Role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice / 中华烧伤杂志

Objective: To investigate the role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice. Methods: The experimental research methods were applied. Eighty-six C57BL/6J male mice (hereinafter briefly referred to as wild-type mice) aged 8-12 weeks were selected for the following experiments. Vγ4 T cells were isolated from axillary lymph nodes of five wild-type mice for the following experiments. Intraperitoneal injection of rapamycin for 42 mice was performed to establish rapamycin-treated mice model for the following experiments. Eighteen wild-type mice were divided into normal control group without any treatment, trauma only group, and trauma+CC chemokine ligand 20 (CCL20) inhibitor group according to the random number table (the same grouping method below), with 6 mice in each group. The full-thickness skin defect wound was made on the back of mice in the latter two groups (the same wound model below), and mice in trauma+CCL20 inhibitor group were continuously injected subcutaneously with CCL20 inhibitor at the wound edge for 3 days after injury. Another 6 rapamycin-treated mice were used to establish wound model as rapamycin+trauma group. On post injury day (PID) 3, the epidermal cells of the skin tissue around the wound of each trauma mice were extracted by enzyme digestion, and the percentage of Vγ4 T cells in the epidermal cells was detected by flow cytometry. In normal control group, the epidermal cells of the normal skin tissue in the back of mice were taken at the appropriate time point for detection as above. Five wild-type mice were used to establish wound models. On PID 3, the epidermal cells were extracted from the skin tissue around the wound. The cell populations were divided into Vγ4 T cells, Vγ3 T cells, and γδ negative cells by fluorescence-activated cell sorter, which were set as Vγ4 T cell group, Vγ3 T cell group, and γδ negative cell group (with cells in each group being mixed with B16 mouse melanoma cells), respectively. B16 mouse melanoma cells were used as melanoma cell control group. The expression of interleukin-22 (IL-22) mRNA in cells of each group was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), with the number of samples being 6. Thirty rapamycin-treated mice were used to establish wound models, which were divided into Vγ4 T cell only group and Vγ4 T cell+IL-22 inhibitor group performed with corresponding injections and rapamycin control group injected with phosphate buffer solution (PBS) immediately after injury, with 10 mice in each group. Another 10 wild-type mice were taken to establish wound models and injected with PBS as wild-type control group. Mice in each group were injected continuously for 6 days. The percentage of wound area of mice in the four groups was calculated on PID 1, 2, 3, 4, 5, and 6 after injection on the same day. Six wild-type mice and 6 rapamycin-treated mice were taken respectively to establish wound models as wild-type group and rapamycin group. On PID 3, the mRNA and protein expressions of IL-22 and CCL20 in the peri-wound epidermis tissue of mice in the two groups were detected by real-time fluorescence quantitative RT-PCR and Western blotting, respectively. The Vγ4 T cells were divided into normal control group without any treatment and rapamycin-treated rapamycin group. After being cultured for 24 hours, the mRNA and protein expressions of IL-22 of cells in the two groups were detected by real-time fluorescence quantitative RT-PCR and Western blotting, respectively, with the number of samples being 6. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, one-way analysis of variance, Bonferroni method, Kruskal-Wallis H test, and Wilcoxon rank sum test. Results: The percentage of Vγ4 T cells in the epidermal cells of the skin tissue around the wound of mice in trauma only group on PID 3 was 0.66% (0.52%, 0.81%), which was significantly higher than 0.09% (0.04%, 0.14%) in the epidermal cells of the normal skin tissue of mice in normal control group (Z=4.31, P<0.01). The percentages of Vγ4 T cells in the epidermal cells of the skin tissue around the wound of mice in rapamycin+trauma group and trauma+CCL20 inhibitor group on PID 3 were 0.25% (0.16%, 0.37%) and 0.24% (0.17%, 0.35%), respectively, which were significantly lower than that in trauma only group (with Z values of 2.27 and 2.25, respectively, P<0.05). The mRNA expression level of IL-22 of cells in Vγ4 T cell group was significantly higher than that in Vγ3 T cell group, γδ negative cell group, and melanoma cell control group (with Z values of 2.96, 2.45, and 3.41, respectively, P<0.05 or P<0.01). Compared with that in wild-type control group, the percentage of wound area of mice in rapamycin control group increased significantly on PID 1-6 (P<0.01), the percentage of wound area of mice in Vγ4 T cell+IL-22 inhibitor group increased significantly on PID 1 and PID 3-6 (P<0.05 or P<0.01). Compared with that in rapamycin control group, the percentage of wound area of mice in Vγ4 T cell only group decreased significantly on PID 1-6 (P<0.05 or P<0.01). Compared with that in Vγ4 T cell only group, the percentage of wound area of mice in Vγ4 T cell+IL-22 inhibitor group increased significantly on PID 3-6 (P<0.05 or P<0.01). On PID 3, compared with those in wild-type group, the expression levels of IL-22 protein and mRNA (with t values of -7.82 and -5.04, respectively, P<0.01) and CCL20 protein and mRNA (with t values of -7.12 and -5.73, respectively, P<0.01) were decreased significantly in the peri-wound epidermis tissue of mice in rapamycin group. After being cultured for 24 hours, the expression levels of IL-22 protein and mRNA in Vγ4 T cells in rapamycin group were significantly lower than those in normal control group (with t values of -7.75 and -6.04, respectively, P<0.01). Conclusions: In mice with full-thickness skin defects, rapamycin may impair the CCL20 chemotactic system by inhibiting the expression of CCL20, leading to a decrease in the recruitment of Vγ4 T cells to the epidermis, and at the same time inhibit the secretion of IL-22 by Vγ4 T cells, thereby slowing the wound healing rate.

