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AIM: To construct the shRNA targeting anterior gradient protein 2 (AGR2) gene for exploring the effect of AGR2 on the biological behavior of nasopharyngeal carcinoma (NPC) cells.METHODS: The expression of AGR2 at mRNA and protein levels in NPC cell lines 6-10B and 5-8F was detected by real-time PCR and Western blot.The pSR-GFP/Neo-AGR2-shRNA expression vector targeting AGR2 was constructed.Based on the interference targeting AGR2, the cell migration and motility were determined by Transwell migration and motility assays.RESULTS: The expression of AGR2 was increased in NPC cell line 5-8F compared with NPC cell line 6-10B (P <0.05).When the AGR2 expression in 5-8F cells was interfered, the cell migration, invasion and tumorigenicity were weakened.CONCLUSION: The expres-sion of AGR2 is up-regulated in NPC cell line 5-8F.pSR-GFP/Neo-CLU-shRNA successfully inhibits the expression of AGR2 in NPC cell line 5-8F.AGR2 inhibits the migration, invasion and tumorigenicity of 5-8F cells in vivo.
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Objective To evaluate the application of DNA image cytometry in screening cervical intraepithe-lial neoplasia and cervical cancer.Methods 80 patients with cervical intraepithelial neoplasia admitted during No-vember 2012 and November 2014 were involved in this study.The samples from all the patients were taken by cervix brushes and prepared for 2 slides:one for liquid-based cytology staining, read by cytologist, and the other for Thi-onin-Feulgen staining, tested by DNA image cytometry.The biopsy was performed with direction of colposcopy if one test was positive to verify the sensitivity and specificity of the two methods of staining.Results 70 cases were positive by DNA image cytometry and 27 ones were positive by liquid-based cytology.There was statistically signifi-cant difference in the sensitivity between the DNA image cytometry and the liquid -based cytology ( 94.8% vs. 38.8%;p<0.05), but no difference in the specificity between them (99.6%vs.99.8%).Conclusion Besides its good specificity, DNA image cytometry is more sensitive in screening cervical intraepithelial neoplasia and cervical cancer.
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Objective To observe the expression and influence of SH2-B in hepatocarcinoma,and to investigate the molecular mechanisms of canceration in hepatocarcinoma.Methods By using SABC imunohis-tochemistry,the expressions of SH2-B were detected in 27 cases of hepatitis,29 cases of hepatocirrhosis and 47 cases of hepatocarcinoma.Hepatocarcinoma cell (HepG)2 with a low-expressed SH2-B was selected using immunofluorescence assay.There were 3 groups:the transfected group (transfected with pcDNA3.1 -SH2-B), the vector group (transfected with pcDNA3.1 )and the blank group (without transfection).After gene transfec-tion,SH2-B expression was detected by Western blotting;cell proliferation was measured by MTT assay;cell colony was counted by colony formation test;and cell cycle was analyzed by flowcy tometer.Results The posi-tive rate of SH2-B in hepatocarcinoma (95.7%)was significantly higher than 55.2% in hepatocirrhosis (χ2 =1 8.64,P 82 ±8 in the vector group (t =-20.33,P <0.01 )and 78 ±9 in the blank group (t =-1 9.64,P <0.01 ), which indicated that the cell colony numbers increased after being transfected with SH2-B.The S stage cells of the transfected group was (45.7 ±5.8)%,significantly higher than (1 9.4 ±4.7)% in the vector group (t =-20.33,P <0.01 )and (20.5 ±5.1 )% in the blank group (t =-34.69,P <0.01 ),which indicated that SH2-B could enhance promote cell cycle of HepG2 cells.Conclusion The expression of SH2-B in hepatocar-cinoma is high,and it may be involved in the canceration of hepatocarcinoma though promoting cell cycle,cell proliferation and cell transformation.
