Objective To explore the expression and significance of chemokine CXC reeeptor (CXCR)3 and CXCR4 and their ligands(CXCL)at the early pregnancy decidua and villi.Methods Decidual mononuclear cells were isolated from the normal decidua of 5-8 weeks pregnant women by lymphocyte separation medium in vitro.CD56+natural killer(NK)cells were purified by dynabeads cell sorter kiL Purity and phenotype of CD56+decidua NK cells were analyzed by fluorescence-activated eell sorter (FACS).Gene expression of CXCR3 and CXCR4 in decidua NK cells and CXCL9,CXCL10 and CXCL12 in early pregnancy decidua and villi was assessed bv RT.PCIZ Protein expression of CXCL9,CXCL10 in normal endometrium and early pregnancy decidua was characterized and quantified by streptavidin-biotin pemxidase chain reaction(SP)immunohistochemistry and computered image analysis system.Correlations between the gray degree of CXCL9 and CXCL10 and the number of CD56+NK cells in upper tissue were analyzed by Spearman's correlation ceefficient rank tesL Results The phenotype of 98.7%decidua NK cells was CD56bright.The genes of CXCR3 and CXCR4 were expressed in decidua NK cells and that of CXCL9 and CXCL1O were expressed in early pregnancy decidua and CXCLI2 in early pregnancy villi.CXCL9 and CXCL10 were expressed in the cytoplasm of surface epithelia,glandular epithelia and stromal cells of early pregnancy deeidua and were not expressed in villi by immunohistochemistry.The gray degree of CXCL9 and CXCL10 in the secretory phase endometrium(56±43,59±47)was stronger than that in the proliferative phase(16±18,8±14,P<0.05)and reached the highest(143±35,158±29,P<0.05)in the early pregnancy decidua.The number of cD+56 NK cell in the secretory phase endometrium(60±20)was more than that in the proliferative phase endometrium(23±4,P<0.05)and was the most in the early pregnancy decidua(114±15,P<0.05).The gray degree of CXCL9 in upper tissue had a positive correlation with the number of CD+56 cells(r=0.88,P<0.05)and that of CXCL10 had a similar pattern to CXCL9(r=0.86,P<0.05).Condusion The interactions between CXCL9,CXCL10 and CXCL12 expressed in decidua and villi and CXCR3,CXCR4 expressed in CD+56 decidua NK cells may influence the CD+56 NK cell recruitment at the maternal-fetal interface.

Objective: To investigate the effect of baicalin on the autophagy of the synovial RSC-364 cells of the rats induced by lipopolysaccharide (LPS) through Pl3k/Akt/mTOR signal pathway, and to clarify its mechanism. Methods: The rat synovial RSC-364 cells in logarithmic growth phase were divided into control group, model group. dexamethasone (DXMS) group. 10/imol • L 1 baicalin group. 20/imol • L 1 baicalin group and 40/imol • L baicalin group. The RSC-364 cells in control group were only supplemented with culture medium, and the RSC-364 cells in the other groups were stimulated with 1 mg • L 1 LPS for 12 h to make the inflammatory cell models. The survival rates of RSC-364 cells were detected by MTT assay, and the expression levels of P13k. Akt, mTOR. Beclinl. and LC3-II mRNA in the RSC-364 cells in various groups were detected by RT-PCR method; Western blotting method was used to detect the expression levels of Beclinl. Atg5. Atg7. Atgl2. microtubule-associated protein-light chain 3-II (LC3-II ). and P62 proteins in the RSC-364 cells in various groups. Results: Compared with control group, the survival rate of RSC-364 cells in model group was significantly increased ( P-'-CO. 01). the expression levels of P13k. Akt. mTOR mRNA and P62 protein in the RSC-364 cells in model group were significantly decreased (P<0. 05 or P<0. 01). and the expression levels of Atg5. Atg7. Atgl2. LC3-II . Beclinl mRNA and proteins were significantly increased ( P<0. 01) ; compared with model group, the survival rates of RSC-364 cells in different concentrations of baicalin groups were significantly decreased (P<0. 05 or P<0. 01). the expression levels of P13k. Akt. mTOR mRNA and P62 protein in the RSC-364 cells in different concentrations of baicalin groups were significantly increased (P-'-CO. 05 or P<0. 01). and the expression levels of Atg5. Atg7. Atgl2. LC3-II and Beclinl mRNA and proteins were decreased ( P<0. 05 or P<0. 01). Conclusion: Baicalin may inhibit the LPS-induced autophagy of the RSC-364 cells by activating the Pl3k/Akt/mTOR signaling pathway.