Analysis of impurities in rifalazil / 中国药学杂志

OBJECTIVE: To analyze the impurities of rifalazil. METHODS: HPLC method was used for related substances, TLC was used to detect the side chain isobutyl piperazine residues, TLC was prepared and column chromatography was used to separate impurities; HPLC was combined with TLC and UV, MS, NMR and other analytical tools was used to speculate the impurity. RESULTS: Impurity 1M-1 was to be 2-amino-3-(tert-butyldimethylsilyloxy) phenol, impurity IM-2 was to be 25-deacetyl-27-demethoxy rifalazil, impurity IM-3 was to be 25-deacetyl-rifalazil, IM-4 was to be rifalazil byproduct of 29 carbon and 15 carbon fracture. CONCLUSION: The analysis of impurities in rifalazil products has important significance for the improvement of the production process of rifalazil.

Structure confirm of impurities of cefatrizine propylene glycolate / 中国药学杂志

OBJECTIVE: To confirm the structures of the impurities of cefatrizine propylene glycolate.

Expression of tissue factor in rabbit pulmonary artery in an acute pulmonary embolism model / 世界急诊医学杂志（英文）

BACKGROUND:Tissue factor (TF) is the initiation factor of the extrinsic coagulation pathway, and plays a critical role in the process of thrombosis. This study aimed to investigate the expression of TF and to explore their clinical effect on the pulmonary artery after acute pulmonary thromboembolism. METHODS:Thirty-four Japanese white rabbits (Level II animals) supplied by Tianjin Medical University were randomly assigned into:group A, specimens of the pulmonary artery taken 3 hours after pulmonary embolism (n=8); group B, specimens of the pulmonary artery taken 8 hours after pulmonary embolism (n=8); group C, specimens of the pulmonary artery taken 24 hours after pulmonary embolism (n=8); and control group, pseudo-operations performed without injection of autologous blood clots (n=10). The animal model of pulmonary thrombo-embolism was established by injection of autologous blood clots into the jugular vein through a 5F catheter, and was confirmed by digital subtraction angiography. The mRNA expression of TF in different parts of the pulmonary artery was accessed by RT-PCR. Theq test was used if there was a significant difference in a given continuous variable among the three groups assessed by ANOVA. The experiment equipment was supplied by the State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, the Chinese Academy of Medical Sciences and Peking Union Medical College. RESULTS:The TF expression in the specimen adjacent to emboli was stable at 3, 8 or 24 hours after embolism. The mRNA expression of TF at 3 and 8 hours after embolism was lower in the specimens taken from the distal end of the morbid pulmonary artery than those adjacent to emboli. While at 24 hours after embolism, there were similar mRNA levels in specimens either adjacent or distal to emboli. CONCLUSION:The high level of TF expression in pulmonary artery tissue adjacent to emboli could lead to locally increased coagulation activity, indicating the necessity of initiating anti-coagulation treatment as soon as possible after acute pulmonary embolism.

