Objective:To study the possible immune escape mechanisms of HCoV-OC43 from human dendritic cells(DC).Methods:HCoV-OC43 was isolated from clinical specimen using BSC-1 cells and identified by Real-time PCR,and the cytopathic effect was observed by phase contrast microscope.DCs were induced in vivo using hu-GM-CSF and IL-4 cytokines,and after 7 days of differentiation,DCs were infected by HCoV-OC43.The morphology of HCoV-OC43 infected DC was observed by transmission electron microscope,and the cytokines related to DC functions were detected by Real-time PCR after infection.DC proportion and function related co-stimulatory molecules were analyzed by flow cytometry.Results:In vitro HCoV-OC43 infected human DC model was successfully built.HCoV-OC43 can infect DC and generate immune response of DC in vitro,but no virus nucleonic acid could be detected in culture supernatant.The DC expression of IFN-α,IFN-β,CCL3 and CCL5 were significant decreased when infected with HCoV-OC43,but the expression of costimulatory molecules including HLA-DR,CD1c and CD86 were not affected by HCoV-OC43 infection.Conclusion:Human DC could be infected by HCoV-OC43 and generate immune response,but could not produce progeny virus.HCoV-OC43 may escape from immune response by suppressing the expression of IFN-α and other inflammatory cytokines and chemokines in DC.

Objective To establish a method for simultaneous determination of HPLC fingerprint and multi-target ingredients in Atractylodis Macrocephalae Rhizoma(AMR),in order to provide reference for its quality control.Methods HPLC-DAD multi-wavelength switching method was used to establish fingerprint of AMR,similarity evaluation combined with hierarchical clustering analysis(HCA),principal components analysis(PCA)and discriminant analysis of partial least squares(PLS-DA)were used to carry out chemometric study.The contents of differential component such as atractylenolide Ⅰ,Ⅱ,Ⅲ and atractylon were determined simultaneously.Results The HPLC fingerprint of 37 batches of AMR was established.Nine common peaks were marked,and 4 of them were identified as atractylon,atractylenolide Ⅰ,Ⅱ,Ⅲ.The similarity degrees were between 0.539 and 0.996,the quality of AMR from different origin and different batches varies greatly.Atractylon,atractylenolide Ⅰ,Ⅱ,Ⅲ and one unknown component(peak 9)are the important factors affecting the quality of AMR.Conclusion The combination methods of HPLC fingerprint and simultaneous determinations of multiple components are simple,stable,accurate and reliable,which can provide reference for the quality evaluation of AMR and the improvement of quality standard,as well as lay a foundation for the basic research of its pharmacodynamic substances and related compound.

OBJECTIVETo investigate a drilling guide in the treatment of acromioclavicular joint dislocation with closed reduction and Kirschner fixation and explore the therapeutic effect.

METHODSFrom June 2008 to December 2009, 36 patients with acromioclavicular joint dislocation (Tossy III) were treated with closed reduction and Kirschner fixation using a self-designed drilling guide as well as percutaneous repair of acromioclavicular joint. Among the patients, 24 patients were male and 12 patients were female,ranging in age from 20 to 61 years, averaged 38.6 years. The duration from injury to operation ranged from 3.5 to 72 h,with a mean of 15.2 h. No clavicle fracture was found in all cases. The operative time, intra-operative bleeding and therapeutic effects were observed.

RESULTSThere were no complications including neurovascular problems. The mean operating time were 20 min,mean blood loss were about 10 ml. According to the observation of postoperative X-ray examination, all Kirschners in acromioclavicular joint were in place. All Kirschners were removed in 6 postoperative weeks. All the patients were followed up ranging from 2 to 26 months (averaged 14.3 months). According to the Karlsson standard,22 patients got an excellent result, 13 good and 1 poor.

CONCLUSIONThis method has following advantages: easy operation and fixation; minimum injuries to articular surface; and which would be widely used in clinical practice.