OBJECTIVE To study the immunomodulatory effect of Guipi pills on D-galactose（D-gal）-induced aging mice. METHODS The immune-related targets and related pathways for Guipi pills to exert immune effects were screened by network pharmacology and verified through pharmacodynamic experiments. Totally 105 male ICR mice were randomly divided into blank control （CON） group， model control （MOD） group， positive control （POS） group， Guipi pills low-dose （GD） group and Guipi pills high-dose （GG） group. Except for the CON group， other groups were subcutaneously injected with 400 mg/kg D-gal to induce the aging model； CON group and MOD group were given distilled water， POS group was given 300 mg/kg pidotimod oral solution intragastrically， GD group and GG group were given Guipi pills 300， 600 mg/kg intragastrically， once a day， for 8 weeks. After medication， the serum and spleen were collected， and the contents of interleukin 2 （IL-2）， IL-4， IL-6 and tumor necrosis factor α （TNF-α）， and the contents of immunoglobulin G （IgG）， IgM and IgA were detected. The spleen index was calculated and the histopathological changes in the spleen were observed. The activities of superoxide dismutase （SOD）， glutathione peroxidase （GSH- Px） and malondialdehyde （MDA）， and the content of 8-hydroxy-2 deoxyguanosine （8-OHdG） in spleen were detected； the expression of TNF/phosphoinositide-3-kinase-threonine protein kinase （PI3k-Akt）-related proteins in spleen was detected except for POS group. RESULTS The results of network pharmacology showed that TNF， IL-6 and Akt1 were core targets. The results of pharmacodynamic study showed that compared with MOD group， the contents of IL-2， IL-4， IgG， IgM and IgA were increased significantly in Guipi pills groups， while the contents of TNF-α and IL-6 were decreased significantly； the spleen index was increased significantly （P＜0.05 or P＜0.01）. The phenomenon of diffuse proliferation of lymphocytes was improved， the spleen cells were closely arranged， and the line between the white pulp and red pulp was clear. The activities of SOD and GSH-Px in spleen were increased significantly， while the activity of MDA， the content of 8-OHdG， and the protein expressions of TNF-α， PI3K and p-Akt were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Guipi pills can regulate the immune function of D-gal-induced aging mice， which is related to regulating the TNF/PI3k-Akt pathway， thereby reducing oxidative stress damage in spleen tissue of mice， and regulating protein expressions of TNF-α， PI3K and p-Akt.

The evolution, structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1ST were carried out using bioinformatics software available online. Its antibacterial mechanism and structural properties were analyzed, and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro. This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides. The software MEGA_X were used to conduct homology alignment and to construct a phylogenetic tree. The online software ProtParam, ProtScale, PeptideCutter, signal, TMHMM Server were respectively used to predict the physicochemical parameters, hydrophilia/hydrophobicity, shear sites, signal peptides, and transmembrane domains of dybowskin-1ST. The online software SOPMA, Jpred4, DNAstar Protean were used to predict the secondary structure of dybowskin-1ST, and SWISS-MODEL, I-TASSER were used to predict the tertiary structure. ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes. The effect of dybowskin-1ST on the wound healing was observed on experimental mice. Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1ST. The dybowskin-1ST consists of 59 amino acid residues, of which leucine accounts for 16.9%, with a molecular formula of C₃₁₈H₅₁₀N₈₀O₉₃S₂. Its theoretical isoelectric point is 5.10 and the charge is -2. The dybowskin-1ST and dybowskin-1CDYa are closely related phylogenetically. The secondary structure of dybowskin-1ST predicted by the three methods were similar, which consisted of α-helix (44.07%), extended strand (16.95%), β-turns (3.39%), and random coil (35.39%). The prediction of tertiary structure showed that dybowskin-1ST was mainly composed of α-helix, and it was regarded as a hydrophilic protein with signal peptide sequence. Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944. Dybowskin-1ST is an extracellular protein with no transmembrane structure region, but contains seven phosphorylation sites, three T-cell epitopes and eight B-cell epitopes. The dybowskin-1ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus. However, it had limited antibacterial activity against fungi and drug-resistant bacteria. Although the structure of dybowskin-1ST is rich in α-helix, the verification experiments showed that its antibacterial ability needs to be enhanced. The reason may be that it is a negatively charged and hydrophilic protein, and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.
Amino Acid Sequence ; Animals ; Mice ; Phylogeny ; Pore Forming Cytotoxic Proteins ; Protein Structure, Secondary ; Ranidae