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Objective To investigate the expressions of Ezrin and phos-ezrin in squamous cell carcinomas (SCC),and to an alyze their clinic significance.Methods Immunohistochemistry was used to detect the expressions of Ezrin and phos-Ezrin in 20 cases of normal skin,31 cases of seborrheic Keratosis (SK),36 cases of basal cell carcinoma (BCC),and 37 cases of SCC.Results (1) The positive rates of Ezrin in normal skin (NS),SK,BCC,and SCC were 20.0％,25.8％,66.7％,and 89.2％,respectively.The positive reates of phos-Ezrin in NS,SK,BCC,and SCC were 10.0 ％,22.6％,77.8％,and 94.6％,respectively.Ezrin and phos ezrin in cancers were higher than that in noncancer tissues (P ＜ 0.01).(2) The expressions of Ezrin,and phos-Ezrin were closely correlated with the cutaneous tumor's type(R1 =0.87,r1 =0.89,P ＜ 0.01),malignant degree (patho-grading of SCC) (R2 =0.80,r2=0.86,P ＜ 0.01),metastasis via lymph node (R3 =0.89,r3 =0.91,P ＜ 0.01).Conclusions Ezrin and phos-Ezrin expressions were closely related to the types of tumor,malignant degrees,and metastasis.The combined-detection of Ezrin and phos-ezrin would provide a novel clue to predicting metastasis and prognosis of cutaneous tumor.
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Objective To investigate the implications of ratio of the CD4+ and CD25+ positive regulatory T cells (CD4+CD25+Tregs) in peripheral blood mononuclear cells (PBMC) and its associated regulatory factors such as forkhead transcription factor 3 (Foxp3) mRNA transcriptional activity in PBMC,serum levels of transforming growth factor beta-1 (TGF-β1),and interleukin 10 (IL-10) in the immunopathology of patients with middle to late staged nasopharyngeal carcinoma (NPC) based on a clinical trial.Methods In this study,18 NPC cases at middle to late stage as observing group and 10 healthy persons as control group were included to detect their ratio of the CD4+CD25+Tregs in the PBMC with flow cytometry (FCM) technique,transcriptional activity of Foxp3 with RT-PCR procedure,and serum levels of TGF-β1 and IL-10 with enzyme-linked immunosorbent assay (ELISA) method.A comparative analysis was used to explore their implications in the immunopathological correlation of NPC cases with their lesion.Results The ratio of the CD4+CD25+Tregs to total CD4+T cells in PBMC was significantly increased [(4.23 ±0.53)％ vs (2.65 ±0.31)％,t =8.60,P ＜0.01],accompanied with significantly elevated levels of Foxp3 transcription in PBMC (3.699 ± 0.309 vs 1.109 ± 0.146,t' =31.08,P ＜ 0.05],and serum contents of TGF-β1 [(645.56 ± 39.61) pg/ml vs (488.82 ± 36.91) pg/ml,t =10.27,P ＜ 0.01] and IL-10 [(1.27 ± 0.21) pg/ml vs (0.68 ± 0.08) pg/ml,t' =10.61,P ＜ 0.05] in these patients,when compared with that of healthy controls.Conclusions It may be true that CD4 + CD25 + Tregs,transcriptional regulatory factor Foxp3,and cytokines TGF-β1 as well as IL-10 altogether were composed of a regulating system in a positive feedback way to promote the developing process of immunotolerance phenomena in the tumor microenvironment and the initiation of immunoescape among patients with middle to late staged nasopharyngeal carcinoma.
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Hepatocellular carcinoma(HCC)shows high malignancy and metastasis. Hepatitis B virus X protein (HBx), a virus protein coded by hepatitis B virus, plays an important role in HCC metastasis. Recently, it was reported that HBx activates metastatic signal-pathways through MMPs, VEGF, uPA etc.,and promotes metastasis of hepatocellular carcinoma.
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Objective To determine the molecular mechanism of berberine (BBR) inhibiting the metastasis and invasion of nasopharyngeal carcinoma 5-8F cells, and identify whether berberine suppresses the tumor-invasive action through inhibiting Ezrin or phosphate-Ezrin. Methods The non-cytotoxic concentration of berberine was detected by MTT assay. Filopodia formation of 5-8F cells was observed by electron microscope. The invasion and motility of 5-8F cells with berberine treatment were measured with Trans-well assay. Western blot was used to investigate the Ezrin and phos-Ezrin expression in 5-8F cells treated by berberine. pcDNA3.1-Ezrin and pcDNA3.1-Ezrin M were transfected into 6-10B cells. The inhibitory effect of berberine on the motility and invasion of 6-10B-pcDNA3.1-Ezrin and 6-10B-pcDNA3.1-Ezrin M was detected, respectively. Results Berberine non-cytotoxic concentration was 0-40 μmol/L. After being treated by berberine, filopodia of 5-8F cells obviously reduced, and the permeating artificial basement membrane cells largely decreased in both time- and concentration-dependent manner. There was significant difference compared with the control group (P<0.05). Berberine suppressed the phos-Ezrin expression of 5-8F cells in both time- and concentration-dependent manner (P<0.05), but the effect of berberine was weaker on 6-10B-pcDNA3.1-Ezrin M than on 6-10B-pcDNA3.1-Ezrin. Conclusion Berberine inhibits nasopharyngeal carcinoma cell invasion through inhibiting phos-Ezrin expression and filopodia formation.
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OBJECTIVE@#To investigate the effect of Dahuang Zhechong pills (DZ) on arterial thrombotic model in vivo.@*METHODS@#Sixty-five rabbits were randomly divided into 7 groups: normal, model (collagen encapsulated thread-drawing),model+aspirin (ASA), model+clopidogrel (CP),model+ASA+CP, model+ low dosage DZ (DZL), and model+high dosage DZ (DZH). All rabbits except the normal group were fed with the drugs repectively for 8 days,and sacrificed at 2 hours after the last feeding, obtained aortae. The pathological changes in the aortae were observed under microscope,and the level of FDP, D-dimer and tissue factor (TF) were measured by enzyme-linked immunosorbent assay (ELISA).@*RESULTS@#The vascular vessels were filled with thrombi in the model group and the elastic membranes of the vessel wall were seriously injured. The arterial thrombi were observed around the vascular wall in the DZL group, but some of the thrombi were dissolved. The number of thrombi was remarkably decreased in the DZH group, and most thrombi were dissolved and the vascular intimal membranes were intact. Compared with the model group, the dry and wet weight of the thrombi and the level of D-dimer, FDP, and TF in the plasma were significantly attenuated (P0.05). The pathological changes in the vascular vessel and the elevation of plasma parameters in the DZL group were similar to those in the ASA and CP groups (P>0.05). The dry and wet weight, D-dimer, FDP, and TF in the plasma in the DZH group were significantly lower than those in the DZL group (P<0.01 or P<0.05, separatively), and closed to those in the ASA+CP group.@*CONCLUSION@#Dahuang Zhechong pills are potential novel anti-thromobotic agent for arterial thrombosis.
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Animals , Male , Rabbits , Carotid Artery, Common , Metabolism , Pathology , Drugs, Chinese Herbal , Therapeutic Uses , Fibrinolytic Agents , Therapeutic Uses , Phytotherapy , Random Allocation , Thromboplastin , Metabolism , Thrombosis , Drug TherapyABSTRACT
Objective To observe the effect of anti-Helicobacter pylori (H.pylori) treatment on patients with idiopathic thrombocytopenic purpura (ITP) and its effect on blood platelet. Methods From April 2006 to April 2008, a total of 31 patients diagnosed as having ITP and H.pylori infection were collected. These patients were given standard antibiotic therapy for H.pylori eradication (omeprazole 20 mg, clarithromycin 0.5 g, and amoxicillin 1.0 g, twice per day for 1 week). The effect of anti-H.pylori treatment was analyzed, and the blood platelets were counted before the treatment and on day 7, 14, and 28 after the treatment. Results Of the 31 ITP patients with H.pylori infection, 21 were cured and 10 were not effective, with the effective rate 67.74 %. Blood platelet increased in the cured group but did not change in the no-effect group. Conclusion Anti-H.pylori treatment could increase the blood platelet, and H.pylori infection may be related to the reduction of blood platelet in ITP patients.