Acromioclavicular Joint ; diagnostic imaging ; injuries ; physiopathology ; surgery ; Adult ; Bone Wires ; Female ; Humans ; Joint Dislocations ; diagnostic imaging ; physiopathology ; surgery ; Male ; Middle Aged ; Tomography, X-Ray Computed ; Treatment Outcome ; Young Adult

Objective To analyse the relationship between Fourier transform infrared （FTIR） spectrum ofrat's spleen tissue and postmortem interval （PMI） for PMI estimation using FTIR spectroscopy combinedwith data mining method. Methods Rats were sacrificed by cervical dislocation, and the cadavers were placed at 20 ℃. The FTIR spectrum data of rats' spleen tissues were taken and measured at different time points. After pretreatment, the data was analysed by data mining method. Results The absorption peak intensity of rat's spleen tissue spectrum changed with the PMI, while the absorption peak position was unchanged. The results of principal component analysis （PCA） showed that the cumulative contribution rate of the first three principal components was 96%. There was an obvious clustering tendency for the spectrum sample at each time point. The methods of partial least squares discriminant analysis （PLS- DA） and support vector machine classification （SVMC） effectively divided the spectrum samples with different PMI into four categories （0-24 h, 48-72 h, 96-120 h and 144-168 h）. The determination coefficient （R2） of the PMI estimation model established by PLS regression analysis was 0.96, and the root mean square error of calibration （RMSEC） and root mean square error of cross validation （RMSECV） were 9.90 h and 11.39 h respectively. In prediction set, the R2 was 0.97, and the root mean square error of prediction （RMSEP） was 10.49 h. Conclusion The FTIR spectrum of the rat's spleen tissue can be effectively analyzed qualitatively and quantitatively by the combination of FTIR spectroscopy and data mining method, and the classification and PLS regression models can be established for PMI estimation.

Objective: To investigate the effects of exosomes from human adipose-derived mesenchymal stem cells (ADSCs) on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice. Methods: The experimental research methods were adopted. The discarded adipose tissue was collected from 3 female patients (aged 10-25 years) who underwent abdominal surgery in the First Affiliated Hospital of Air Force Medical University. ADSCs were extracted from the adipose tissue by collagenase Ⅰ digestion and identified with flow cytometry. Exosomes were extracted from the human ADSCs by differential ultracentrifugation, the morphology of the exosomes was observed by transmission electron microscopy, the particle diameter of the exosomes was detected by nanoparticle tracking analyzer, and the protein expressions of CD9, CD63, tumor susceptibility gene 101 (TSG101), and β-actin were detected by Western blotting. The human ADSCs exosomes (ADSCs-Exos) and RAW264.7 cells were co-cultured for 12 h, and the uptake of RAW264.7 cells for human ADSCs-Exos was observed. The RAW264.7 cells were divided into phosphate buffer solution (PBS) group stimulated with PBS for suitable time, endotoxin/lipopolysaccharide (LPS) stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group stimulated with LPS for corresponding time, with 3 wells in each group, and the mRNA expressions of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), IL-6, and IL-10 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) method. The RAW264.7 cells were divided into PBS group, LPS alone group, and LPS+ADSCs-Exos group, with 3 wells in each group, which were dealt correspondingly for the time screened out in the previous experiment, the mRNA expressions of IL-1β, TNF-α, IL-6, IL-10, trasforming growth factor β (TGF-β,) and vascular endothelial growth factor (VEGF) were detected by real time fluorescence quantitative RT-PCR method, and the protein expressions of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) were detected by Western blotting. Twenty-four 8-week-old male BALB/c mice were divided into PBS group and ADSCs-Exos group according to the random number table, with 12 mice in each group, and a full-thickness skin defect wound with area of 1 cm×1 cm was inflicted on the back of each mouse. Immediately after injury, the wounds of mice in the two groups were dealt correspondingly. On post injury day (PID) 1, the concentration of IL-1β and TNF-α in serum were detected by enzyme-linked immunosorbent assay, and the mRNA expressions of IL-1β, TNF-α, and IL-6 were detected by real time fluorescence quantitative RT-PCR method. On PID 3, 6, 9, 12, and 15, the wound healing was observed and the wound non-healing rate was calculated. On PID 15, the defect length of skin accessory and collagen volume fraction (CVF) were detected by hematoxylin eosin staining and Masson staining, respectively, the CD31 expression and neovascularization were detected by immunohistochemistry, and the ratio of Ki67 positive cells, the ratio of iNOS and Arg1 double positive cells, and the ratio of iNOS positive cells to Arg1 positive cells and their fluorescence intensities were detected by immunofluorescence method. The number of samples in animal experiments was 6. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: At 12 h of culture, the cells exhibited a typical spindle shape, which were verified as ADSCs with flow cytometry. The exosomes with a vesicular structure and particle diameters of 29-178 nm, were positively expressed CD9, CD63, and TSG101 and negatively expressed β-actin. After 12 h of co-culture, the human ADSCs-Exos were endocytosed into the cytoplasm by RAW264.7 cells. The mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group were significantly higher than those in PBS group (with t) values of 39.10, 14.55, 28.80, 4.74, 48.80, 22.97, 13.25, 36.34, 23.12, 18.71, 29.19, 41.08, 11.68, 18.06, 8.54, 43.45, 62.31, 22.52, 21.51, and 37.13, respectively, P<0.01). The stimulation 12 h with significant expressions of all the inflammatory factors was selected as the time point in the following experiment. After stimulation of 12 h, the mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS alone group were significantly higher than those in PBS group (with t values of 44.20, 51.26, 14.71, and 8.54, respectively, P<0.01); the mRNA expressions of IL-1β, TNF-α, and IL-6 of RAW264.7 cells in LPS+ADSCs-Exos group were significantly lower than those in LPS alone group (with t values of 22.89, 25.51, and 8.03, respectively, P<0.01), while the mRNA expressions of IL-10, TGF-β, and VEGF were significantly higher than those in LPS alone group (with t values of 9.89, 13.12, and 7.14, respectively, P<0.01). After stimulation of 12 h, the protein expression of iNOS of RAW264.7 cells in LPS alone group was significantly higher than that in PBS group and LPS+ADSCs-Exos group, respectively (with t values of 11.20 and 5.06, respectively, P<0.05 or P<0.01), and the protein expression of Arg1 was significantly lower than that in LPS+ADSCs-Exos group (t=15.01, P<0.01). On PID 1, the serum concentrations of IL-1β and TNF-α and the mRNA expressions of IL-1β, TNF-α, and IL-6 in wound tissue of mice in ADSCs-Exos group were significantly those in lower than PBS group (with t values of 15.44, 12.24, 9.24, 7.12, and 10.62, respectively, P<0.01). On PID 3, 6, 9, 12, and 15 d, the wound non-healing rates of mice in ADSCs-Exos group were (73.2±4.1)%, (53.8±3.8)%, (42.1±5.1)%, (24.1±2.8)%, and 0, which were significantly lower than (82.5±3.8)%, (71.2±4.6)%, (52.9±4.1)%, (41.5±3.6)%, and (14.8±2.5)% in PBS group, respectively (with t values of 4.77, 8.93, 5.54, 7.63, and 7.59, respectively, P<0.01). On PID 15, the defect length of skin accessory in wounds of mice in PBS group was significantly longer than that in ADSCs-Exos group (t=9.50, P<0.01), and the CVF was significantly lower than that in ADSCs-Exos group (t=9.15, P<0.01). On PID 15, the CD31 expression and the number of new blood vessels (t=12.99, P<0.01), in wound tissue of mice in ADSCs-Exos group were significantly more than those in PBS group, and the ratio of Ki67 positive cells was significantly higher than that in PBS group (t=7.52, P<0.01). On PID 15, the ratio of iNOS and Arg1 double positive cells in wound tissue of mice in PBS group was (12.33±1.97)%, which was significantly higher than (1.78±0.29)% in ADSCs-Exos group (t=13.04, P<0.01), the ratio of iNOS positive cells and the fluorescence intensity of iNOS were obviously higher than those of ADSCs-Exos group, and the ratio of Arg1 positive cells and the fluorescence intensity of Arg1 were obviously lower than those of ADSCs-Exos group. Conclusions: The human ADSCs-Exos can alleviate inflammatory response of mouse RAW264.7 cells, decrease macrophage infiltration and secretion of the pro-inflammatory cytokines, increase the secretion of anti-inflammatory cytokines to promote neovascularization and cell proliferation in full-thickness skin defect wounds of mice, hence accelerating wound healing.
Animals ; Exosomes ; Female ; Humans ; Male ; Mesenchymal Stem Cells ; Mice ; Skin ; Vascular Endothelial Growth Factor A ; Wound Healing

Objective: To investigate the molecular evolution characteristics of human coronavirus (HCoV) subtypes in patients with fever and respiratory tract infection in Guangzhou from 2010 to 2012. Methods: Partial fragments of NP, RdRp and S genes of HCoV-OC43, HCoV-229E and HCoV-NL63 positive samples were amplified by RT-PCR and sequencing. Bioinformatics software, including Bio-edit, Mega4.0 and Clustal1.83 were used for comparison and analysis of NP, RDRp and S gene sequences. Molecular evolutionary tree of different gene regions of HCoV-OC43, HCoV-229E and HCoV-NL63 were built. Results: No remarkable variation or recombinant strain of HCoV-OC43, HCoV-229E and HCoV-NL63 was found in Guangzhou during 2010—2012. The HCoV-OC43 substrains were genetically closest to the strains found in Belgium and Hong Kong (GenBank accession number JN129834 and AY903460). HCoV-229E substrains were genetically closest to those found in Amsterdam (GenBank accession number JX503060) and HCoV-NL63 most genetically close to those in Amsterdam and Beijing (GenBank accession number JX104161 and DQ445911). The NP and RDRp genes of all subtypes were highly conserved, while S gene was more variable. Conclusions There were at least 3 substrains of HCoV-OC43, HCoV-229E and HCoV-NL63 epidemic in Guangzhou during 2010—2012, and no remarkable variation or recombinant viral strain was found. The NP and RDRp genes of all subtypes were highly conserved and can be used in virus detection, while S gene was more variable and suitable for phylogenetic and variation study.

