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.

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.

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.

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.

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.

Objective: To analyze the effectiveness of unicompartment allografts replacement for reconstructing bone defect after bone tumor resection around knee.

This study aimed at investigating the drug preparation of precise powder decoction pieces (PPDP) system,Fallopia multiflora radix preparata (FMRP) was employed in this study.Different specifications of PPDP were prepared,their extract rates were in contrast with the original pieces.Compared the quality uniformity of three batches between FMRP original slices and its PPDP extraction,the similarity of the chemical fingerprints was evaluated,and the contents of common peaks and quality uniformity were compared by relative peak areas.ITS2 sequence was taken as a DNA barcode to identify F.multiflora radix (FMR).As a result,the extract rate of PPDP was 2.5 times as much as the original slices.The average content of stilbene glucoside from the three original slices and the PPDP extraction were 3.56 ± 2.61 and 13.23 ± 0.37 mg·g-1,respectively;while the RSD were 73.28％ and 2.82％.The similarity of the fingerprints of the PPDP extraction was almost the same as that of the original slices,but the content and the uniformity of the common peaks of the PPDP extraction were significantly improved.Thus,FMR was accurately identified using ITS2 sequences.It was concluded that the PPDP considerably improve the decocting rate and quality uniformity,indicating that PPDP could save resources and improve the clinical efficacy.

This study aimed at comparing the precise powder decoction pieces and market raw TCM slices of P.cacumen over the decocting quality.ITS2 sequence was adopted as a DNA barcode to identify P.cacumen.The chemical composition of the medicinal materials was characterized by HPLC fingerprints for the evaluation of the similarity of precise powder decoction pieces and market TCM slices.The concentrations of quercitrin were determined using UPLC,and the characteristic common peaks were identified.In addition,the extraction efficiency between the market TCM slices and the precise powder decoction pieces was also compared by standard decoction method.It was found that P.cacumen was accurately identified by ITS2 sequences.HPLC fingerprints showed that the extraction efficiency and similarity of the precise powder decoction pieces increased compared with the market TCM slices.However,the extraction yield rate of the precise powder decoction pieces was improved by 20％ increased in accordance with the standard decoction method,while the contents of the index component,quercitrin,presented rare increase and the decocting rates of the other chemical components little change in the study.In conclusion,it was indicated that precise powder decoction pieces improved the extraction efficiency and uniformity in comparison with TCM slices.

Objective To detect the relationship between the molecular defects and their phenotypes in children with growth hormone insensitivity syndrome (GHIS).Methods 21 patients defined as GHIS were enrolled in the study.4 candidate genes (GHR,IGFALS,JAK2,and STAT5B) were analyzed by genomic DNA sequence screening and clinical relevance analysis.Results The statistical descriptions of the patients were showed as an average height standard deviation (SDS)-4.33 ± 1.91 (-9.17 to-2.21),average serum peak values of GH (22.67 ±20.98) tg/L (11.33 to 104.21 μg/L),basal serum insulin-like growth factor-Ⅰ SDS-2.65 ± 0.53 (-3.57 to -1.79),insulin-like growth factor-binding protein 3 SDS-1.77 ± 1.64 (-4.13 to 0.96).Bone age of backward difference (chronological age-bone age) (43.10 ± 19.54) months (6 to 82 months).One of two children with severe growth failure and mid-face hypoplasia was found to a homozygote for G to A gene mutation in the intron 6 splice donor consensus sequences (IVS6 ds+ 1 G-A) in the GHR gene,causing its functional defect.3 cases with mild dwarf were found gene variations as novel finding:c.1097T＞C c.1098C＞T p.V366A pathogenic variant,c.1229C＞T p.S410L and nt1843707 A→G of 5' UTR region in the IGFALS gene.JAK2 and STAT5b genes mutations were not found.Conclusion Molecular pathology of GHIS is considered as involving the defects of GHR and its signal pathway.The mutation of intron 6 splice donor sequences in GHR gene has been reported which affect the function of GHR.The 3 novel type base variants in IGFALS gene,causing non severe dwarfism,might be suspected with pathogenic roles of GHIS.

Alemtuzumab (Campath) was successfully injected in 21 kidney transplant patients,7 islet transplant patients and 1 simultaneous kidney and islet transplant patient for either prevention or treatment of graft rejection.Prophylactic administration was successfully completed in all patients without discontinuation.Adverse events were not observed in 11 patients (38%),but hypertension in 18 patients (62%),shivering in 3 patients (10.3%),high fever in 3 patients (10.3%),and bronchospasm in 1 patient (3%),respectively.All complications alleviated after proper therapy.During the prophylactic administration of alemtuzumab,strict,timely and proper ward-management was needed.Care for lung,perineum,skin,diet and psychological nursing were necessary.Neither graft acute rejection nor graft chronic rejection episode occurred in all patients during 6 months to 2 years follow-up.Therefore,long term effects of Alamtuzumab and consequences of lymphocytopenia need further observation.