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Objective: To analyze the effectiveness of unicompartment allografts replacement for reconstructing bone defect after bone tumor resection around knee.
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Objective:To investigate the values of immunophenotype and the Collagen type1 alpha1/Proto-oncogene Proteins c-sis (COL1A1/PDGFB) fusion gene in the diagnosis of dermatofibrosarcoma protuberans (DFSP). Methods:IHC markers and the COL1A1/PDGFB fusion gene were detected by IHC staining and interphase fluorescence in situ hybridization (FISH) in 73 cases previously diagnosed as DFSP. A total of 85 and 10 non-DFSP cases were also included as controls for IHC staining and FISH, respectively. Results:In the 73 DFSP cases, the positive detection rates for immunohistochemical marker vimentin, CD34, CD99, S100, desmin and SMA were 100%, 91.78%, 61.64%, 0, 0, and 6.85%, correspondingly. Protein expression levels in these cases varied from the control group, and CD34 ex-pression was significantly different among the differential diagnoses. The positive detection rate for the COL1A1/PDGFB fusion gene was 86.96%(60/69), whereas the gene expression in the control group was negative. Conclusion:The COL1A1/PDGFB fusion gene is a highly specific and sensitive marker in the diagnosis of DFSP. CD34 is a suitable marker for DFSP.
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Objective To study the effects of a miRNA family member,let-7e,on the LPS-induced expression of TNF-α in primary monocytes and the possible mechanism.Methods Peripheral blood mononuclear cells (PBMCs) were isolated form human blood sample by using density gradient centrifugation for further isolation of primary monocytes.Flow cytometry analysis was used to measure the purity of isolated primary monocytes.The efficiencies of transfection were evaluated by qRT-PCR and immunofluorescence assay after transfecting the primary monocytes with let-7e mimic or miRNA mimic negative control (NC) for 24 h,36 h and 48 h.To screen out the optimal stimulation time,ELISA was performed to detect the concentrations of TNF-α in the supernatants of cell culture after stimulating the primary monocytes with 1 mg/L of LPS for 0 h,1 h,3 h,6 h and 12 h,respectively.ELISA and qRT-PCR were used to measure the expression of TNF-α in the transfected cells with the interference of LPS.Western blot assay was used to detect the level of enhancer of zeste homolog 2 (EZH2) in let-7e mimic-transfected primary monocytes and the levels of NF-κB p65,ADP-ribosylation factor GTPase-activating protein 1 (ARFGAP1) and Arfaptin2 in the siEZH2-transfected monocytes.Results More than 70％ of the isolated cells were CD14+ cells.The miRNA mimics could transfect the primary monocytes effectively and the transfection rate was about 70％.High levels of let7e were detected in let-7e mimic-transfected primary monocytes 24 hours after the transfection.High levels of TNF-α were observed in the primary monocytes after stimulated with LPS for 12 h,which was considered as the optimal LPS stimulation time.Results of the ELISA indicated that let-7e mimic significantly inhibited the LPS-induced expression of TNF-α in primary monocytes at both mRNA and protein levels.Western blot assay showed that the levels of EZH2 in the let-7e mimic transfected primary monocytes were significantly lower than that in mimic NC transfected primary monocytes.Silenced expression of EZH2 significantly inhibited the expression of NF-κB p65 in nucleus as well as the expression of ARFGAP1 and Arfaptin2.Conclusion let-7e mimic significantly inhibited the LPS-induced expression of TNF-α in primary monocytes.It is possible that Let-7e regulates the expression of NF-κB p65,ARFGAP1 and Arfaptin2 by targeting EZH2 directly to inhibit the expression of TNF-α.
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Objective To investigate the effects of a miRNA family member, let-7e, and a combi-nation of miR-106b and miR-20a on the expression of cytokines by THP-1 cells with Luminex xMAP technol-ogy.Methods The efficiency of transfection was evaluated by immunofluorescence assay after transfecting THP-1 cells with micrONTM mimic negative control (Cy3) for 24 h, 36 h and 48 h.The three miRNA mim-ics (let-7e, miR-106b and miR-20a) were respectively used to transfect the THP-1 cells for 24 h, 36 h and 48 h and the expression of each miRNA was analyzed by qRT-PCR analysis for screening out the optimal transfection time.The transfected THP-1 cells were stimulated with1 mg/L of LPS for 1 h.The Luminex xMAP technology was used to detect the expression of IL-8, interferon-inducible protein-10 (IP-10), mono-cyte chemotactic protein 1 (MCP-1), IL-1α, IL-6, IL-10, TNF-α, IFN-αand IFN-βin the supernatants of cell culture.A statistical analysis was performed to analyze the data obtained by using SPSS16.0 software. Results More than 90% of the transfected THP-1 cells were labeled with red fluorescence.The optimal transfection times for let-7e mimic and miR-106b/miR-20a mimics were 48 h and 24 h, respectively.Com-pared with the corresponding negative control (NC), the expression of IL-8, IP-10 and MCP-1 by THP-1 cells were enhanced after the transfection with let-7e mimic, but were inhibited after the co-transfection with miR-106b and miR-20a mimics.Conclusion The expression of IL-8, IP-10 and MCP-1 were enhanced in let-7e transfected THP-1 cells, but were inhibited in miR-106b and miR-20a co-transfected THP-1 cells.
