OBJECTIVETo evaluate the impact of nicotine- and tar-free cigarette smoke extract (fCSE) on the serum testosterone (T) level and erectile function of male rats.

METHODSWe randomized 30 male SD rats to three groups of equal number to receive subcutaneous injection of PBS (1.0 ml / 300 g body weight per day), fCSE (1.0 ml/300 g body weight per day), and reduced glutathione hormone (GSH, 200 mg per kg body weight per day) in addition to fCSE (fCSE + GSH), respectively, all for 8 weeks. Then we evaluated the erectile function of the rats by measuring the maximal intracavernous pressure (MICP), mean arterial pressure (MAP), ICP/MAP ratio, time of stimulation to MICP (Tmax), and cavernosal filling fate (CFR). We determined the serum T level, the activities of superoxide dismutase (SOD) , malondialdehyde (MDA), and nitric oxide synthase (NOS) in the cavernosal tissue, and also observed the morphological changes of the corpus cavernosum.

RESULTSCompared with the controls, the rats of the fCSE group showed obvious decreases in the levels of serum T ([5.37 ± 1.43] vs [3.22 ± 1.11] μg/L), NOS ([2.90 ± 0.27] vs [1.67 ± 0.18] U/mg) , and SOD ([18.41 ± 1.09] vs [13.36 ± 1.18] U/mg prot) and erectile function-related indexes MICP ([85.92 ± 6.36] vs [58.99 ± 10.76] mmHg), MICP/MAP (0.86 ± 0.09 vs [0.56 ± 0.08]), and CFR (2.14 ± 0.44 vs 0.89 ± 0.44), but markedly increased Tmax ([29.90 ± 5.78] vs [42.90 ± 8.56]s), with a positive correlation between the serum T level and CFR (r = 0. 364, P < 0.05). Masson staining revealed a lower ratio of the corpus cavernosum smooth muscle tissue to collagen fiber in the fCSE group (0.27 ± 0.04) than in the control (0.98 ± 0.12). Compared with the fCSE group, the fCSE + GSH group exhibited significantly improved MICP ([58.99 ± 10.76 ] vs [77.95 ± 7.71] mmHg), MICP/MAP (0.56 ± 0.08 vs 0.77 ± 0.09), and CFR (0.89 ± 0.44] vs 1.76 ± 0.42) and shortened Tmax ([42.90 ± 8.56 ] vs [32.10 ± 5.84 ] s). The ratio of the corpus cavernosum smooth muscle tissue to collagen fiber was higher in the fCSE + GSH than in the fCSE group (0.77 ± 0.09 vs 0.27 ± 0.04) but still lower than in the control (0.98 ± 0.12).

CONCLUSIONNicotine- and tar-free cigarette smoke extract reduces the serum T level and erectile function of rats, which is related to oxidative stress. Antioxidant therapy can improve erectile function but has a limited value for morphological protection of the penile tissue.

Animals ; Erectile Dysfunction ; chemically induced ; Male ; Malondialdehyde ; metabolism ; Muscle, Smooth ; pathology ; Nicotine ; Nitric Oxide Synthase ; metabolism ; Penile Erection ; drug effects ; Penis ; pathology ; Rats ; Rats, Sprague-Dawley ; Smoke ; adverse effects ; Superoxide Dismutase ; metabolism ; Tars ; Tobacco ; adverse effects

Red in vivo recombination is a new kind of genetic engineering technique based on homologous recombination. In this work, plasmid pKD46 which expresses Red recombination proteins is transferred into Escherichia coli strain DH5?.The kanamycine resistant gene is generated by PCR by using primers with homology to hisDCB gene of E.coli chromosome. Thus, the hisDCB gene was replaced with kanamycine resistant gene by the plasmid recombination system, then the resistant gene was eliminated by a helper plasmid encoding the FLP recombinase. At last, a E.coli histidine auxotroph which is sensitive to kanamycine was got. The results indicate that Red in vivo recombination is a convenient method to construct auxotrophs.

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibody Specificity ; CHO Cells ; Cricetulus ; Female ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Thrombomodulin ; immunology ; Transfection

OBJECTIVETo measure the vertical height of mesio-distal marginal ridge to cusp in posterior teeth, which may be helpful to brackets positioning.

METHODSThe study groups comprised of 60 patients (30 men, 30 women, mostly aged 12-14 years) who underwent orthodontic treatment without tooth extraction and matched the Andrews normal occlusion standard after treatment. Study model of each patient was made. Three-dimensional laser measurer was used to evaluate the vertical height of mesio-distal marginal ridge to mesial cusp in posterior teeth. The data were stored in a personal computer and submitted to statistical analysis of paired t test.

RESULTSNo statistical significant difference was found in the same teeth between men and women. Not only in maxilla but also in mandible, there was no significant difference between the left and the right (P>0.05). The average vertical height of maxillary first premolars was (1.70+/-0.50) mm, the maxillary second premolars was (1.24+/-0.45) mm, and for maxillary first molars, the result was (0.83+/-0.40) mm. The difference between each result was statistically significant (P9< 0.01). The average vertical height of mandibular first premolars was (2.25+/-0.45) mm, the mandibular second premolars was (1.55+/-0.45) mm, and for mandibular first molars, the result was (1.18+/-0.40) mm. The difference between each result was statistically significant (P<0.0 1).

CONCLUSIONThe vertical height of brackets position in posterior teeth should be considered to guarantee that mesio-distal marginal ridges of deferent posterior teeth located in the same plane, so that satisfying goal could be achieved, If the vertical height in the first molar was X mm, the vertical height in the second premolar should be (X+0.5) mm, and (X+1.0) mm might be suit for the first premolar.

Bicuspid ; Dental Occlusion ; Female ; Humans ; Male ; Mandible ; Maxilla ; Molar ; Tooth ; Tooth Extraction ; Tooth Movement Techniques

OBJECTIVETo explore the distribution characteristics of the types of M protein gene (emm) in group A streptococcus (GAS) isolated from children in Beijing in the year 2011.

METHODSDuring May to July in 2011, a total of 3315 patients who were diagnosed scarlet fever or pharyngeal infection by doctors in pediatric outpatient and emergency units of 36 hospitals, were selected as subjects. Their throat swab samples were collected and isolated the strains of GAS. Gene emm was then amplified and sequenced by PCR method, and the differences in types of gene emm between different populations and diseases were compared.

RESULTSA total of 633 strains of GAS were isolated from the 3315 throat swab samples, 610 strains out of which were gene emm positive and were recruited in the study. Out of the 610 recruited strains, 448 (73.4%) were isolated from scarlet fever patients, the other 162 (26.6%) were isolated from pharyngeal infection patients; 397 (65.1%) were from urban, the other 213 (34.9%) were from suburb; 240 (39.4%) were from patients aging between 1 - 5 years old, the other 369(60.6%) were from patients aging 6 - 18 years old. A total of 8 types of gene emm (scarlet fever: 6 types, pharyngeal infection: 4 types) and 21 subtypes of gene emn (scarlet fever: 16 subtypes, pharyngeal infection: 10 subtypes) were identified. Three new subtypes were found in the study, naming emm1.63, emm12.62 and st5144.20. Among them, emm1.63 was found both in scarlet fever and pharyngeal infection patients, while emm12.62 and st5144. 20 were only found in pharyngeal infection patients. Among all the types of gene-emm, emm12 accounted for the highest percentage as 80.5% (491/610) and then followed by emm1 (18.0% (110/610)). Among all the subtypes, the dominant subtype was emm12.00, accounting for 69.0% (421/610), following by emm1.00 (16.9% (103/610)) and emm12.19 (6.1% (37/610)). All the above types and subtypes of gene emm were the most prevalent strains in scarlet fever patients and pharyngeal infection patients. Significant differences in the distribution of prevalent strains were observed among various aging patients and regions. The constituent ratios of emm1, emm1.00 and emm12.19 were higher in patients from suburb (emm1: 22.1% (47/213), emm1.00: 19.2% (40/213), emm12.19: 8.0% (17/213)) than those in urban areas (emm1: 15.9% (63/397), emm1.00: 15.6% (62/397), emm12.19: 5.0% (20/397)). The difference showed statistical significance (P < 0.05). The constituent ratio of emm1.00 was higher among patients aging 6-18 years old (19.2% (71/369)) than those aging 1 - 5 years old (13.3% (32/240)). The difference also showed statistical significance (χ(2) = 8.45, P < 0.05).

CONCLUSIONAmong the types of gene emm in GAS isolated from children in Beijing in year 2011, the most prevalent two were emm12 and emm1, and the most prevalent emm subtypes were emm12.00, emm1.00 and emm12.19. A significant difference in their distribution between various aging patients and isolated places can be obviously found.

