Chicken embryo fibroblasts(CEFs)are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus(AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR(QPCR)analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4(RPL4)and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide(YWHAZ)are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene(ACTB)and the ribosomal protein L4(RPL4)gene are the best references.

OBJECTIVEThis study measures the glutaredoxin (Grx) gene and protein expression in umbilical vein endothelial cells upon exposure to Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). The involvement of the Akt-signaling pathway is also determined.

METHODSEA-hy926 cells were pretreated with 1,000 ng · mL⁻¹ P. gingivalis LPS for 4, 12, 18, and 24 h, and then real-time reverse transcription polymerase chain reaction was employed to detect Grx1 expression. The effect of Grx on Akt activity was investigated using Western blot for the control, LPS (1,000 ng · mL⁻¹ LPS), and carmus- tine (BCNU) groups (1,000 ng · mL⁻¹ LPS, and the EA-hy926 cells were pretreated with 25 μmol · ml⁻¹ BCNU for 30 min).

RESULTSGene expression of Grx1 significantly increased in LPS group compared with that in the control group. The Grx1 expression reached the peak level in 12 h, and the variation between the expression in 4 and 12 h was significant (P < 0.05). After 12 h, the protein levels of Grx and phosphorylated-Akt (p-Akt) significantly increased in the LPS group (P < 0.05), whereas the BCNU group showed a considerable decrease in both Grx and p-Akt expression levels (P < 0.05). Moreover, a slight difference was observed in the total Akt protein levels in the three groups (P > 0.05).

CONCLUSIONGrx expression increased upon exposure of EA-hy926 cells to the LPS. Akt activity could be inhibited by BCNU (a Grx inhibitor), which indicated that Akt might act as a downstream regulator of Grx.

Endothelial Cells ; Glutaredoxins ; genetics ; Humans ; Lipopolysaccharides ; pharmacology ; Oxidative Stress ; drug effects ; Phosphorylation ; Porphyromonas gingivalis ; pathogenicity ; Proto-Oncogene Proteins c-akt ; drug effects ; Signal Transduction ; drug effects ; Umbilical Veins

Objective To analyze the variations of glycerol-3-phosphate dehydrogenase 1 like gene (GPD1-L) and address the association with sudden m anhood death syndrom e (SMDS). Methods The genom ic DNA was extracted from blood sam ples of the SMDS group and the norm alcontrolgroup.The exons, exon-in-tron boundaries and 3′-U TRs of coding region of GPD1-L w ere PCRam plified and DNAsequenced di-rectly to confirm the types of variations. The genotype frequency and allele frequency w ere analyzed statistically. Results There w ere tw ovariants in the SMDS group, c.465C>Tand c.*18G>T, the latter existed certain degree difference of genotype distribution and allele frequency betw een the SMDS group and the control group, but there was no statistically significant (P>0.05). Conclusion The relation be-tw een gene m utation of GPD1-L and the occurrence of Chinese SMDS deserves a further research.

Objective To investigate the location and proliferation of human hair follicle stem cells. Methods The expression of keratin 19(K19) in the human hair follicles on the occiput was detected by immunohistochemical staining, and the positive area was located. The follicle epithelium containing the K19-positive area above the hair bulb were cultured with or without mesenchymes (dermal papilla cells, DPCs) at the air-liquid interface of the collagen gel. When the proliferating colonies formed, the distance from the colony to the bottom of hair follicles was measured and the relationship between the K19-positive position and the proliferation colonies was analyzed. The ultrastructure of the proliferation colony was observed by transmission electron microscopy. Results Two portions were found positive for K19 in the outer root sheath of the human hair follicles, the upper one being at the bulge region and the lower one at the outer root sheath above the hair bulb. The proliferating colony formed only when the epithelium of the hair follicle was co-cultured with mesenchymes cells. The statistical analysis suggested the lower portion positive for K19 and the proliferation colonies in the culture were traced to locate at the same site of the human hair follicles. Under electron microscopy, juvenile cells, mature keratinocytes and apoptotic cells were found in the proliferating colonies. Conclusions The human hair follicles may contain two distinct reservoirs for stem cells, which locate in the bulge area and in the region of the outer root sheath above the hair bulb, respectively. Mesenchymes are needed for the proliferation of stem cells. New stem cells, mature keratinocytes and apoptotic cells may be three endings of the stem cells′ proliferation.

