lin affer acute myocardial infarction(AMI) of the pateins .Methods 200 pateints with AMI and 100 normal controls(age ,sex matched) were enrolled .Blood samples were obtained from the normal controls and patients with AMI on the 3 days ,7 days and 14 days .Real‐time PCR was developed to detect the expression of miR‐21 and TGF‐β1 in plasma .Results The expression of miR‐21 was significantly up‐regulation in the 3 days ,7 days and 14 days in MI group than that cntrol group ,0 .74 ± 0 .21 vs .2 .62 ± 0 .23 , vs .3 .67 ± 0 .25 ,vs .4 .13 ± 0 .27 up‐regulation in the 3 days ,7 days and 14 days in MI group than that cntrol group ,0 .98 ± 0 .18 vs .2 .35 ± 0 .24 ,vs .3 .67 ± 0 .25 ,vs .4 .13 ± 0 .27 ,P<0 .05 ,respectively .The expression of miR‐21 and TGF‐β1 were up‐regulation with the change of cardiac function .Positive relationship between miRNA‐21 expression and LVDd (r=0 .757 ,P<0 .05);Positive relationship between TGF‐β1 mRNA expression and LVDd(r=0 .701 ,P<0 .05) .Conclusion The expression of miR‐21 and TGF‐β1 were up‐regulation in cardiac remodelin affer AMI of the pateins ,which involved in regulation in cardiac remodelin affer AMI .
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BACKGROUND:Because macrophages play an important role in the body’s inflammatory response, the detection of the impact of biological materials on the behavior of macrophages can assess the immunogenicity of materials. OBJECTIVE: To analyze the activation effect of decelularized extracelular matrix materials on macrophages. METHODS: The peritoneal macrophages of BALB/c mouse were obtained and cultured by dividing into five groups. Control group was simple cel culture group, experimental group 1 was acelular matrix membrane material directly contacting with macrophage for culture, experimental group 2 was fresh pericardial material directly contacting with the macrophage for culture, experimental group 3 was acelular matrix membrane material indirectly contacting with macrophages for culture, experimental group 4 was fresh pericardium material indirectly contacting with macrophages for culture. After 24 hours of culture, the secretion of nitric oxide and cytokines in cel culture supernatant was determined. After 48 hours of culture, the absorbance value was determined by MTT method and the toxicity grading was determined. RESULTS AND CONCLUSION: The toxicity grading in experimental groups 1-4 was respectively grades 2, 4, 0, 2. The nitric oxide level in experimental groups 1 and 2 was higher than that in the control group (P < 0.05), and the nitric oxide level in the experimental group 2 was higher than that in the experimental group 1 (P < 0.05). There were no significant differences in interleukin-2, interleukin-4,interferon γ, interleukin-17A and interleukin-10 levels between these five groups. The interleukin-6 level in the experimental group 2 was higher than that in the control group (P < 0.05); The expression levels of tumor necrosis factors in experimental group 1, 2 and 4 were higher than those in the control group (P < 0.05), and experimental group 2 higher than the experimental group 1 (P < 0.05), experimental group 1 higher than the experimental group 4 (P < 0.05). These results show that acelular matrix material can activate macrophages in direct contact.
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[ ABSTRACT ] AIM: To observe the effect of rapamycin on the apoptosis of mouse astrocytes in vitro.ME-THODS:The astrocytes from C57BL/6J newborn mouse pups were isolated and primarily cultured.The effect of rapamycin on the viability of astrocytes was assessed by MTT assay.The mean fluorescence intensity of SYTOX?Green stain in the astrocytes was detected by fluorescence microplate reader in order to analyze the effects of rapamycin on the cell death in-duced by H2 O2 , ionomycin and/or deferorxamin.DiOC6 (3) staining was used to analyze the mitochondrial membrane po-tential of the astrocytes induced by H2 O2 .Flow cytometry analysis was used to determine the production of ROS in the as-trocytes and mitochondria by staining with H2 DCFDA and MitoSOXTM Red reagent, respectively.RESULTS: Rapamycin at concentration of 0.5 μmol/L protected the astrocytes against cell death induced by H2 O2 or deferoxamine plus ionomy-cin.Rapamycin protected the mitochondrial membrane potential of astrocytes from the injury of H2 O2 .It also reduced the production of ROS in the astrocytes and decreased the level of ROS in the mitochondria.CONCLUSION:Rapamycin re-duces the ROS overload in the mitochondria, keeps mitochondrial membrane potential safety and protects the astrocytes a-gainst apoptosis in vitro.
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Before the technique of advanced high-throughput sequencing comes up , less is known about the human gut microbiota .It has been understood that trillions of microbes , in which 99% are bacteria , inhabit the human gut, forming a complicated ecological community .The gut microbiota has a great impact on human physiology and suscepti -bility to disease through its integrative metabolic activities and interactions with the host .In physiology , gut microbiota con-tributes to the host acquisition of nutrition and energy from diets , promoting development and maturation of gastrointestinal tract and immune system , and protecting host from invasion of enteropathogens .In pathology , dysbiosis underlying altered gut microbiota is associated with the susceptibilities to various diseases , including inflammatory bowel disease , type 1 dia-betes, asthma, obesity, metabolic syndrome , autism and cancer .Understanding of the factors that underlie alterations in the composition and function of gut microbiota will be helpful in the development of drugs and the design of therapies that target it.This goal is formidable .It is because that the compositions of gut microbiota are immensely diverse , varying be-tween individuals in a population and fluctuating over time in an individual , especially during early development and disea-ses.Viewing the gut microbiota with an ecological perspective will provide new insights into how to improve our health by targeting this microbial community in clinical treatments .
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Nude mice can be used as important animal models to study thyroid cancer because of their deficiency of immunitv,The model is helpful study the sensitivity of thyroid cancer to drugs.The tumor cells may come from primary tumor tissue,or from the cell lines existed.There are three methods to transplant the tumor,including orthotopic transplantation,heterotopic transplantation and tail vein injection.
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Objective:The protein of missing in metastasis (MIM), a novel discovered actin-binding scaffold protein, is involved in actin cytoskeleton rearrangements, signal transduction and transcriptional activation, and has close association with tumor growth, invasion, and metastasis. Recently, much focus has been placed on the role of MIM performed in tumor progression. In recent years, more and more attention is focused on its action mechanism in various kinds of tumors, which has a wide foreground of investigation. In this paper, we make a comprehensive review of the association of MIM with cancer development, in order to provide the theoretical basis and new strategies for application of MIM proteins in cancer diagnosis and treatment.
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Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.
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Synaptic injury exists in the early period of vascular cognitive impairment (VCI), and it is closely correlated with the cognitive dysfunction, however, its specific mechanism remains unclear. To study the plasticity of synaptic morphological structure, the plasticity of the efficiency of synaptic transmission as well as the role and mechanism of synapic proteins in the onset of VCI will help to further clarify the pathogenesis of VCI, and thus more effectively combat VCI.
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The study investigated the effects of red clover extract (RCE) on mouse T macrophages and lymphocytes in vitro. The cell toxic effect of RCE was estimated by MTT assay. Multiple-fluorescence staining plus flow cytometry were used to detect the effect of RCE on CD69/CD25/CD71 expression of mouse T lymphocytes stimulated by Con A; CFDA-SE staining plus flow cytometry were used to analyze the effect of RCE on proliferation of T lymphocytes activated by Con A; The effect of RCE on nitric oxide(NO) secretion of mouse macrophages stimulated by lipopelysaccharide (LPS) for 24 h was assayed by Griess reagent system. We found that RCE had potent anti-inflammatory effects on mice. RCE had littl ecell toxic effect on mouse lymphocytes and macrophages. RCE strongly inhibited the excessive production of inflammatory mediators ( NO, CD69, CD25, CD71 ), in a dose-dependent manner, like cyclosporine A injection. RCE could inhibit proliferation of CD3<'+> T lymphocytes. These data suggested that RCE migh texhibit anti-inflammatory effect by inhibiting the activation and proliferation of mouse lymphocytes and the NO secretion of mouse macrophages.
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Objective To investigate the character of real-time gray-scale contrast-enhanced ultrasonography (CEUS) and its clinical value in diagnosing hepatic focal lesion. Methods One hundred and three patients with 142 focal hepatic lesions were examined by CEUS after an intravenous administration of the contrast agent, then the characters of the images were analyzed. Results The initial contrast-enhanced signal patterns were classified into 5 modes, peak contrast-enhanced signal patterns into 4 modes, and contrast agent perfusion patterns into 7 modes. Different lesions had different characters of contrast-enhanced phases. The accuracy rate of the CEUS in diagnosing focal hepatic lesion was 93.0%. which was significantly higher than that of conventional ultrasound and contrast-enhanced CT (X2=47.430, P<0.05). Conclusions The characteristic initial contrast-enhanced pattern and contrast agent perfusion pattern are helpful in the differential diagnosis of hepatic focal lesion, while peak contrast-enhanced signal pattern is relatively unreliable. Compared with conventional ultrasound and contrast-enhanced CT, CEUS can dramatically improve the accuracy of qualitative diagnosis of hepatic focal lesion.
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Objective: To observe the effect of Trifoliumpratense Leguminosae extract (TLE) on mouse allogenetic skin transplantation. Methods: Recipient BALB/c was divided into physiologic saline (PS) group and TLE group, full-thickness skins were transplanted through back to back method from donor C57BL/6. The allogenetic transplanted skin growth condition was observed. The proliferation of lymphocytes of recipient mice in vitro were detected by CFDA-SE stain and mixed lymphocyte reaction respectively. Results: The allogenetic transplanted skin injected with TLE 25g/kg per day by vena caudalis growed better than that in PS group. The proliferation of lymphocyte in TLE group was smaller than that in PS group. Conclusion: TLE maybe participate in the regulation of mouse immune system and induce its tolerance to the allogenetic transplanted skin.
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AIM: The aim of the present study is to explore the effects and mechanisms of HIV-1 glycoprotein gp120 on calcium-influx and ERK-phosphorylation of human microglia.METHODS: The level of intracellular calcium of human microglia grown on coverslip,which was loaded by calcium-probe,Fluo-4,and then treated in various experimental processing,was detected by confocal microscopy with time resolution mode.The binding of gp120 to human microglia was determined with confocal microscopy or flow cytometry after treatment with gp120 and stained with anti-gp120-FITC antibody.Phosphorylation of ERK within human microglia with or without gp120 stimulation was analyzed with confocal microscopy following the direct immuno-staining with anti-phosphorylated ERK antibody.RESULTS: The results of confocal microscopy showed that calcium-influx response was triggered by HIV-1 glycoprotein gp120 in human microglia.The results from analysis by confocal microscopy and flow cytometry showed that gp120 was able to bind to human microglia.ERK phosphorylation was enhanced in human microglia stimulated with gp120.CONCLUSION: HIV-1 glycoprotein gp120 induces calcium-influx in human microglia and enhances ERK phosphorylation in human microglia,indicating that gp120 is an activator of human microglia.So gp120 may be involved in the pathogenesis of HIV-associated dementia.
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AIM:To investigate the effects of AT-2-inactivated HIV-1 particles on human CD4+T cell activation and cytokine secretion in whole blood (WB) in vitro. METHODS: HIV-1ⅢB particles were inactivated by AT-2 chemical and the concentration of p24 antigen was determined by p24 ELISA. AT-2-inactivated HIV-1ⅢB particles were added to human WB culture system in serial concentrations to stimulate the cells. PHA was used as positive control. After 24 h, all the cultural supernatants were harvested and the concentrations of Th1 (IL-2, IFN-? and TNF-?) and Th2 (IL-4, IL-6 and IL-10) cytokines released to the supernatants were detected by cytometric bead array (CBA). The percentage of CD69 expression on CD4+T cells from WB was detected by immuno-fluorescence staining plus flow cytometry. RESULTS: The concentration of p24 antigen in the AT-2-inactivated specimen was 85.5 ?g/L. 24 h later, the percentage of CD69 expression on CD4+T cells from control group was (1.62?0.63) %, whereas it was (38.82?6.00)%, (3.83?1.07)%, (5.94?0.85)% and (9.30?1.22)% in PHA group, HIV-1 (1/500) group, HIV-1 (1/50) group and HIV-1 (1/5) group, respectively. Cytokines secreted by WB in control group were mainly TNF-? and IL-6. However, all the six cytokines tested were strikingly increased in PHA group, as well as in HIV-1ⅢB groups. CONCLUSION: AT-2-inactivated HIV-1ⅢB particles activate CD4+T cells from WB, and up-regulate both Th1 and Th2 cytokine secretion in WB. Besides the effects of viral proteins, other mechanisms may be proposed that HIV-1 particles act as antigen presenting cell (APC) because many host-derived immune molecules are incorporated into HIV-1 envelop when it is released from infected cells by budding, and exert immune modulation.
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A breakthrough has recently been made in the studies on pathogenesis of HIV disease,which result in some new theories.New strategies for HIV drug and vaccine development are emerging in the impact of these new understandings.The intestinal infection hypothesis proposes that HIV disease can be regarded as some kind of infectious disease of gut immune system as the major HIV infection is located in intestine.The acute catastrophe hypothesis suggests that the subsequent pathological changes are the fallout from a mucosal catastrophe of acute intestinal HIV infection,in which the majority of CD4+ T cells are deleted due to infection.The general immune overactivation hypothesis proposes that the general immune overactivation is harmful to patient as it increases the cell susceptibility to HIV infection and supportiveness of HIV replication.The host proteins related to HIV replication can be new targets for HIV drug discovery.The mucosal vaccine strategies,which attempt to induce HIV specific neutralizing antibodies on mucosal surface may be the first choice in development of prophylactic HIV vaccines.Induction of tolerance may be one of the strategies for HIV therapeutic vaccines because HIV disease can be regarded as autoimmune disease caused by HIV infection.
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Object To study the effects of different growth regulators and organic additives on rooting of regenerated shoots from Dendrobium huoshanense C Z Tang et S J Cheng protocorm like bodies Methods In order to induce root development, the regenerated shoots were cultured on MS medium containing 3% sucrose and 0 8% agar and supplemented with NAA, IBA, BA, KT, banana mud or amino acids at different concentrations During rooting induction, the development of roots was observed and the frequency of rooted shoots was scored Results The frequency and the process of root initiation were significantly influenced by different growth regulators or organic additives For initiation and development of roots, the suitable concentration of NAA, IBA, BA, KT, banana mud or amino acids was 0 2 , 0 1, 0 8, 0 8 mg/L, 20% and 0 1%, respectively Conclusion In all media tested, NAA, IBA or banana mud display greater effects on rooting of regenerated shoots of D huoshanense than others and KT enables shoots to keep green
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AIM:To explore a method of isolation,purification,culture and identification of human microglia.METHODS:The brain tissue from abortive fetus was sterilely obtained,then chopped and triturated gently.The suspension was filtered and centrifuged to separate and isolate mixed glia cells.The cell density was adjusted to 2?1010 cells/L with DMEM/F12 complete medium and cultured in 75 cm2 flask at 37 ℃ in CO2 incubator(marked as first 0 d).After 7-10 days culture,floating cells including microglia and oligodendrocytes appeared in the flask.The first time purification was carried out to obtain highly purified microglia,for which the flasks were shaken gently.The floating cells were collected and transferred into a new flask(marked as second 0 d).4-5 days later,when microglia and oligodendrocytes grew in confluent state,the second purification was performed,for which the mixed cultured cells were digested with trypsin-EDTA and cell suspension was centrifuged,then the cell pellet was suspended by DMEM/F12 complete medium and cultured in flask(marked as 0 h).After 2 h,non-microglial cells including oligodendrocytes and cell debris were removed,and fresh medium was added into the adherent cells.In this way,the whole procedure of microglia purification was completed and the highly purified microglial cells were obtained.After purification,the expression rate of CD45,CD11b and CD68 on/in microglia were detected by laser-confocal microscopy and flow cytometry on the basis of immuno-fluorescence staining to identify the purity of microglial cells.Microglial phagocytotic function was evaluated by phagocytosis of fluorescent microspheres.The level of activation was identified by the phenomena of membrane ruffling in combination with fluorescent phalloidin staining.RESULTS:More than 98% of the microglias that we isolated and purified expressed CD45,CD11b and CD68.Almost all of the microglias could phagocytize fluorescent microbeads CONCLUSION:We succeed in isolating and purifying human microglias,which express CD45,CD11b and CD68.Almost all of the cells exhibit phagocytic function.So these cells will be useful for further functional and proteomic studies.
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AIM: To study the effects of [8-(diethylamino) octyl-3, 4, 5 -trimethoxybenzoate] (TMB-8), an intracellular Ca2+ antagonist, on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (Con A) in vitro. METHODS: After stimulated with Con A, T cells were treated with different concentrations of TMB-8 alone and its combination with cyclosporine A (CsA). The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation-related index was determined by carboxyl fluorescin diacetate succinmidyl ester (CFDA-SE) flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining.RESULTS: After 6 h culture, the activation rate of CD69+ T cell in Con A group was (74.88?1.88)%. 10, 20 and 40 ?mol/L of TMB-8 inhibited the expression of CD69 (P
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AIM: To explore the inhibitory effect of oxymatrine (OMT) on the allergic contact dermatitis (ACD) stimulated by dinitrofluorobenzene (DNFB) and its effects on the proliferation of the lymphocytes. METHODS: ① An ACD mouse model was established by stimulation with DNFB, and then the mice were injected intraperitoneally with different dosages of OMT, PBS and hydrocortisone (HCT) respectively, the swelling degree of their auricles was examined. ② Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) dye and flow cytometer were used to examine the fluorescence intensity changes of lymphocytes stimulated by polyclonal stimulator ConA and OMT. RESULTS: ① compared with PBS group, OMT possessed the strong inhibitory effect on the ACD caused by DNFB in a dose-dependent manner, and its inhibitory effect was equivalent to the HCT of the same dosage with fewer side effects. ② In vitro experiments proved that OMT (500, 125 and 31 mg/L) had the ability to restrain the proliferation of lymphocytes of mouse. CONCLUSION: OMT possesses an inhibitory effect on the ACD induced by DNFB, and OMT is a kind of immunosuppressor.
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Aim To investigate the effects of caffeic acid phenethyl ester (CAPE) on activation and proliferation of murine CD3+ T lymphocytes,and to study the mechanisms of its immunomodulative effects. Methods Lymphocytes suspension was isolated and prepared sterilely from murine lymph nodes, and was incubated with different concentrations of CAPE for 2 h, then polyclonal activator Con A was added to activate the lymphocytes; double-fluorescence staining and flow cytometry were used to detect CD69 and CD25 expressions to evaluate activation level of CD3+ T lymphocyte, and CFDA-SE staining plus flow cytometry were used to analyze CD3+ T lymphocyte proliferation. Results CAPE (0.5, 1, 5, 10 mg?L-1) could inhibit the expression of CD69 and CD25 of CD3+ T lymphocytes in a dose-dependent manner, and it also could inhibit T lymphocyte proliferation stimulated by Con A in a dose-dependent manner. Conclusion In vitro, CAPE could exert notably inhibition on activation and proliferation of murine CD3+ T lymphocytes,and its immunomodulative effects might result from affecting PLC-? signaling and MAPK-signaling pathways. So CAPE was a potentially effective immunoinhibitory agent.
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AIM: To explore the characteristics of T cell activation and regulatory T cells derived from murine Peyer's patches through comparative studies on Peyer's patches, mesenteric lymph nodes and inguinal lymph nodes. METHODS: Signal cell suspendsions were prepared from murine mesenteric lymph nodes (MLNs), the Peyer's patches (PPs) and inguinal lymph nodes (ILNs), respectively. The percentage of cell subpopulations such as CD3+ T cells, CD3+CD4+ helper T cells and regulatory T cells (Treg, CD4+CD25+) were analyzed. Lymphocytes were activated by polyclonal stimulators such as concanavalin (Con A), phorbol 12, 13-dibutyrate (PDB) only, and PDB plus ionomycin (Ion). The expression of CD69 (the early marker of CD3+ T cell activation) was measured by FACS. RESULTS: A lower ratio of CD3+ T cells was seen in PPs than those in MLNs and ILNs. The ratios of CD3+ CD4+ T cells to CD3+ T cells in PPs, MLNs and ILNs were almost the same. A higher rate of Treg was seen in CD4+ T cells from the PPs as compared with those from MLNs and ILNs. A higher percentage of activated CD3+ T cells derived from the PPs cultured without polyclonal stimulators were detected as compared to MLNs and ILNs, while lower responsiveness of CD3+ T cells from the PPs stimulated by Con A was seen as compared with those from MLNs and ILNs. CONCLUSIONS: The lower rate of CD3+ T cells as well as higher rate of Treg in PPs was due to its desensitization. The higher rate of basic activated state in CD3+ T cells from the PPs indicated that the T cells were activated by enteric antigens in physiological conditions. The lower responsiveness of activation to some polyclonal stimulators probably reveals that the T cells are in a state of anergy. All the characteristics mentioned above contribute to prevent pathological inflammations and maintain tolerance to enteric antigens such as food proteins and commensal bacteria but simultaneously retain proper immune responses to pathogenic microbes.