Objective To observe the effect of ethanol extract and ethyl acetate extract of Capparis Spinosa on the thickness of dermis,synthesis of collagen type Ⅰ,type Ⅲ,and expression of transforming growth factor-β1 in mouse models of scleroderma.Methods Mouse models of scleroderma were established through local injection of bleomycin on the back once a day for 4 weeks.After confirmation of model establishment,72 mouse models were equally and randomly divided into three groups.Two groups received topical treatment with ethanol extract of Capparis Spinosa and ethyl acetate extract of Capparis Spinosa,respectively,no treatment was given to the rest of the control group.After 2-,4-,6-week treatment,8 mice were sacrificed and tissue samples were obtained from the back,and subiected to the measurement of dermal thickness by HE staining,as well as to the analysis of expression of collagen type Ⅰ,collagen type Ⅲ and transforming growth factor-β1 by immunohistochemical staining.Results On week 2,4,6,the thickness of dermis was 23.22,24.94,19.97 μm respectively in mice treated with ethanol extract of Capparis Spinosa,27.66.26.15,22.13 μm respectively in those treated with ethyl acetate extract of Capparis Spinosa.Compared with the mouse models without treatment,the thickness of dermis significantly decreased(F=12.99,P＜0.01),the expression of collagen type Ⅰ(F=7.47,P＜0.01)and transforming growth factor-β1(F=11.76,P＜0.01)were also inhibited in those receiving treatment.However,the expression of collagen type Ⅲ was not affected obviously by the treatment.Conclusion The ethanol extract and ethyl acetate extract of Capparis Spinosa have the effect against skin fibrosis.

Objective To observe the levels of Foxp3+ CD+ CD25+ regulatory T cells in the peripheral blood of condyloma acuminatum (CA) patients and investigate their roles in the pathogenesis of CA. Methods The peripheral blood was collected from 30 CA patients (including 15 with relapsing and 15 with first onset) and 20 healthy controls. Peripheral blood mononuclear ceils (PBMC) were isolated and stained with anti-human CD4-PE-Cy5 and anti-human CD25-fluorescein isothiocyanate (FITC) monoclonal antibodies on cell membrane, followed by intraeellular staining with anti-human Foxp3-PE. The percentage of Foxp3+ CD4+-CD25+ regulatory T cells was detected by three-color flow cytometry. Comparison between groups was done by ANOVA test. Results The percentages of Foxp3+ CD4+ CD25+ regulatory T cells among total CD4 + T cells in CA patients and relapsing CA group were (3.4 ± 1.0) % and (4.7 ±+ 1.2) %, respectively, which were both significantly higher than that in healthy control group [(1.2±0. 5)%, P<0.01]. Furthermore, that in first onset CA group was (2. 1 ± 1.0) %, which was higher than that in healthy control group, but without statistical significance; but that in relapsing CA group was significantly higher than that in first onset group (P<0.05). Conclusions The number of Foxp3+ CD4+ CD25+ regulatory T cells increases in the peripheral blood of CA patients. The disorder of cellular immunity may be involved in the immunopathogenesis of CA.

Objective To screen and identify the predicted epitopes of synthesized HLA-A*0201restricted CTL derived from HPVll E7 antigen.Methods Five HPVll E7 CTL epitope peptides and terramers consisting of HLA-A*0201 were selected by way of computer and synthesized by Sanquin company,including HPVllE7 7-15(TLKDIVLDL),15-23(LQPPDPVGL),47-55(PLTQHYQIL),81-89(DLLLGTLNI)and 82-90(LLLGTLNIV).These peptides binding to human peripheral blood-derived DCs were tested for their ability to activate T cells isolated from peripheral blood lymphocytes of HLA-A*0201 healthy individuals.the number of specific tetramer+CD8+T cells by flow cytometry,the level of the section of IFN-γ by ELISA,and the ability of the CTL to kill the target cells were observed.Results The immature DCs could be fully activated by all the five HPV11 E7 peptides.Peptide-loaded mature DCs were able to stimulate the epitope-specific T cells responses in vitro.An increased frequency(P<0.05)of T ceils specific for the E7 7-15 epitope compared to other epitopes of HPV11E7.The epitope-specific CTL of E7 7-15 induced by the activated DCs specifically killed HPV11E7 expressing 293 cell line,and in a ratio of 50:1,the specific cytolytic activity was the strongest than the others(P<0.05).Conclusion DCs loaded with HPV11 E7 7-15(TLKDIVLDL)peptide can induce highly effective and specific ectogenic processed epitopespecific CTL responses in vitro.This peptide may be the candidate for development of CTL based vaccine in the treatment of HPV infeetions.