The successful retrieval of ancient mitochondrial DNA (mtDNA) from Neanderthals provides powerful experimental evidence that clarifies the arguments between the out-of-Africa and multiregional models of evolution. However, the lack of nuclear DNA from Neanderthal fossils and mtDNA of early modern human fossils dating back to approximately the same time in the Pleistocene constitutes a limitation that may compromise the significance of mtDNA phylogenetic analysis. In this report, we introduce a mitochromic analysis using Neanderthal mtDNA as a foreign transgene and humans as a naturally occurring transgenic species. Forty Neanderthal mtDNA retrievable nuclear fragments were identified by blasting human genome data with Neanderthal mtDNA. Five of the 40 fragments exhibited higher correlation with Neanderthal mtDNA than those with modern human mtDNA. Furthermore, these five nuclear fragments harbor Neanderthal mtDNA-unique haplotypes. Based on the 98%+ identity between Neanderthal and modern human mtDNA when compared by groups, we suggest that some of the modern human nuclear fragments retrieved using Neanderthal mtDNA may aid in decoding Neanderthal genetic information, and also may simultaneously demonstrate a close genetic evolutionary relationship between modern humans and Neanderthals.

OBJECTIVES: To analyse the Fourier transform infrared （FTIR） spectral data of renal tissue at different temperatures in rats after death, and to explore the effects of temperature on the FTIR spectral characteristics of renal tissue. METHODS: The rats were sacrificed by cervical dislocation and placed at 4 ℃, 20 ℃ and 30 ℃. The FTIR spectral data of renal tissue were collected at different time points and analysed by data mining method. RESULTS: The principal component analysis （PCA） results showed that there were significant trends of clustering in the samples of partial time point at 4 ℃, 20 ℃ and 30 ℃. Partial least square （PLS） regression models were established with the spectral data at three temperature groups. The performance of PLS regression models in 20 ℃ and 30 ℃ groups were more superior than that in 4 ℃ group, and the stability of the model in 20 ℃ group was better than that in 30 ℃ group. CONCLUSIONS There are differences in the FTIR spectral characteristics of renal tissue of rats after death at different temperatures. Temperature has a major impact on the performance of FTIR spectral PLS regression model. Therefore, in order to improve the accuracy of postmortem interval estimation, the effects of temperature on the model should be considered in the related study by spectral method.
Animals ; Autopsy ; Death ; Postmortem Changes ; Rats ; Spectroscopy, Fourier Transform Infrared/methods* ; Temperature

Objective To analyze the differences among electrical damage, burns and abrasions in pig skin using Fourier transform infrared microspectroscopy （FTIR-MSP） combined with machine learning algorithm, to construct three kinds of skin injury determination models and select characteristic markers of electric injuries, in order to provide a new method for skin electric mark identification. Methods Models of electrical damage, burns and abrasions in pig skin were established. Morphological changes of different injuries were examined using traditional HE staining. The FTIR-MSP was used to detect the epidermal cell spectrum. Principal component method and partial least squares method were used to analyze the injury classification. Linear discriminant and support vector machine were used to construct the classification model, and factor loading was used to select the characteristic markers. Results Compared with the control group, the epidermal cells of the electrical damage group, burn group and abrasion group showed polarization, which was more obvious in the electrical damage group and burn group. Different types of damage was distinguished by principal component and partial least squares method. Linear discriminant and support vector machine models could effectively diagnose different damages. The absorption peaks at 2 923 cm^-1, 2 854 cm^-1, 1 623 cm^-1, and 1 535 cm^-1 showed significant differences in different injury groups. The peak intensity of electrical injury's 2 923 cm^-1 absorption peak was the highest. Conclusion FTIR-MSP combined with machine learning algorithm provides a new technique to diagnose skin electrical damage and identification electrocution.
Algorithms ; Animals ; Fourier Analysis ; Least-Squares Analysis ; Machine Learning ; Swine