The mechanism of HBV infection of human trophoblast cell / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the changes of human trophoblast cells after infected with hepatitis B virus.</p><p><b>METHODS</b>HBV positive serum was used to infect human trophoblast cells in vitro. HBsAg in cell culture medium were detected by ELISA method and HBV DNA in cell culture medium and cells were detected by PCR method. HBV fluorescence polymerase chain reaction diagnose kit were used to detect the HBV DNA concentration. Ultra structure of trophoblast cells were observed with transmission electron microscopy (TEM).</p><p><b>RESULTS</b>HBsAg could be detected in infection group by ELISA. Infection group cell culture medium and infection group cells were HBV DNA positive. HBV DNA concentrations in HBV infection cell culture medium in 0, 12, 36, 60, 84 h after extensively PBS washed were < 10(3), 3 x 10(4), 6 x 10(5), 5 x 10(5), 3 x 10(5) copies/mL. HBV infected trophoblast cells were found many forms of endosomes, some of which contents virus like particle.</p><p><b>CONCLUSION</b>HBV might take advantage of clathrin-mediated endocytosis to enter trophoblast cell, which might lead to cell infection or across the cell bar by transcytosis.</p>

An investigation on hepatitis E virus infection status among livestock in Xi'an area / 中华流行病学杂志

<p><b>OBJECTIVE</b>To explore the carrier state of hepatitis E virus(HEV) in livestock in Xi'an area.</p><p><b>METHODS</b>Bile samples from swine, canine, sheep and cow were collected from a local slaughtering house. Reverse transcriptase nested polymerase chain reaction (RT-nPCR) was employed to amplify the ORF2 region in HEV RNA genome. All positive samples were sequenced and compared with data from GenBank. Homology analysis was conducted based on the outcome of sequencing.</p><p><b>RESULTS</b>194, 178, 79 and 191 bile samples from swine, canine, cow and sheep were collected. Positive rates with RT-nPCR method in these domestic animals were 4.10%, 0%, 0% and 0% respectively. Genetic distance analysis indicated that strains being identified were close to genotype IV of HEV, then genotype I, II and III in nucleic acid. Same outcome was shown by the same analysis on amino acid.</p><p><b>CONCLUSION</b>Swine was the only reservoir of HEV in livestock and genotype IV was the prevalent genotype.</p>

A retrospective study on the association of sexual behavior during pregnancy with intrauterine infection of hepatitis B virus / 中华流行病学杂志

<p><b>OBJECTIVE</b>Case-control study was employed to explore the association of sexual behavior during pregnancy and hepatitis B virus (HBV) intrauterine infection.</p><p><b>METHODS</b>212 HBsAg positive pregnant women were consecutively collected and investigated as objects. Those neonates detected for HBsAg with S/N value > or = 5 by Abbott reagents in periphery sera were selected as cases, others as controls. Information on sexual behavior during pregnancy, maternal HBeAg status and other factors was collected, and were analyzed with univariate analysis, multivariate logistic regression analysis, etc, to explore the association of factors with HBV intrauterine infection.</p><p><b>RESULTS</b>Ten of the 214 neonates were validated as occurrence of HBV intrauterine infection. Sexual behavior in the second trimester during pregnancy, with odd ratios 9.15 (95% CI: 1.10 - 76.28), as well as maternal positivity for HBeAg and HBV DNA, was significantly associated with HBV intrauterine infection, and sequently affirmed by multiple logistic regression analysis. The strength of association increased with frequency of sexual behavior. Interaction analysis suggested that there was synergistic interaction between maternal positivity of HBeAg and sexual behavior in the second trimester.</p><p><b>CONCLUSION</b>Sexual behavior was a newly discovered risk factor for HBV intrauterine infection, which need to be estimated in future studies. Inhibition of virus replication and moderate control of sexual behavior would be helpful to prevent HBV intrauterine infection.</p>

