Objective To investigate the effect of penehyclidine (PHCD) pretreatment on Toll-like receptor 4 (TLR4) mRNA expression in the lung following acute lung injury (ALI) induced by hemorrhagic shock in rats. Methods Forty healthy SD rats of both sexes weighing 200-250 g were randomly divided into 5 groups( n = 8 each): group Ⅰ sham operation (group S); group Ⅱ ALI (group ALI); group Ⅲ, Ⅴ, PHCD 0.3,1.0, 3.0 mg/kg were given iv respectively at 30 min before hemorrhagic shock (P1-3). Hemorrhagic shock was induced by exsanguinations. MAP was maintained at 35-45 mm Hg for 60 min. The animals were killed at 4 h after resuscitation. Their lungs were removed for microscopic examination, W/D lung weight ratio and determination of TLR4 mRNA expression in the lung tissue (by RT-PCR). NF-κB p65 protein expression in the lung tissue was determined (by immuno-histochemical staining). Results Hemorrhagic shock significantly increased TLR4 mRNA and NF-κB p65 protein expression in the lung tissue and W/D lung weight ratio. Pretreatment with PHCD 1.0 or 3.0 mg/kg significantly inhibited hemorrhagic shock-induced increase in TLR4 mRNA and NF-κB p65 protein expression in the lung and W/D lung weight ratio. The lung injury was significantly ameliorated in group P2,3 as compared to group ALI. Conclusion PHCD pretreatment can attenuate ALI induced by hemorrhagic shock through down-regulation of TLR4 mRNA expression and decreasing NF-κB activity in the lung.

Objective To study on glycogen assay,polymerae chain reaction,and cell culture for diagnostic value of chlamydia infection of vervical smeat.Methods 106 specimens were examined by using glycogen assay,PCR and cell culture.Results Compared with cell culture,the sensitivity and specifity of glycogen assay are 80.0％ and 95.8％ ,and the sensitivity and specifity of PCR are 90.0％ and 97.9％ ,respectively.Conclusion The glycogen assay possesses diagnostic value for chlamydia trachomatis infection of vervical smear.

Objective:To analyze the activities of human NKG2D ligand MICA/B promoter induced by 5-Fu.Methods:The 5'-end flanking regions of MICA /B promoter and their different truncated fragments were amplified from A549 genome by PCR. The resulting amplicons were cloned into pGL3-Basic vector to generate the MICA/B luciferase reporter plasmids. All the constructs were transiently transfected A549 cells. The promoter region activities were determined by dual-luciferase reporter assays. The effect of 5-Fu on the promoter activities of MICA/B was also tested.Results and Conclusion:The 5'-end flanking regions of MICA /B promoter and five of their different truncated fragments were successfully obtained. The normalized luciferase reporter gene activities driven by the above promoters and fragments were 3.61,2.26,1.63,0.313,0.711 and 0.663 for MICA and 17.49,10.11,7.398,0.822,0.997 and 0.49 for MICB,respectively. Promoter activities in transiently transfected A549 cells treated by 20,40,80,160 and 320 μg/m of 5-Fu increased 1.69,1.48,1.62,1.55 and 1.78 fold for MICA and 1.44,1.87,1.38,1.19 and 1.25 fold for MICB. Our results suggest that 5-FU can significantly up-regulate the promoter activity of both MICA and MICB.

