OBJECTIVE: To extract sperm DNA from mixed stain by the modified differential lysis method combined with silicon bead method and to evaluate its application value. METHODS: Fifty-two mixed stains containing female STR genotypes detected by differential lysis method were collected. The sperm DNA was extracted by the modified method combined with silicon bead method, then genotyped with the Identifiler Kit, and compared with the results of genotyping by the conventional differential lysis method as control. RESULTS: Of the 52 samples, 38 samples with sole male STR genotypes in all loci were detected. The detection rate of male STR genotypes was 98.08% through the modified method combined with silicon bead method. CONCLUSION The modified differential lysis method combined with silicon bead method can be used in extraction of sperm DNA from mixed stain.
DNA/isolation & purification* ; DNA Fingerprinting ; Female ; Genotype ; Humans ; Male ; Polymerase Chain Reaction ; Silicon ; Spermatozoa

Objective To investigate the transcription and protein expressions of chemokines CL16, CL12 and their receptors CR6, CR4 in first-trimester human cytotrophoblast cells and human choriocarcinoma cell line JAR. Methods Transcriptions of CR6, CL16, CR4, CL12 in purified first-trimester human trophoblast cells and JAR line were assessed by semi-quantitative RT-PCR, and protein expressions of CR6, CL16, CR4, CL12 were analyzed in primary cultured villous cytotrophoblasts (VCT), extravillous cytotrophoblasts (EVCT), JAR line and placentas by immunostaining. Results CR6 and CR4 were highly transcribed in primary cultured trophoblast cells with mRNA relative level of 1.12?0.25 and 1.08?0.11 respectively, and their ligands CL16 and CL12 were transcribed moderately with mRNA relative level of 0.89?0.11 and 0.78?0.10 respectively. It was demonstrated that CL16, CL12, CR6 and CR4 were expressed in primary cultured VCT, EVCT, JAR line and placentas by immunostaining. Conclusion The co-expression of CL16/CR6 and CL12/CR4 in trophoblast cells may play a role in the proliferation and differentiation of first-trimester trophoblast cells in a manner of autocrine.

OBJECTIVE: To study the percentages of polymorphonuclear leukocytes (PMN), mononuclear cells (MNC) and fibroblastic cells (FBC) in different post-traumatic intervals after skeletal muscle mechanical injury in rats. METHODS: The rat model of skeletal muscle mechanical injury was established. The rats were divided into injured groups (6 h, 12 h, 1 d, 3 d, 7 d, 10 d and 14 d after injury) and control group. The percentages of PMN, MNC and FBC in different post-traumatic intervals after skeletal muscle mechanical injury were assessed with HE staining and image analysis. RESULTS: At post-injury 6-12h, the percentages of PMN and MNC infiltration appeared in injured sites and that of PMN reached peak. At 1 d, the percentage of MNC infiltration appeared and reached peak, while that of PMN decreased. At 3-7 d, the percentage of FBC gradually increased, while that of PMN and MNC decreased. At 10-14d, the percentage of FBC reached peak. CONCLUSION The percentages of PMN, MNC and FBC in injured zones showed time-dependent changes, which might be used as reference index for determination of age of skeletal muscle injury.
Animals ; Fibroblasts ; Muscle, Skeletal/injuries* ; Neutrophils ; Rats ; Time Factors

AIMTo identify the drug-metabolizing enzymes involved in the hydroxylation of the new anti-inflammatory and anodyne imrecoxib.

METHODSImrecoxib was incubated with heterologous expression human cytochrome P450 (rCYPs) in vitro, and metabolites and remained parent drug were detected with liquid chromatography-multistage mass spectrometry. The contribution of 4 CYPs in the hydroxylation metabolism of imrecoxib was evaluated by total normalized rate (TNR) method.

RESULTSImrecoxib is metabolized by CYP2C9, CYP2D6 and CYP3A4, with the rate of 62.5%, 21.1% and 16.4%, respectively.

CONCLUSIONCYP2C9 is the major enzyme involved in imrecoxib hydroxylation metabolism.