A kind of adjustable external fixation device for lower extremity is designed. The circuit is mainly composed of TEC1-00703 semiconductor refrigeration chip, HZC-30A pressure sensor, STC89C52RC single chip microcomputer and other electrical components. It can realize the timing intelligent temperature control and meet the local fixed-point refrigeration. The design of adjustable structure and the application of intelligent air cushion can satisfy the full fixation of lower limbs of different individuals. Its operation does not need much medical knowledge. It can solve the problem of emergency transportation and follow-up treatment of lower limb injury in ice and snow sports. It has a good application prospect and universality.
External Fixators ; Fracture Fixation ; Humans ; Lower Extremity ; Refrigeration ; Semiconductors

ObjectiveThe prognosis of patients with advanced liver cancer is poor and there is no effective treatment so far. This paper observed the effect of hepatoma cells HepG2 exosomes on its own methylation and attempted to explore its mechanism.MethodsThere was experimental group and control group in the research. The medium has been changed when the cells grow to 60% in a DMEM culture dish with 10% serum. Cells in the control group were cultured in serum-free DMEM medium while the experimental group were cultured in serum-free DMEM medium added 100 L (0.5mg/mL) exosomes, and the supernatant was retained after incubation for 48h. HepG2 cells were cultured and exosomes were extracted by overspeed differential centrifugation, and identified by particle size analysis, transmission electron microscopy, Western blot and other methods. The effect of exosomes of hepatocellular carcinoma cells HepG2 on cell proliferation was analyzed by scratch test. Fluorescence antibody staining was used to observe the change of automethylation level. Fluorescence quantitative PCR was used to detect mRNA expression levels of methyltransferase-related genes DNMT3A, DNMT3B, DNMT1, and apoptosis-related genes Bax and BcI2.ResultsUnder inverted fluorescence microscope, red fluorescent exosomes could be seen entering the cell, surrounding the blue fluorescent nucleus or on the nucleus, indicating that DiI entered the cell membrane or cytoplasm. The area ratio of 6 and 12 h in the experimental group [(57.25±2.06, 83.92±3.17) %] was significantly higher than that in the control group [(28.32±1.22, 40.03±1.74) %] (P<0.05). The genes expressions of Bax, DNMT3A and DNMT3B in the experimental group were higher than those in the control group (P<0.05). The expression of BcI2 was lower than that of the control group (P<0.05).ConclusionThe exosomes of hepatoma cell HepG2 can enhance DNA methylation level by changing the transcriptional expression of DNMT3A, DNMT3B and other genes to affect the expression of apoptose-related genes Bax and BcI2, and to promote the proliferation and growth capacity of hepatoma cell HepG2.