BACKGROUND:316L-Cu antibacterial stainless steel is made by adding a certain amount of copper into the stainless steel fol owed by a special heat treatment to uniformly disperse copper-rich precipitates in stainless steel substrate, thereby harvesting the antibacterial properties. OBJECTIVE:To evaluate the antibacterial activity of the Cu ions released from 316L type Cu-bearing antibacterial stainless steels against Porphyromonas gingivalis, thereby providing biomedical evidence for its clinical application. METHODS:The medical 316L stainless steel samples at a surface area to volume ratio of 0.1 cm2/L were soaked in simulated body fluids at 37 ℃ for 1-10 days. A graphite furnace atomic absorption spectrophotometer was employed to detect the amount of Cu release in the simulated body fluids each day and then the rate of Cu release per day could be determined. The antibacterial activities of the steel samples were evaluated by a standard film-covered method under a scanning electron microscope. RESULTS AND CONCLUSION:The daily Cu releasing amount from the 316L-Cu stainless steel within 10 days was significantly higher than that of 316L stainless steel, and al the values remained nearly constant. With time, the sterilizing rate of 316L-Cu stainless steel was gradual y increased, and reached 100%until the 10th hour. Porphyromonas gingivalis showed some morphological changes at 3 hours after treated with 316L-Cu stainless steel, appeared with cleavage at 6 hours, and mostly disintegrated into pieces at 9 hours. The results indicated that the 316L-Cu antibacterial stainless steel showed excel ent antibacterial property against Porphyromonas gingivalis, slowly release Cu irons, and alter the surrounding microenvironment, which is a highly promising biomaterial and has good clinical value.

Objective To observe the effect of nitrous oxide (N2O) on the content of serum free hemoglobin ,and intercellular adhesion molecule‐1 (ICAM‐1) of patients with HIFU Therapy ,and investigate its action of tissue damage mechanism .Methods 50 patients with primary liver cancer undergoing HIFU surgery (ASA Ⅰ - Ⅱ class) were randomly divided into control group (group C) and experimental group(group N) ,25 patients of each group .General anesthesia method was used in both two groups , group C was by total intravenous anesthesia ,group N was adopted intravenous‐inhalation anesthesia .both two groups was adopted the same anesthesia induction method .anesthesia maintain of group N was joined N2 O on the basis of group C .both two groups were draw blood from the radial artery at the points of before anesthesia (T1 ) ,before operation (T2 ) ,1 h (T3 ) ,2 h (T4 ) ,3 h (T5 ) after intraoperative ,and 24 h after operation (T6 ) ,peroxidase reaction test and double antibody sandwich ELISA method were a‐dopted to detect the content of Fhb value and ICAM‐1 ;ultrasonography system of HIFU therapeutic instrument was used to meas‐ure the abdominal wall thickness of patients before and after operation .Results The content of FHb and ICAM‐1 in serum were significantly increased after operation than before with the anesthesia time (P<0 .05);compared with group C ,group N increased obviously at the same point in time (P<0 .05);preoperative and postoperative abdominal wall thickness value of group N was in‐creased significantly (P< 0 .05) .Conclusion It may be connected with N2 O enhanced ultrasound cavitation effect that the body produces more FHb and ICAM‐1 of group N in HIFU treatment ,and induces abdominal wall skin markedly swollen .

Objective To study the influence of fluoride on the expression of Runx2 in suckling rat osteoblasts. Methods Osteoblasts obtained from calvarium of suckling Wistar rats were cultured in the media supplemented with NaF at different doses(0, 1,2 and 4 rag/L), and Runx2 Mrna expression and protein expression were evaluated by RT-PCR and ELISA, respectively. Results Runx2 Mrna expression in suckling rat osteoblasts cultured in vitro significantly increased after exposure to NaF for 48 h at different doses (0.613±0.055, 0.773±0.070 and 0.775±0.070 for 1,2 and 4 mg/L,respecfively) compared to the control (0.482±0.043 ,P< 0.05). Runx2 Mrna expression further increased after 72 h exposure to NaF(0.969±0.048,1.229±0.061,1.255± 0.063 for 1,2 and 4 mg/L, respectively) ,which is significantly higher than the control(0.724±0.036,P<0.05) and corresponding groups at 48 h. NaF doses and exposure time exhibited a significant synergistic effect on Runx2 Mrna expression (P<0.05). Similarly, NaF also enhanced Bunx2 protein expression in suckling rat osteoblasts cultured in vitro. Significant differences were observed between groups exposed to NaF (1,2 and 4 rag/L) and control at48 h post-exposure (0.141±0.007, 0.143±0.008, 0.143±0.011 vs 0.129±0.012, P<0.05) as well as 72 h post-expesure(0.156±0.014, 0.168±0.018, 0.162±0.0100 vs 0.137±0.016, P<0.05). In addition, Runx2 protein expression at 72 h post-exposure was significantly higher than that at 48 h. Conclusions The results suggested that NaF could increase Runx2 expression in suckling rat osteoblasts with a synergistic effect between the doses and exposure time.

ObjectiveTo clarify the influence of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 V4 region with mutations at amino acid locuses on its abilities to enter target cells.Methods Based on the facts that ADA strains was a CCR5-tropic strain,only had the ability to infect CCR5 cells; that HXB2 strains was a CXCR4-tropic strain,only had the ability to infect CXCR4 cells,serial glycoprotein 120 mutants with alanine substitution in V4 region of ADA and HXB2 strains,were constructed by overlaping PCR.Eukaryotic expression vectors of mutants and expression vectors of HIV framework gene with luciferase reporter gene were cotransfected into eukaryotic cells to produce pseudoviruse.Concentration of HIV-1 gag P24 in pseudoviruses was detected by enzyme-linked immunosorbent assay(ELISA).U87.CD4.CCR5 and U87.CD4.CXCR4 cells were infected with 20 and 40 ng pseudoviruses,with wild ADA and HXB2 strains as control groups,respectively.The ability to infect cells of pseudovirus of each mutant with HIV-1 V4- region mutated at amine acid locuses 386-417 was measured by detecting the luciferase activity (relative light unit,RLU).ResultsTen mutants with alanine substitution in V4 region of HIV-1 ADA and HXB2 strains were successfully constructed,respectively.Mutants of pseudoviruse with 20 ng and 40 ng at locuses 389-391 and 414-417 with alanine substitution of V4 region in both ADA and HXB2 strains lost completely the abilities to enter CCR5 and CXCR4 expressing cells[ (0 ± 0)％].It was found that introduction of alanine to ADAs 400-403 and ADAs 408-410 increased the ability to infect cells to (124 ± 35)％,(182 ± 29)％ and (127 ± 8)％,( 134 ± 16)％ with pseudoviruse of 20 ng and 40 ng,respectively.Likewise,the ability to infect CXCR4 expressing cells also increased to (144 ± 42 )％ and (121 ± 18 )％ with pseudoviruse of 20 ng and 40 ng,respectively by introduction of alanine to HXB2s 395-397.However,other mutants in V4 region of ADA and HXB2 only maintained partial entry abilities( 15％- 84％).ConclusionsMutants of V4 region of HIV-1 envelope glycoprotein 120 with alanine substitution at locuses 389-391 and 414-417 in both ADA and HXB2 strains have been constructed successfully.They completely lost the ability to enter target cells.

OBJECTIVETo explore the renal protective effect of Tongxinluo (TXL) and its mechanism of action.

METHODSEight-week old SD rats were divided into the sham-operated group (A), the model group (B) and the TXL group, 6 rats in each group. Angiotensin II (Ang II) was administered slowly (200 ng/kg per min) to rats in group B and C via subcutaneously embedded osmotic pump to make them stimulative model of renal injury, while to rats in group A, pump embedding with saline infusion. After modeling, TXL was given to group C by gastric perfusion in dosage of 0.8 g/kg per day. And the following indexes were observed 14 days later: configuration of renal arterial endothelium by transmission electron microscope; pathologic figure of kidney with HE stain; renal apoptosis by TUNEL; expression of p53 and p22phox by RT-PCR;and level of reactive oxygen species (ROS) in kidney.

RESULTSDifferent degree of congestion, swelling, denudation of endothelial cell in renal arterial endothelial cell; glomerular matrix proliferation and partial glomerular atrophy with tendency of fibrosis; increased renal parenchymal apoptosis; enhanced expression of p53 and p22phox; and elevated ROS were found in model animals. All the above-mentioned abnormalities, including glomerular injury, renal cell apoptosis, as well as the increased p53, p22phox expressions and ROS production were all alleviated in group C after TXL treatment.

CONCLUSIONTXL could protect renal against Ang II injury, and it may be realized by inhibiting NADPH-ROS/p53 pathways and suppressing cell apoptosis in renal parenchyma.

Angiotensin II ; metabolism ; Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Endothelium, Vascular ; drug effects ; metabolism ; Kidney ; blood supply ; pathology ; Male ; NADPH Oxidases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo investigate the effect of 2,3,4',5-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component extracted from the root of Polygonum multiflorum, on angiotensin II (Ang II)-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and to identify the potential mechanism.

METHODSCell proliferation and cell cycle were determined by cell counting, 5-bromo-2'-deoxyuridine incorporation assay, proliferating cell nuclear antigen protein expression and flow cytometry. Levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), mitogenic extracellular kinase 1/2 (MEK1/2) and Src in VSMCs were measured by Western blot. The expression of c-fos, c-jun and c-myc mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). Intracellular reactive oxygen species (ROS) was measured by fluorescence assay.

RESULTSTSG significantly inhibited Ang II-induced VSMCs proliferation and arrested cells in the G /S checkpoint (P<0.05 or P<0.01). TSG decreased the levels of phosphorylated ERK1/2, MEK1/2 and Src in VSMCs (P<0.05 or P<0.01). TSG also suppressed c-fos, c-jun and c-myc mRNA expression <0.05 or P<0.01). In addition, the intracellular ROS was reduced by TSG (P<0.01).

CONCLUSIONSTSG inhibited Ang II-induced VSMCs proliferation. Its antiproliferative effect might be associated with down-regulation of intracellular ROS, followed by the suppression of the Src-MEK1/2-ERK1/2 signal pathway, and hence, blocking cell cycle progression.

Angiotensin II ; pharmacology ; Animals ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Glucosides ; pharmacology ; Intracellular Space ; metabolism ; Male ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Phosphorylation ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Stilbenes ; pharmacology ; Superoxide Dismutase ; metabolism

OBJECTIVETo establish a quantitative method to detect circulating tumor cells (CTC) in patients with small cell lung cancer, and analyze its sensitivity and stability.

METHODSA specific primer and probe for prepro-gastrin-releasing peptide (preproGRP) was designed and a quantitative RT-PCR method was established to detect preproGRP mRNA. Cell incorporation method was used to evaluate the sensitivity. Magnetic cell sorting (MACS) was used to isolate and purify CTC from peripheral blood, and the MACS in combination with morphological diagnosis were used for cell counting.

RESULTSThe isolation rate of CTC by MACS was 30% and the lower detection limit was 5 cells per ml blood. The sensitivity of quantitative RT-PCR in detection of preproGRP mRNA in CTC was 0.64 cells per reaction, and the lower detection limit was 50 cells per ml blood, which was lower than that of MACS. However, the cell numbers calculated by Ct value was in greater accordance (about 80%) with actual cell numbers than that obtained by MACS.

CONCLUSIONSPreproGRP quantitative RT-PCR and MACS have both advantages and disadvantages in detecting CTC of SCLC patients. MACS has a higher sensitivity, and is more favorable when CTC count is below 50 per ml blood. Meanwhile, preproGRP mRNA quantitative RT-PCR is more reliable in calculating actual cell numbers.

Humans ; Immunomagnetic Separation ; Lung Neoplasms ; blood ; metabolism ; pathology ; Neoplastic Cells, Circulating ; Peptides ; genetics ; metabolism ; Protein Precursors ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Small Cell Lung Carcinoma ; blood ; metabolism ; pathology