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The molecular techniques were used to analyse tyrosine phosphorylation of JAK2 and STAT3 in leptin receptor overpression cell lines and SH2-Bβ knockout (SH2-Bβ-/-) mice. The serum level of leptin in SH2-Bβ mice was measured by ELISA. The results showed that SH2-Bβ dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and STAT3 in vitro. Leptin-stimulated activation of JAK2 and phosphorylation of STAT3 were significantly impaired in hypothalamus of SH2-Bβ-/- mice. The fasting and postprandial serum levels of leptin and body weight were markedly increased in SH2-Bβ-/- mice. Therefore, SH2-Bβ is an endogenous enhancer of leptin sensitivity and regulates body weight via leptin/ JAK2/STAT3 pathway.
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Objective To investigate the expression of ezrin in seborrheie keratosis(SK),basal cell carcinoma (BCC).squamous cell carcinoma (SCC)and its relation to elinical pathology parameters.Methods Skin samples were collected from 36 patients with SCC,27 patients with BCC,20 patients with SK and 10 normal human controls.Immunohistochemistry was performed to detect the expression of ezrin in these samples.The relationship between ezrin expression and prognosis of SCC was assessed using COX regression analysis.Results The positivity rate of ezrin in SK,BCC and SCC samples was 25.0%,66.7%,91.3%,respectively,compared to 20.0%in normal controls.Compared with the controls,a significant increase was observed in the expression of ezrin in patients with BCC and SCC(both P＜0.05).but not in those with SK(P＞0.05).Moreover.increased expression level of ezrin was correlated with a high degree of tumor malignancy,advanced pathological stage,and occurrence of lymph node metastasis of SCC (r=0.87,0.80,0.89,respectively,all P＜0.01).COX regression analysis revealed that the expression level of ezrin was an independent factor for the prognosis of SCC.Conclusion The expression level of ezrin is closely related to the malignancy of skin tumors,pathological grading and lymph node metastasis of SCC.
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Objective: To investigate the inhibitory effect of Dahuangzhechong pill to platelet activation.Methods: The blood platelet were incubated with the medicated blood plasma with Dahuangzhechong pill,and then activated with ADP and marked with PAC-1 monoclonal antibody.The activation rate of blood platelet was analyzed by flow cytometer.The patients with coronary heart disease or cerebral infarction took Dahuangzhechong pill,after one course of treatment,the patients,blood platelet were separated and then incubated with PAC-1 monoclonal antibody.The activation of blood platelet was detected by ? ow cytometer.Results: Compared with aspirin group(51.7%),the activation rate(20.82%) of blood platelet in Dahuangzhechong pill group decreased(P
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<p><b>OBJECTIVES</b>To identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-kappaB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC).</p><p><b>METHODS</b>A stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining.</p><p><b>RESULTS</b>LMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMP1. Moreover, LMP1-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the kappaB site kappaB1 or kappaB5 was disrupted, whereas mutation of kappaB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene.</p><p><b>CONCLUSION</b>LMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-kappaB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.</p>
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Humans , Apoptosis , Physiology , NF-kappa B , Physiology , Nasopharyngeal Neoplasms , Protein Biosynthesis , Signal Transduction , Physiology , TNF Receptor-Associated Factor 1 , Tumor Cells, Cultured , Viral Matrix Proteins , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization.</p><p><b>METHODS</b>The morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining.</p><p><b>RESULTS</b>Morphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture.</p><p><b>CONCLUSIONS</b>Nasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells. Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.</p>
Subject(s)
Humans , Cell Transformation, Viral , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p16 , Epithelial Cells , Physiology , Virology , Herpesvirus 4, Human , Physiology , Nasopharynx , Cell Biology , Virology , Tetradecanoylphorbol Acetate , PharmacologyABSTRACT
AIM: To investigate the mechanism of the AP-1 signal transduction pathway inhibited by JIP in nasopharyngeal carcinoma cells. METHODS: AP-1 activity was triggered by Dox-induced LMP1 expression in Tet-on-LMP1-HNE 2 cells (L7). The retention of phospho-JNK in the cytoplasm caused by JIP was examined with immunofluroscence assay. RESULTS: 24 h after transfection of L7 cells with the JIP expression plasmid, the translocation of activated JNK was inhibited, which resulted in the retention of phospho-JNK in the cytoplasm and down-regulation of the AP-1 activity. CONCLUSION: JIP down-regulates the activity of AP-1 through the inhibition of the translocation of JNK.