ObjectiveTo observe the effect of polysaccharides from root， stem， leaf and fruit of Schisandra chinensis on exercise endurance in the aging mice induced by D-galactose. MethodMale ICR mice were randomly assigned into six groups： blank control group， model group， root polysaccharide group， stem polysaccharide group， leaf polysaccharide group and fruit polysaccharide group. The mice were administrated with distilled water or root， stem， leaf and fruit polysaccharide （total sugar content of 35 mg·kg^-1） by gavage. Thirty minutes after the administration， the blank control group was subcutaneously injected with normal saline， and the other groups with D-galactose （300 mg·kg^-1）， once daily for 6 weeks. The anti-fatigue effects were evaluated by rotarod test， forelimb grip strength test， and weight-loaded swimming test. The fatigue and oxidation indicators such as blood urea nitrogen （BUN）， serum lactic acid （LD）， lactic dehydrogenase （LDH）， creatine kinase （CK）， superoxide dismutase （SOD）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px）， and reactive oxygen species （ROS） were measured by chemical colorimetry. The protein levels of pro-apoptotic protein B cell lymphoma-2 （Bcl-2）-associated X protein （Bax）， anti-apoptotic Bcl-2 and cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3） in mouse skeletal muscle were detected by Western blot. ResultIn the rotarod test， the time on rod was shorter in the model group than in the blank control group （P<0.01） and the root， stem and fruit polysaccharide groups （P<0.01）. In the forelimb grip strength test， the forelimb grip strength in the model group was lower than that in the blank control group （P<0.01） and the root， stem， leaf and fruit polysaccharide groups （P<0.01）. In the weight-loaded swimming test， the weight-loaded swimming time in the model group was shorter than that in the blank control group （P<0.01） and the root， stem， leaf and fruit polysaccharide groups （P<0.01）. Compared with those in the blank control group， the BUN， LD， LDH and CK levels significantly increased in the model group （P<0.05， P<0.01）. The increases in BUN and LDH levels were decreased by the root， stem and fruit polysaccharides （P<0.05， P<0.01） and those in LD and CK by the root， stem， leaf and fruit polysaccharides （P<0.05， P<0.01）. Compared with the blank control group， the model group showed decreased SOD and GSH-Px activities （P<0.01） and increased MDA and ROS content （P<0.01）. Compared with the model group， the root， stem， and fruit polysaccharide increased the SOD activity （P<0.05， P<0.01） and decreased ROS content （P<0.01）. The root and stem polysaccharides decreased the MDA content （P<0.01） and increased the GSH-Px activity （P<0.05， P<0.01）. Compared with the blank control group， the model group showed up-regulated protein levels of Bax and cleaved Caspase-3 and down-regulated protein level of Bcl-2 （P<0.01）. Compared with the model group， the root， and stem polysaccharides down-regulated the protein levels of cleaved Caspase-3 （P<0.05） and up-regulated protein level of Bcl-2 （P<0.01）. ConclusionThe polysaccharides from the root， stem， leaf， and fruit of S. chinensis have anti-fatigue effect in D-galactose-induced aging mice. The polysaccharides may exert such effect by improving the antioxidant capacity and inhibiting the apoptosis of skeletal muscle cells.

Objective: To explore the enhanced cell killing effect of HSV tk using VP22 intercellular traffciking. Methods: The chimeric genes were constructed by fusing a marker gene for the green fluorescent protein (GFP) or a prodrug enzyme gene for the Herpes simplex virus thymidine kinase (HSV tk) with that of VP22. After being sequenced, the fusion genes were transferred into 293T or COS7 cells. The transfection efficiency and intercellular trafficking were certified using Western blot and immunofluorescence.The cell proliferation was detected through MTT method in the different concentration of GCV and under indicated between transfected cells and untransfected cells. The supernatant of transfected cells was used to culture the untransfected cells to test whether the bystander effect could transferred by media. Results: The gene insertion was proved correct using PCR and DNA sequencing. When the fusion genes were transferred into 293T or COS7 cells at transfection efficiency of 25%～30%, fusion proteins were expressed and efficient intercellular trafficking was demonstrated.The VP22 HSV tk, as a prodrug enzyme fused with VP22, showed an amplified cell killing effect in the presence of GCV as low as 0.1 ?g/ml. Further quantification of the bystander effect showed that cell killing increased with higher proportion of VP22 HSV tk expressing cells. The bystander effect could not be transferred through media. Conclusion: These results clearly indicate that VP22 enhanced intercellular trafficking promotes tumor cell killing effect of HSV tk/GCV.