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Objective To investigate the effects of a microRNA family member , let-7e, on mono-cytic cell line THP-1 with regard to cell apoptosis and cytokine secretion and to analyze the possible mecha -nism.Methods THP-1 cells were transfected with mimic negative control (cy3) and observed with immu-nofluorescence microscopy for the evaluation of transfection rate .The expression of let-7e in THP-1 cells re-spectively transfected with let-7e mimic, mimic negative control, let-7e inhibitor and inhibitor negative con-trol were detected by qRT-PCR.MTT assay and flow cytometry analysis were used to detect the activities and apoptosis of transfected THP-1 cells.Western blot assay was performed to measure the expression of the genes encoding interferon alpha-inducible protein 6( IFI6 ) , enhancer of zeste homolog 2 (EZH 2 ) and caspase -3 that were target genes of let-7e predicted by bioinformatics analysis .THP-1 cells were transfected with let-7e mimic and mimic negative control for 48 h and then stimulated with LPS for 2 h for further detec-tion.The supernatants of cell culture were collected for the detection of secreted cytokines by Human Cyto -kine Array.Results The monocytic THP-1 cells were transfected with mimic negative control with a trans-fection efficiency of about 75%.There were 8.551±0.365, 83.893±15.941, 38.858±2.743 and 0.594± 0.174, 2.427±1.229, 3.053±0.207 fold increases in let-7e expression after the transient transfection of THP-1 cells with let-7e mimic and let-7e inhibitor for 12 h, 24 h and 48 h, respectively.The transfection of let-7e mimic into THP-1 cells enhanced the cell activities and inhibited the apoptosis of the transfected cells . Bioinformatics analysis showed that let-7e bound to the genes encoding EZH 2, IFI6 and caspase-3 with the mirSVR scores of -0.1608,-0.5693 and-0.9423, suggesting them as the predicted target genes of let-7e. The expressions of IFI6, EZH2 and caspase-3 in let-7e mimic transfected THP-1 cells were decreased as in-dicated by Western blot assay .The results of Human Cytokine Array showed that the expression of LPS-in-duced cytokines including CD154, G-CSF, CD54, IL-13, IL-1RA and IL-23 were inhibited in let-7e mimic transfected THP-1 cells. Con clusion Let-7e had an anti-apoptosis effect on monocytic THP-1 cells and in-fluenced the secretion of LPS-induced cytokines in THP-1 cells.Let-7e might regulate the biological function of THP-1 cells through inhibiting the expression of target genes encoding caspase -3, IFI6 and EZH2.
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<p><b>OBJECTIVE</b>To evaluated HER2 status using immunohistochemistry (IHC) assay and fluorescence in situ hybridization (FISH) at two different time points of tissue fixation after surgical resection of gastric cancer, emphasizing the importance of standard operation and quality control in HER2 testing.</p><p><b>METHODS</b>Forty-one resection specimens of advanced gastric cancer were collected with tissue fixation periods of < 30 min or > 30 min after surgical resection. HER2 status was evaluated by immunohistochemistry (IHC) assay and fluorescence in situ hybridization (FISH).</p><p><b>RESULTS</b>The frequency of HER2 expression by IHC in the samples with fixation time of < 30 min was higher than that in those of > 30 min (P < 0.05). However, no significant difference was observed by FISH (P > 0.05) between the two groups. Samples of < 30 min fixation time had high concordant results between IHC and FISH (100.0% for both positive and negative cases, Rho = 0.724, P < 0.05). In addition, HER2 expression by IHC was significantly correlated with Lauren classification, histologic differentiation, TNM stage and gender (P < 0.05).</p><p><b>CONCLUSION</b>The time to tissue fixation after surgical resection of more than 30 min has deleterious effect on the detection of HER2 by IHC although FISH testing is not affected.</p>
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Aged , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Receptor, ErbB-2 , Stomach Neoplasms , Chemistry , Pathology , General Surgery , Time Factors , Tissue Fixation , MethodsABSTRACT
In order to explore the expansive effect of human placental cell-free suspension (HPCFS) on bone marrow hematopoietic stem/progenitor cells, and to compare the effect of HPFCS with some cytokines and their combination, human marrow CFU-GM, CFU-GEMM and BFU-E were assayed in a semisolid methyl cellulose culture system using HPCFS, IL-3, GM-CSF, and IL-3 + IL-6 + GM-CSF + EPO as colony stimulating factors, respectively. The results showed that HPCFS stimulated the growth of CFU-GM, CFU-GEMM and BFU-E and the optimal concentrations for stimulating effect were 200 - 300 micro g protein/L, and the yield of 3 kinds of colony in HPCFS group was higher than that in IL-3, GM-CSF and IL-3 + IL-6 + GM-CSF + EPO groups. The expansive effect of HPCFS on marrow progenitors was superior to IL-3, GM-CSF and IL-3 + IL-6 + GM-CSF + EPO. Human placental cell-free suspension contained a variety of cytokines to stimulate proliferation of hematopoietic stem/progenitor cells, and it could be used as an efficacious and inexpensive agent to expand hematopoietic stem/progenitor cells in vitro.
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AIM:To investigate apoptosis and the expression of death receptors of TRAIL, TNF and Fas on hepatic veno-endotheliocyte ED25 cell strain induced by dengue virus type 2(DV2).METHODS: Flow cytometric analysis was used to detect the number of apoptotic cells and the expression levels of TRAILR1-4 ,TNFR1-2,Fas on ED25 cells before/after DV2 infection. RESULTS: The numbers of apoptotic cells of ED25 increased after DV2 infection, there were only about 5.7%?1.2% of apoptotic cells before virus infection while there were approximately 27.3%?1.6% of apoptotic cells after virus infection. At the same time the expression level of Fas also increased, before virus infection about 44.3%?2.2% of ED25 cells expressed Fas while 63.0%?2.3% of ED25 cells expressed Fas after virus infection. CONCLUSION: DV2 infection can induce apoptosis of ED25 cells, and it suggests strongly that Fas/FasL may be involved in the apoptotic signal transduction.