In vitro infection of human hepatoma (Hep G2) cell line by hepatitis B virus positive serum / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a culture system of HBV positive serum infected Hep G2 cells in vitro.</p><p><b>METHODS</b>Hep G2 cells were seeded into six-well cluster dishes, at 1 x 10(-6) cells per well and incubated with 3 ml 10% fetal calf serum/ Dulbecco's modified Eagle's medium (10% FCS/DMEM) at 37 degrees in 5% CO2 air. At 24 h after plating, infection group Hep G2 cells were cultured with 0.5 ml HBV positive serum, in control group HBV negative serum was used, 24 h later the inoculums was removed. The cells were then extensively washed with 0.01 mol/L phosphate-buffered saline (PBS). After washing with PBS, 4 ml 2% FCS/DMEM were added to each well and the medium was collected every 12 h. ELISA method was used to detect HBsAg in culture medium. HBV DNA in cells and culture medium was detected by PCR.</p><p><b>RESULTS</b>In infection group, HBsAg could be detected from cell culture medium from 12 h (after PBS washed) to 84 h. HBV DNA could be detected by PCR in culture medium and cells.</p><p><b>CONCLUSION</b>Infection of Hep G2 cells by HBV positive serum is feasible.</p>

Screening of cellular proteins binding to the core region of hepatitis C virus RNA by ultraviolet cross-linking assay / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen cellular proteins binding to the core region of hepatitis C virus (HCV) from human hepatoma cells.</p><p><b>METHODS</b>Unlabeled and labeled RNA transcripts were prepared by in vitro transcription. Cytoplasmic extracts were prepared from human hepatoma cells HepG2. Ultraviolet (UV) cross-linking was used to screen the cellular proteins that would bind to the core region of HCV. Competition experiment was performed to confirm the specificity of the binding in which excess unlabeled RNA of HCV core region and plasmid RNA were used as competitors.</p><p><b>RESULTS</b>Two cellular proteins of 6.6 x 10(4) and 5.5 x 10(4) were found binding to the core region of HCV RNA by UV cross-linking assay. The unlabeled core region of HCV RNA could compete out this binding whereas the unlabeled plasmid RNA could not.</p><p><b>CONCLUSION</b>The cellular proteins from HepG2 cells could bind to the core region of HCV RNA.</p>

Association of 4G/5G polymorphism in PAI1 promoter with PAI1 level in deep vein thrombosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To reveal the association of 4G/5G polymorphism in the promoter region of the plasminogen activator inhibitor 1 gene (PAI1) with plasma PAI1 level in deep vein thrombosis (DVT) in Chinese Han ethnic group.</p><p><b>METHODS</b>One hundred and twenty Chinese DVT patients and 120 healthy controls were recruited. The PAI1 promoter 4G/5G polymorphism was detected using polymerase chain reaction (PCR). The antigen of tissue-type plasminogen activator (tPA) or PAI1 was quantified by a commercially available enzyme-linked immunosorbent assay (ELISA) in DVT cases and health controlsì respectively.</p><p><b>RESULTS</b>Neither in the distribution of PAI1 promoter 4G/5G polymorphism nor in the frequencies of 4G and 5G allele was there a difference between two groups. The levels of PAI1 antigen in the carriers of the 4G/4G genotype were significantly higher than those either in the 4G/5G genotype or in the 5G/5G genotype; In the 4G/5G genotype or in the 5G/5G genotype the TG levels are an independently determinant factor of PAI1 antigen levels.</p><p><b>CONCLUSION</b>There is a close relationship of the PAI1 4G/5G polymorphism to its plasma level in deep vein thrombosis in Chinese Han ethnic group, although lack of association between this genetic variation and risk of DVT suggest no major cause-effect pathogenic role of this polymorphism by itself.</p>