Objective To improve the effect of clinical diagnosis and treatment of acetabular fracture with a femoral nerve injury by analyzing the causes of femoral nerve injury following acetabular fractures.Methods From January 1996 to November 2004,146 cases of acetabular fractures were treated operatively.Six cases of them were complicated with femoral nerve injury.The causes of femoral nerve injury were analyzed on the basis of clinical manifestations,CT scan and 3-dimensional reconstruc- tion.All the cases were classified according to Letournel and Judet classification.Three cases had hema- toma compression (2 cases with double column fractures and 1 with transverse-posterior wall fractures);2 cases had femoral nerve injury caused by fracture fragments (1 case with anterior wall fracture following anterior hip dislocation,the another with old fracture of anterior column combined with fracture of superior ramus of pubis);and one case had anterior column fracture combined with fracture of wing of ilium,and the femoral nerve was injured by traction in operation.Clearance of hematoma,nerve tract decompression and epineuria solution were performed in 5 cases,and 1 case was treated conservatively.Results The average follow-up period was 1.8 years(ranging from 1 to 3 years).The muscle power of quadriceps fem- oris recovered from 1-2 grade before operation to 4-5 grade after operation in 5 cases.The function of ex- tensor knee and gait was normal.The function of sensory completely recovered in 4 cases.One case was followed up for 2 years,which showed the patient still suffered from hypoesthesia in the lower 2/3 of the thigh and the medial of the leg.One ease of traction injury was followed up for 1.2 years,showing the muscle power recovered to normal,but still presented with sensory disability.Conclusion Acetabular fractures associated with femoral nerve injury are rare.For complex acetabular fractures and severe trau- ma,attention should be given to the possibility of femoral nerve injury.Fragment stabbing and compres- sion of hematoma around iliopsoas muscle are the common causes of femoral nerve injury following ace- tabular fractures.Iatrogenic injury should not be ignored.

Objective:To investigate the role of celecoxib in inhibiting liver cancer and to explore the potential molecular mech-anisms.Methods:A total of 65 cases of patients with primary hepatocellular carcinoma(HCC) underwent surgical treatment in our hospital from February 2012 to June 2016 were recruited as study object.Another 15 cases of normal liver tissue offered by the department of hepatobiliary surgery were selected as control.Immunohistochemical staining(IHC) was used to detect the expression level of COX-2 and PD-1 in tumor and normal tissue samples.Pearson correlation analysis were used to evaluate the correlation between COX-2 and PD-1 in HCC patients.Construction of H22 hepatoma cells bearing BALB/c mice model,randomly divided them into control group,celecoxib group and PD-1 antibody group(15 mice for each group).Sacrificed all mice alive at the end of the 4th weeks after the treatment and removed the tumors then.The tumor growth curves and survival curves were drawn to observe the anti-tumor effect.IHC were used to evaluate the expression of COX-2,PD-1 as well as the number of CD8+T and foxp3 positive Treg cells in tumor tissue.Flow cytometry were used to determine the number of CD8+T and CD4+CD25+Treg cells in peripheral blood.The peripheral blood mononuclear cell(PBMC) of BALB/c mice were separated and scramble or PD-1 siRNA were transfected then,Western blot analysis were used to detected the level of COX-2,PD-1,CD8 and Foxp3 after the treatment of celecoxib.Results:IHC results showed that the expression level of COX-2 and PD-1 in tumor tissue of HCC patients were significant higher than control(P<0.001).Pearson correlation analysis showed COX-2 were both positive correlated with PD-1 in the tumor tissue of HCC patients(R2=0.673,P<0.001).The tumor growth curves and survival curves in celecoxib or PD-1 antibody groups were significant better than in control group (P<0.05).There were no significant difference of tumor growth curves and survival curves between celecoxib group and PD-1 antibody group(P>0.05).IHC and flow cytometry analysis showed that celecoxib treatment significant decreased the expression level of PD-1(P<0.05).Both celecoxib and PD-1 antibody treatment significant increased the number of CD8+T cells while decreased the number of Treg cells in the tissue or the peripheral blood(P<0.05).Western blot analysis showed that celecoxib significant decreased the level of COX-2,PD-1,CD8 and Foxp3 in PBMCs,while it did not affect the level of CD8 and Foxp3 in PD-1 siRNA transfected PBMCs.Conclusion:The expression level of COX-2 and PD-1 were increased in the tumor tissue of HCC patients.Celecoxib may inhibit liver cancer by regulating PD-1 mediated tumor immune via inhibiting COX-2.

Background and purpose:Diffusion-weighted whole-body imaging with background body signal suppression (DWIBS) can be used for MR imaging systemic examination, especially the lymph node and bone diseases can be clear, and the imaging result is similar with PET. The aim of this study was to compare the value of clinical application in the diagnosis of malignant metastatic osteopathic between DWIBS and bone scintigraphy mapping. Methods:Thirty-six specimens conifrmed with malignant tumors by the pathology of operation or biopsy underwent both DWIBS imaging and bone scintigraphy mapping, chi-square test was used for comparing the detection results of bone metastasis by this two imaging methods. Results:Thirty (165 positions in all) of 36 malignant tumor patients were conifrmed as having bone metastasis, compared that 26 patients (143 positions) with DWIBS method and 23 patients (132 positions) with bone scintigraphy mapping were detected, but there was no statistical signiifcance between this two imaging methods (χ2=1.002, P=0.506). The sensitivity, positive predictive value (PPV) and accuracy of the detection rate of bone metastasis were similar in DWIBS and bone scintigraphy, with 86.7%, 96.3%, 86.1%and 76.7%, 88.5%, 72.2%, respectively;but the speciifcity and negative predictive value (NPV) in DWIBS (83.3%and 55.6%) was higher than that of in bone scintigraphy (50.0%and 30.0%). The detection rates of different bone metastasis with DWIBS and bone scintigraphy were 86.7%(143/165) and 80.0%(132/165), and it was no signiifcant difference (χ2=2.640, P=0.104);DWIBS method was better than bone scintigraphy in the detection of osseous metastasis on pelvis and limbs long bone, and there was different signiifcant (χ2=6.783 and 7.636, P=0.023 and 0.016). Conclusion:DWIBS could detect bone metastatic lesions effectively, and there is ifne consistency with bone scintigraphy. Therefore, DWIBS is to hope to be extended and applicated clinically.

OBJECTIVETo explore surface roughness of bone cement and surround tissue on histological characteristic of induced membranes.

METHODSBone cements with smooth and rough surface were implanted in radius bone defect, intramuscular and subcutaneous sites of rabbits, and formed induced membranes. Membranes were obtained and stained (HE) 6 weeks later. Images of membrane tissue were obtained and analyzed with an automated image analysis system. Five histological parameters of membranes were measured with thickness,area,cell density,ECM density and microvessel density. Double factor variance analysis was used to evaluate the effect of the two factors on histological characteristics of induced membranes.

RESULTSMembranes can be induced by each kind of bone cement and at all the three tissue sites. In histological parameters of thickness,area and micro vessel,there were significant differences among the membranes induced at different tissue sites (P = 0.000, P = 0.000, P = 0.000); whereas, there were no significant differences in histological parameters of cell density and ECM density (P = 0.734, P = 0.638). In all five histological parameters of membranes, there were no significant differences between the membranes induced by bone cements with different surface roughness (P = 0.506, P = 0.185, P = 0.883, P = 0.093, P = 0.918).

CONCLUSIONSurround tissue rather than surface roughness of bone cements can affect the histological characteristics of induced membranes. The fibrocystic number, vascularity, mechanical tension and micro motion of the surround tissue may be closely correlated with the histological characteristics of induced membranes.

Animals ; Bone Cements ; Female ; Membranes ; cytology ; Rabbits ; Radius ; cytology ; Surface Properties ; Tissue Engineering ; methods ; Tissue Scaffolds

The active ingredients in traditional Chinese medicine have been reported to possess significant pharmacological activity and played an important role in clinical treatments. However, lots of the active ingredients in traditional Chinese medicine suffer from disadvantages such as low solubility, high melting point and low stability that results in low bioavailability and limit its clinical application. Crystal structure plays an important role in improving physicochemical properties and efficacy of the active ingredients in traditional Chinese medicine. This review concludes the research advances of several crystal forms used in the active ingredients in traditional Chinese medicine in terms of polymorph, cocrystal, amorphous/coamorphous and nanocrystal. And the effects of crystal forms on the physicochemical properties and efficacy of the active ingredients in traditional Chinese medicine were reviewed. This research may be useful for the formulation preparation and development of the active ingredients in traditional Chinese medicine.

OBJECTIVETo investigate the gene mutation in the areas of pre core/core (Pre C/C) and basic core promotor (BCP) of HBV DNA and its clinical significance.

METHODSThe nt 1 735-1 965 segment of HBV DNA was amplified with PCR in 54 cases with chronic hepatitis B and 10 cases with post-hepatitis cirrhosis. Then the PCR product was sequenced.

RESULTSThere were 168 site mutations in 48.5% (33/68) cases with hepatitis B. The first ten mutation sites were nt 1 764 (58.8%), 1 762 (48.5%), 1 799 (21.0%), 1 766 (14.7%), 1 896 (13.2%), 1 754 (8.8%), 1 899 (8.8%), 1 768 (7.4%), 1 814 (7.4%) and 1 913 (7.4%). Three rare mutations of nt 1907, 1 922 and 1 923 were also detected. The mutations of nt 1 896, 1 764 and 1 762 were found in 16.7%, 35.2% and 35.2% of chronic hepatitis, and in 30.0%, 60.0% and 60.0% respectively of post-hepatitis cirrhosis cases. There was statistical significance between the two groups (P < 0.01).

CONCLUSIONThe mutations in the areas of Pre C/C and BCP of HBV DNA might possibly be associated with liver fibrosis. There are many mutation sites in HBV DNA and mutation occurs frequently, therefore gene sequencing is helpful to the design of gene chip and to clinical application.

DNA, Viral ; blood ; genetics ; Gene Frequency ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; pathology ; virology ; Humans ; Mutation ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length