Objective: To explore the expressions of Foxp3 ' Tregs and CDllc mDCs in the colorectal cancer (CRC) tissue and their infiltrations in the tumor draining lymph node (TDLN) tissue, and to explore the clinical significances. Methods: Fifty-two samples of surgical resection of the CRC patients were collected The expressions of Foxp3 + Tregs and CDllc mDCs in the specimens of the CRC patients and TDLN tissue were detected by immunohistochemistry, and the relationship between the expression frequencies of Foxp3 + Tregs and CD11c+ mDCs and the clinicopathological characteristics of the patients were discussed Results: The positive expression frequency of Foxp3 + Tregs in cancer tissue of the CRC patients was higher than that in adjacent normal mucosa tissue (P< 0. 01); The expression frequency of Foxp3 + Tregs in cancer tissue in the patients with positive lymph node metastasis was higher than that in the patients with negative lymph node metastasis (P<0. 01); the expression frequency of Foxp3 + Tregs in the patients in TNM stage III + IV was higher than that in TNM stage I + II (P< 0. 01); the positive expression frequency of Foxp3 Tregs in metastatic TDLN (mTDLN) tissue was higher than that in metastatic free TDLN (mfTDLN) tissue (P<0. 01). The positive expression frequency of CD11c+ mDCs in cancer tissue of the CRC patients was higher than that in adjacent normal mucosa tissue (P<0. 01); the positive expression frequency of CD11c+ mDCs in the patients with positive lymph node metastasis was lower than that in the patients with negative lymph node metastasis (P<0. 01); the positive expression frequency of CDllc1 mDCs in the patients in TNM stage IE + IV was lower than that in the patients in TNM stage I + II (P<0. 01); the positive expression frequencies of CD11c+ mDCs in cancer tissue with different depths invasion were significantly different (P<0. 05); the positive expression frequency of CD11c+ mDCs in mTDLN tissue was lower than that in mfTDLN tissue (P < 0. 01). There were no significant correlations between the expressions of Foxp3 Tregs and CDllc mDCs in the CRC and TDLN tissues (P>0. 05). Conclusion: The occurrence and development of CRC are related to the changes of Foxp3 Tregs and CDllc mDCs expression frequencies in the local microenvironment of cancer tissue. Foxp3+ Tregs and CD11c+ mDCs play a role in inhibiting the cellular immunity in tumor microenvironment.

Objective: To explore the expressions of B7-H1 and B7-H4 in colorectal cancer (CRC) tissue and their relationships with the Foxp3 regulated T-cell (Treg) infiltration in tumor tissue, and to provide the basis for judging the biological behaviours and prognosis of CRC. Methods: Immunohistochemistry was used to detect the expressions of B7-H1 and B7-H4 and the infiltration of Treg in 56 cases of CRC tissue samples and adjacent normal tissue samples, and their relationships with the clinicopathological features of CRC patients were analyzed. Results: The positive expression rate of B7-H1 in CRC tissue of the CRC patients was higher than that in adjacent normal tissue (χ2= 72. 34, P<0. 01). The differences of expression frequencies of B7-H1 in CRC tissues with different infiltration depths were significant (P<0. 01). The high expression frequency of B7-H1 in CRC tissue of the patients with positive lymphnode metastasis was higher than that in the patients with negative lymphnode metastasis (P<0. 01). The high expression frequency of B7-H1 in CRC tissue of the patients at Duke' s stage (C+D) was higher than that of the patients at Duke' s stage (A+B) (P<0. 01). The positive expression rate of B7-H4 in CRC tissue was higher than that in adjacent normal tissue (χ2=78. 92, P<0. 01). The differences of expression frequencies of B7-H4 in CRC tissues with different infiltration depths were significant (P<0. 05). The high expression frequency of B7-H4 in CRC tissue of the patients with positive lymphnode metastasis was higher than that of the patients with negative lymphnode metastasis (P<0. 05). The number of Treg positive expression cells in CRC tissue was significantly higher than that in adjacent normal tissue (P<0. 01), the number of Treg postive expression cells in CRC tissues with different differentiation degrees were different (P<0. 01), and the number of Treg positive expression cells in CRC tissue of the patients with positive lymphnode metastasis was higher than that of the patients with negative lymph node metastasis (P<0. 01). The expression of B7-H1 was correlated with Treg infiltration (P<0. 01, Cramer ' s = 0. 463); the expression of B7-H4 was correlated with Treg infiltration (P<0. 05, Cramer' s = 0. 320). Conclusion: B7-H1 and B7-H4 are highly expressed in human CRC tissue, and they are associated with the increasing of Treg infiltration; they can be used as the indicators to judge the prognosis of CRC.