Adolescent ; Antigens, Bacterial ; classification ; genetics ; Bacterial Outer Membrane Proteins ; classification ; genetics ; Carrier Proteins ; classification ; genetics ; Child ; Child, Preschool ; China ; Female ; Genes, Bacterial ; Genotype ; Humans ; Infant ; Male ; Microbial Sensitivity Tests ; Streptococcus pyogenes ; genetics ; isolation & purification

OBJECTIVETo study the inhibitory effect of CD133 monoclonal antibody labeled with ¹³¹I (¹³¹I-CD133mAb) on Huh-7 human liver cancer cell line overexpressing CD133 antigen in vitro and in mouse models bearing the tumor cell xenograft.

METHODS¹³¹I-CD133mAb was prepared by chloramines-T method and evaluated for its stability. Flow cytometry and immunohistochemistry were used to detect the expression of CD133 in Huh-7 cells and in Huh-7 cell-derived tumors, respectively. Huh-7 cells treated with ¹³¹I-CD133mAb plus cisplatin (DDP), ¹³¹I -CD133mAb, DDP, or no treatment (blank control) were examined for cell proliferation suppression by MTT assay with the IC₅₀ calculated. BALB/c mice bearing subcutaneous Huh-7 cell xenograft in the right forelegs were treated with ¹³¹I -CD133mAb, DDP, or both every two days for two weeks. The tumor size and volume were measured twice a week, and pathological examination of the tumor was carried out after the treatments. The tumor inhibition rate was calculated and tumor cell apoptosis observed with HE staining.

RESULTSThe labeling ratio of ¹³¹I-CD133mAb was 90.25% and the radiochemical purity was 97.78%. Huh-7 cells showed obviously higher CD133 expression than HepG2 cells. ¹³¹I-CD133mAb combined with DDP group resulted in a significantly higher tumor inhibition rate than other treatments in the tumor-bearing mice.

CONCLUSION¹³¹I-CD133mAb can inhibit the growth of liver cancer cells with a high CD133 expression both in vivo and in vitro.

AC133 Antigen ; Animals ; Antibodies, Monoclonal ; pharmacology ; Antigens, CD ; immunology ; Apoptosis ; Carcinoma, Hepatocellular ; drug therapy ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Cisplatin ; pharmacology ; Glycoproteins ; immunology ; Hep G2 Cells ; Humans ; Liver Neoplasms ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Peptides ; immunology ; Xenograft Model Antitumor Assays

OBJECTIVETo explore the effects of surgical procedure combined with the intravascular minimal invasive technique for the treatment of deep vein thrombosis (DVT) of lower extremity.

METHODSAt the curse of disease from six hours to ninety days, one hundred and two patients with DVT including one hundred and three lower extremities had received surgical procedure and intravascular minimal invasive treatment.

RESULTSThere were not procedure-related morbidities in 102 cases, and symptoms disappeared, all procedures were successful based on angiography. The detecting head for the intravascular ultrasound ablation was entered to inferior vena cava (IVC) in 74 cases (78%), Forgarty catheter was entered to IVC in 21 cases (21%), the stenosis in the confluence of the common iliac vein and IVC was dilated by sacculus rotundus catheter in 89 cases (88%), including 9 patients underwent percutaneous transluminal stenting. One hundred and two patients followed up for twenty months, follow-up by angiography showed no restenosis in 91 cases, restenosis in ilio-femoral vein in 1 cases, and thrombus recontouring in 4 cases, as well as 6 cases died caused by primary disease.

CONCLUSIONSurgical procedure combined with the intravascular minimal invasive technique is a safe and effective therapeutic method for DVT.

Adult ; Aged ; Aged, 80 and over ; Angioplasty, Balloon ; Catheter Ablation ; methods ; Combined Modality Therapy ; Female ; Follow-Up Studies ; Humans ; Lower Extremity ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; Stents ; Ultrasonic Therapy ; Venous Thrombosis ; surgery ; therapy

OBJECTIVETo summarize experiences of serious perioperative complications management of obstructive sleep apnea hypopnea syndrome (OSAHS), and evaluate the effect of intervention in decreasing the incidence of serious complications.

METHODSRetrospective analysis of clinical data in Shenyang General Hospital of PLA and Liaoning Province Jinqiu Hospital of OSAHS surgery cases from January 1995 to December 2009 were included in this study, patients were divided into two groups according to with or without intervention. Experience and lessons were analyzed.

RESULTSPatients without and with intervention were 402 and 521 respectively, and uvulopalatopharyngoplasty (UPPP) cases in each group were 387 and 390. Five patients in the first group who accepted UPPP had breathing difficulty and were all successfully rescued, while no one in the second group had breathing difficulty. The difference was significant (P < 0.05). Sixteen patients in the first group had severe bleeding after UPPP, while only 5 patients had the severe bleeding in the second group. The difference was significant, too P < 0.05. No breathing difficulty cases in the second group, and serious bleeding cases in each group was 5 and in 1, there was no significant difference (P > 0.05).

CONCLUSIONSBreath difficulty and serious bleeding are serious perioperative complications of OSAHS surgery, and with systemic intervention the incidence of the complications can be decreased.

Adolescent ; Adult ; Aged ; Female ; Humans ; Intraoperative Complications ; prevention & control ; Male ; Middle Aged ; Palate, Soft ; surgery ; Retrospective Studies ; Sleep Apnea, Obstructive ; surgery ; Uvula ; surgery ; Young Adult

OBJECTIVETo determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase.

METHODSTwo-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated.

RESULTSExo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase.

CONCLUSIONThese data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.

DNA Primers ; chemistry ; genetics ; Exonucleases ; metabolism ; Humans ; Phosphorothioate Oligonucleotides ; chemistry ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

Objective To study the influence of targeted silencing of DEK on the proliferation and cell cycle of human hepatoma cell lines.Methods The human hepatoma cells line HepG2 were routinely cuhured and the cells were divided into blank control group,siRNA control group and DEK siRNA group when the cells grew to 90％ tusion.The cells in blank control group were cultured normally without any treatment;the cells in siRNA control group and DEK siRNA group were transfected with siRNA expression vector and DEK siRNA expression vector mediated by LipofectamineTM2000 liposomes,respectively.The expression of DEK mRNA in HepG2 cells was detected by real-time polymerase chain reaction;the expression of DEK and CyclinD1 protein in HepG2 cells was detected by Western blot;the proliferation of HepG2 cells was detected by methyl thiazolyl tetrazolium method,and the cell cycle was observed by flow cytometry.Results The expression of DEK mRNA in the blank control group,siRNA control group and DEK siRNA group was 0.826 ±0.052,0.776 ±0.051 and 0.420 ±0.050 respectively;the expression of DEK protein in the blank control group,siRNA control group and DEK siRNA group was 0.691 ± 0.073,0.726±0.061 and 0.311 ±0.038 respectively;the expression of CyclinDl protein in the blank control group,siRNA cuntrol group and DEK siRNA group was 0.712 ± 0.069,0.780 ± 0.074 and 0.434 ± 0.039 respectively.The expressions of DEK mRNA,DEK protein and CyclinD1 protein in DEK siRNA group were significantly lower than those in the blank control group and siRNA control group (P ＜ 0.05);there was no statistic difference in the expression of DEK mRNA,DEK protein and CyclinD1 protein between the blank control group and siRNA control group(P ＜0.05).The proliferation ability of HepG2 cells in DEK siRNA group after transfection of 24,48,72,96,120 h was significantly lower than that in the blank control group and siRNA control group(P ＜0.05);there was no statistic difference in the proliferation ability of HepG2 cells between the blank control group and siRNA control group at each time point(P ＜ 0.05).The proportion of G0 + G1 phase cells in DEK siRNA group was significantly higher than that in the blank control group and siRNA control group(P ＜ 0.05);the proportions of S phase and G2 + M phase cells in DEK siRNA group were significantly lower than those in the blank control group and siRNA control group(P ＜ 0.05);there was no statistic difference in the proportion of G0 + G1 phase,S phase and G2 + M phase cells between the blank control group and siRNA control group (P ＜ 0.05).The result of Pearson correlation analysis showed that the expression of CyclinD1 protein was positively correlated with the expression of DEK mRNA and protein(r =0.909,0.899;P ＜ 0.05).Conclusion DEK siRNA can inhibit the proliferation of HepG2 cells,and change the cell cycle distribution through down regulating the expression of DEK gene in HepG2 cells.This process may be related to the down regulation of the expression of CyclinD1.