Aryl Hydrocarbon Hydroxylases ; metabolism ; Cyclooxygenase 2 Inhibitors ; metabolism ; Cytochrome P-450 CYP2C9 ; Cytochrome P-450 CYP2D6 ; metabolism ; Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme System ; metabolism ; Hydroxylation ; Pyrroles ; metabolism ; Spectrometry, Mass, Electrospray Ionization ; Sulfides ; metabolism

Seven acylated triterpene saponins were isolated from the roots of Securidaca inappendiculata by means of various chromatographic techniques such as silica gel, MPLC, preparative HPLC, and semi-preparative HPLC. Their chemical structures were identified as securioside A(1), securioside B(2), 3-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(1-->4)-α-L-rhamnopyranosyl-(1-->2)-[β-D-glucopyranosyl-(1-->3)]-4-O-[(E)-3,4-dimethoxycinnamoyl]-β-D-fucopyranosyl ester(3), 3-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(1-->4)-α-L-rhamnopyranosyl-(1-->2)-[β-D-glucopyranosyl-(1-->3) ] -4-O-[(E/Z)-3, 4-dimethoxycinnamoyl]-β-D-fucopyranosyl ester(3/4), 3-O-β-D-glucopyranosyl presenegenin 28-O-α-L-arabinopyranosyl-(1-->3)-β-D-xylopyranosyl-(1-->4)-α-L-rhamnopyranosyl-(1-->2)-4-O-[(E)-3,4-dimethoxycinnamoyl]-β-D-fucopyranosyl ester(5), polygalasa- ponin XLV(6), and polygalasaponin XLVI (7) on the basis of spectroscopic data analysis and physicochemical properties. Among them, compounds 5-7 were isolated from the plants in genus Securidaca for the first time and compounds 3, 3/4 were isolated from the species for the first time. The cytotoxicity assay showed that compounds 2, 3/4, 5 have moderate cytotoxic activities against Lewis lung carcinoma LLC cells with IC50 values of 41.10, 38.17, and 48.92 µmol · L(-1), respectively; compound 2 exhibited moderate cytotoxic activities against human breast cancer MCF-7 cells with an IC50 value of 47.93 µmol · L(-1).
Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Humans ; MCF-7 Cells ; Plant Roots ; chemistry ; Saponins ; chemistry ; isolation & purification ; pharmacology ; Securidaca ; chemistry

OBJECTIVE: To research the relation between the time-dependent appearances of myotibroblasts during the repair of contused skeletal muscle in rat and wound age determination. METHODS: A total of 35 SD male rats were divided into the control and six injured groups according to wound age as follows: 12 h, 1 d, 5 d, 7 d, 10 d and 14 d after injury. The appearances of myofibroblasts were detected by HE staining, immunohistochemistry and confocal laser scanning microscopy. Masson's trichrome staining was utilized to examine collagen accumulation in the contused areas. RESULTS: Immunohistochemical staining showed that α-SMA+ myofibroblasts were initially observed at 5 d post-injury. The average ratio of myofibroblasts was highest at 14 d post-injury, with all samples, ratios more than 50%. In the other five groups, the average of α-SMA positive ratios were less than 50%. The collagen stained areas in the contused zones, concomitant with myofibroblast appearance, were increasingly augmented along with advances of posttraumatic interval. CONCLUSION The immunohistochemical detection of myofibroblasts can be applied to wound age determination. The myofibroblasts might be involved in collagen deposition during the repair of contused skeletal muscle in rat.
Animals ; Collagen/metabolism* ; Contusions/metabolism* ; Immunohistochemistry ; Male ; Microscopy, Confocal ; Muscle, Skeletal/metabolism* ; Myofibroblasts/metabolism* ; Rats ; Time Factors ; Wound Healing