ObjectiveTo observe the effect of acetylcholine (ACh) on lipopolysaccharide (LPS) induced inflammatory model of rat alveolar macrophages, and to observe the effect of the acetylcholinesterase inhibitor physostigmine (Phy) on the anti-inflammatory effect of ACh.Methods The rat alveolar macrophages NR8383 were cultured in vitro, which were divided into five groups: blank control group, LPS group (stimulated with 1 mg/L LPS for 12 hours), LPS+ ACh group (0.01, 0.1, 1, 10, 100μmol/L of ACh were added for 5 minutes before LPS stimulation), LPS+ Phy group (1 mmol/L Phy was added for 5 minutes before LPS stimulation), and LPS+ ACh+ Phy group (1 mmol/L Phy and 10μmol/L ACh were added for 5 minutes before LPS stimulation). The supernatants were collected in each group, the enzyme-linked immunosorbent assay (ELISA) was used to assay the contents of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, and IL-6). The activity of acetylcholine esterase (AChE ) in the supernatant was also determined.Results① The contents of TNF-α (ng/L: 605.09±57.13 vs. 34.07±8.62), IL-1β (ng/L: 377.09±28.55 vs. 32.33±10.62) and IL-6 (ng/L: 558.04±77.45 vs. 42.62±11.21) in the LPS group were significantly higher than those in the blank control group (allP< 0.05). These results indicated that the inflammatory model of rat alveolar macrophages was constructed successfully.② ACh with the final concentrations of 0.01, 0.1, and 1μmol/L had less influence on the production of TNF-α, IL-1β and IL-6 in the culture supernatants of alveolar macrophages stimulated with LPS compared with LPS group (allP> 0.05). Nevertheless, 10μmol/L and 100μmol/L ACh notably reduced the production of TNF-α (ng/L: 451.19±30.67, 332.19±32.19 vs. 604.96±22.56), IL-1β(ng/L: 261.08±24.78, 143.98±28.39 vs. 367.06±10.44) and IL-6 (ng/L: 342.75±54.60, 235.48±29.75 vs. 562.69±63.34) in the culture supernatants compared with the LPS group (allP< 0.05).③ The activity of AChE in the LPS group was significantly higher than that in the blank control group (kU/L: 5.21±0.63 vs. 3.09±0.10,P< 0.05). The activity of AChE was successfully inhibited by 1 mmol/L acetylcholinesterase inhibitor Phy pretreatment compared with that in the LPS group (1.51±0.12 vs. 5.21±0.63,P< 0.05).④ The level of TNF-α (ng/L: 183.17±35.44 vs. 451.19±30.67), IL-1β (ng/L: 91.49±12.27 vs. 261.08±24.78) and IL-6 (ng/L: 108.17±22.82 vs. 342.75±54.60) in the culture supernatants of LPS+ ACh+ Phy group was significantly decreased as compared with LPS+ ACh group (allP< 0.05).Conclusions ACh with the final concentrations of 10μmol/L and 100μmol/L can inhibit the LPS induced inflammatory reaction in alveolar macrophages. The acetylcholinesterase inhibitor Phy can reinforce the ACh-mediated anti-inflammatory effect on alveolar macrophages inflammatory model.

h Kaposi' s sarcoma in Xingjiang, which have a high homology with those strains from Africa and Europe. A5 and C7 genotypes of HHV-8 have been first isolated in China.

AIM:To investigate the role of microRNA-132 (miR-132) on alveolar macrophage inflammation. METHODS: Rat alveolar macrophage cell line NR8383 was transfected with miR-132 mimic, mimic negative control ( NC) , miR-132 inhibitor, or inhibitor NC.The cells were divided into transfection group, transfection +lipopolysaccha-ride ( LPS) group, and transfection +LPS +acetylcholine ( ACh) group.The mRNA expression of acetylcholinesterase ( AChE) was detected by real-time PCR.The protein levels of AChE, signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (p-STAT3) in the cells, and nuclear factor-κB (NF-κB) in the cytoplasm and nu-cleus were analyzed by Western blot.The activity of AChE in the culture supernatant was measured by AChE activity assay kit.The nuclear translocation of NF-κB was detected by immunofluorescence assay.RESULTS: Up-regulation or down-regulation of miR-132 had no effect on the mRNA expression of AChE.However, up-regulation of miR-132 decreased the protein level of AChE compared with mimic NC group (P<0.05).Transfection with miR-132 inhibitor increased the pro-tein expression of AChE compared with inhibitor NC group ( P<0.05 ) .In the alveolar macrophages treated with LPS+ACh, the inhibition of nuclear translocation of NF-κB p65 in miR-132 mimic group was more effective than that in mimic NC group ( P<0.05) .The inhibitory effect in miR-132 inhibitor group was weaker than that in inhibitor NC group ( P<0.05 ) .The inhibitory effect of miR-132 mimic on the protein levels of STAT3 and p-STAT3 was stronger than that of mimic NC (P<0.05).CONCLUSION:miR-132 in LPS-stimulated alveolar macrophages reinforced ACh-mediated anti-inflam-matory reaction by targeting AChE to suppress ACh hydrolyzation, which was related to the suppression of NF-κB and STAT3 activation.

Cytosine methylation is one of the major types of DNA epigenetic modifications and plays an important role in maintaining normal cell function and regulating gene expression. Bisulfite sequencing PCR (BSP) based cloning and sequencing is a general method for detecting DNA methylation at specific sites, which can clarify the methylation status of each CpG site in the target fragment. However, this method requires large amounts of single-clonal sequencing, which is complicated to operate, time consuming and expensive. Therefore, the development of an accurate, efficient and convenient DNA methylation detection technology is of great significance to improve the efficiency of epigenetic research. Based on the high-throughput mutation detection platform Hi-TOM (high-throughput tracking of mutations) developed by our group, we further established a site-specific DNA methylation high-throughput detection platform Hi-Meth (High-throughput Detection of DNA Methylation). After bisulfite treatment of DNA samples, the specific site-specific DNA methylation analysis results could be obtained through the Hi-Meth platform by performing only one round of PCR amplification. Using the Hi-Meth platform, the DNA methylation status of two promoter regions of rice were detected. The DNA methylation results from Hi-Meth were consistent with the results from BSP-based method. Thus, site-specific DNA methylation analysis results could be obtained accurately and conveniently through the Hi-Meth platform. In conclusion, Hi-Meth provides an important methylation detection platform for specific DNA regions, which has important significance for epigenetic research.
DNA Methylation ; Epigenesis, Genetic ; Epigenomics ; High-Throughput Nucleotide Sequencing/methods* ; Polymerase Chain Reaction ; Sequence Analysis, DNA/methods*