Objective To explore the causes,clinical manifestation and therapy of frontal sinusitis after transfrontal craniotomy.Methods Thirty-three patients with frontal sinusitis after transfrontal craniotomy were included in the study.Among them,7 cases had frontal sinus abscess and 4 cases had frontal sinus fistula.Twenty-three patients were treated with traditional frontal sinus surgery with facial incision.The nasofrontal dilatation tube was positioned for more than 3 months.Nine patients were treated with endoscopic frontal sinus surgery,and 1 patient was treated with combined endoscopic and traditional frontal sinus surgery,with nasofrontal dilatation tube positioned for less then 1 month.In the revision surgery,the bone wax and phlogistic acestoma were cleaned out in both operational methods.The causes of frontal sinusitis after transfrontal craniotomy were discussed by studying the frontal sinus CT image,and prior surgical data.Results All patients were followed up for more than 6 months after the nasofrontal dilatational tube was removed.Among 33 patients,two cases with traditional frontal sinus surgery were operated twice due to nasofrontal dilatation tube fall off in 1 month.In all 33 patients,30 cases cured and 3 cases got better.There were no curative difference between two operational methods.Conclusions The causes of frontal sinusitis after transfrontal craniotomy were inadequate sinus management in craniotomy and bone wax tamping in frontal sinus.There was more frontal sinus abscess and fistula occurring in frontal sinusitis after transffontal craniotomy than that in ordinary frontal sinusitis.The therapy included cleaning out bone wax and phlogistic acestoma,and expanding the frontal sinus ostium.The satisfying curative effect was obtained in both operational methods,but endoscopic frontal sinus surgery Was better because it is minimally invasive,no facial incision and quick recovery with less nasofrontal dilatational tube posting time.

Objective To 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. Methods Retrospective 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. Results Patients 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 ). Conclusions Breath difficulty and serious bleeding are serious perioperative complications of OSAHS surgery, and with systemic intervention the incidence of the complications can be decreased.

BACKGROUNDThe classic glycine receptor (GlyR) in the central nervous system is a ligand-gated membrane-spanning ion channel. Recent studies have provided evidence for the existence of GlyR in endothelial cells, renal proximal tubular cells and most leukocytes. In contrast, no evidence for GlyR in myocardial cells has been found so far. Our recent researches have showed that glycine could protect myocardial cells from the damage induced by lipopolysaccharide (LPS). Further studies suggest that myocardial cells could contain GlyR or binding site of glycine.

METHODSIn isolated rat heart damaged by LPS, the myocardial monophasic action potential (MAP), the heart rate (HR), the myocardial tension and the activities of lactate dehydrogenase (LDH) from the coronary effluent were determined. The concentration of intracellular free calcium ([Ca(2+)](i)) was measured in cardiomyocytes injured by LPS and by hypoxia/reoxygenation (H/R), which excludes the possibility that reduced calcium influx because of LPS neutralized by glycine. Immunohistochemistry was used to detect the GlyR in myocardial tissue. GlyR and its subunit in the purified cultured cardiomyocytes were identified by Western blotting.

RESULTSAlthough significant improvement in the MAP/MAPD(20), HR, and reduction in LDH release were observed in glycine + LPS hearts, myocardial tension did not recover. Further studies demonstrated that glycine could prevent rat mycordial cells from LPS and hypoxia/reoxygenation injury (no endotoxin) by attenuating calcium influx. Immunohistochemistry exhibited a positive green-fluorescence signaling along the cardiac muscle fibers. Western blotting shows that the purified cultured cardiomyocytes express GlyR beta subunit, but GlyR alpha1 subunit could not be detected.

CONCLUSIONSThe results suggest that glycine receptor is expressed in cardiomyocytes and participates in cytoprotection from LPS and hypoxia/reoxygenation injury. Glycine could directly activate GlyR on the cardiomyocytes and prevent calcium influx into the cardiomyocytes.

Animals ; Blood Pressure ; drug effects ; Blotting, Western ; Calcium ; metabolism ; Cytoprotection ; Glycine ; pharmacology ; Heart ; drug effects ; physiology ; Heart Rate ; drug effects ; Immunohistochemistry ; L-Lactate Dehydrogenase ; secretion ; Lipopolysaccharides ; toxicity ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Glycine ; analysis ; physiology

BACKGROUNDO(6)-methylguanine-DNA-methyltransferase (MGMT) is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells.

METHODSMammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells (PBMCs) via liposome, then assayed for gene expression at RNA and protein levels. MTT assay was used to check the drug resistance of cells transfected with MGMT gene.

RESULTSMGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively.

RESULTSof Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC(50) values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC.

CONCLUSIONThe alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.

Blotting, Western ; Cell Survival ; drug effects ; genetics ; physiology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Enzymologic ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; cytology ; drug effects ; metabolism ; Microscopy, Fluorescence ; Nitrogen Mustard Compounds ; pharmacology ; O(6)-Methylguanine-DNA Methyltransferase ; genetics ; metabolism ; physiology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection