AIM:To investigate whether Smad pathway participates the process of extracellular signal regulated kinase (ERK) induced the proliferation of vascular smooth muscle cells (VSMCs). METHODS: Human umbilical artery smooth muscle cells (hUASMCs) were divided into four groups: control group, PDGF (platelet derived growth factor) group, ERK blocking agent group and PDGF+ERK blocking agent group. MTT assay was used to detect the proliferation of hUASMCs (A value). Immunohistochemical technique was used to detect the expression of PCNA, phosphorylated ERK (p-ERK) and phosphorylated Smad2/3 (p-Smad2/3) protein in hUASMCs. The expression of Smad2/3 mRNA in hUASMCs was detected by RT-PCR. RESULTS: The proliferation of hUASMCs and the expression of PCNA, p-ERK and p-Smad2/3 proteins in hUASMCs in PDGF group were increased obviously than those in other groups (P

OBJECTIVETo investigate the mothod and therapeutic efficacy of total hip anthroplasties (THA) for osteoarthritis secondary to Crowe type IV developmental dysplasia of hip in adults.

METHODSFrom May 2006 to December 2013, THA was performed on 15 adult patients (17 hips) with Growe type IV acetabular dysplasia, including 13 females and 2 males, with a mean age of 30.9 years old (22 to 58 years old) and an average preoperative Harris score of (34.0 ± 6.5) points. Traction of the affected limb was not performed before surgery. After extensive release and lengthening of soft tissues, sub-trochanteric osteotomy of the femur was performed, hip joint center was rebuilt and the abduction function was restored.

RESULTSThe patients were followed up with a mean period of 33 months (ranged from 6 months to 5 years). The postoperative Harris score was 85.0 ± 7.3,higher than the preoperative score. The extended length of limb ranged from 1.6 to 5.4 cm, with a mean of (3.42 ± 0.65) cm. The shortening and malformation of the affected limb were corrected in the most patients,with the difference in length of the two legs less than 1.5 cm. After surgery, 1 patient experienced partial sciatic nerve injury, which was largely recovered after 3 months of conservative treatment. One patient experienced complete sciatic nerve injury, which was partially recovered after 6 months of conservative treatment; a foot-drop varus deformity was formed in the distal end of the affected limb, which was improved after tendon transposition and transplantation. Joint pain was relieved, and the joint function was restored significantly. Over the follow-up period, no severe complications such as dislocation, infection, prosthesis loosening, or subsiding occurred.

CONCLUSIONSatisfactory efficacy can be achieved for adult Growe type IV acetabular dysplasia associated with osteoarthritis by THA, with proper soft tissue release and lengthening, sub-trochanteric osteotomy of femur, joint functional restoration, appropriate choice of prosthesis, and careful protection of nerves and vessels.

Adult ; Arthroplasty, Replacement, Hip ; methods ; Female ; Hip Dislocation, Congenital ; complications ; Humans ; Leg Length Inequality ; therapy ; Male ; Middle Aged ; Osteoarthritis, Hip ; surgery

OBJECTIVE: To study the depletion of cardiac troponin T in Rats of early myocardial ischaemia. METHODS: Immunohistochemical method (LSAB) was used and the depletion area of CTnT in myocardial ischaemia were measured by the computer image analysis. RESULTS: There is obviously depletion of CTnT after 15 min in myocardial ischaemia of rats, the mean of CTnT depletion of myocardial ischaemic group is significant difference compared with that of the control group (P < 0.01), and the depletion area increased with the prolongagtion of ischaemic time. CONCLUSION The depletion of CTnT is one of sensitive signs of early myocardial ischaemia.
Animals ; Female ; Forensic Medicine ; Image Processing, Computer-Assisted ; Immunohistochemistry ; Male ; Myocardial Ischemia/metabolism* ; Myocardium/metabolism* ; Rats ; Rats, Sprague-Dawley ; Troponin T/metabolism*

Objective To observe the efficacy and safety of applying sirolimus-eluting stents in emergency percutaneous coronary intervention (PCI) for the patients with acute myocardial infarction (AMI).Methods In total,220 patients with AMI were enrolled in this study at Shanghai Tongji Hospital, divided into two groups,one with bare-metal stent and the other with sirolimus-eluting stent.Cardiovascular fatality,major adverse cardiac events (MACE) and target vessel revascularization (TVR) were observed one and six months after PCI in the two groups.Results There was no significant difference in overall fatality and MACE in the 1~(st) or 6~(th) months after PCI between the two groups.Three cardiogenic deaths occurred in bare-metal stent group with a fatality of 2.8 percent,and five deaths in sirolimus-eluting stent group with a fatality of 4.5 percent in six months after PCI.However,rate of restenosis in those with sirolimus-eluting stents was significantly lower than that of bare-metal stents (6.0 percent vs 16.1 percent,P

OBJECTIVETo investigate the relationship between point mutation of penicillin-binding protein gene (pbp) and amoxicillin resistance (AMOgamma) of Helicobacter pylori (H. pylori) as well as to compare the protein profiles under proteomic technology to get the candidate resistance-related proteins.

METHODS(1) AMOgamma strains were selected from the sensitive H. pylori strain 26695 by serial passage technique in vitro. (2) Point mutations of five putative resistance genes (HP0597, HP1565, HP1542, HP1556, and HP0160) were analyzed by denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. (3) Proteins samples were separated by two-dimensional electrophoresis (2-DE). Protein profiles were compared between the AMOgamma strain obtained in vitro and its sensitive parent strain 26695. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed to identify the proteins of interest. The proteins were searched by software MASCOT and identified by peptide fingerprint map using the program MS-FIT of Protein Prospect.

RESULTS(1) An AMOgamma strain (MIC 8 microg/ml) was obtained. Complete loss of the resistant phenotype was observed after cultivation in the absence of AMO or storage at - 80 degrees C. (2) DHPLC and Sequencing result showed no point mutations in five pbp genes in the AMOgamma strain when compared with the corresponding PCR products from its parent strain 26695. (3) Protein profiling showed that eleven protein spots were differently expressed between 26695 and the AMOgamma strain. Of these protein spots in the AMOgamma strain, two new spots (Spot 1 and Spot 2) were observed with one (Spot 3) was up-regulated three-fold and the remained ones (Spot 4-11) were down-regulated.

CONCLUSIONAMO resistance of H. pylori might be resulted from, unstable phenotype change rather than point mutations of pbp genes. These differentially regulated proteins in AMOgamma strain might play a role in development of resistance to AMO in H. pylori.

Amoxicillin ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Chromatography, High Pressure Liquid ; Drug Resistance, Bacterial ; genetics ; Electrophoresis, Gel, Two-Dimensional ; Helicobacter pylori ; drug effects ; genetics ; metabolism ; Point Mutation ; genetics ; physiology ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of budesonide in dog plasma. Budesonide and the internal standard triamcinolone acetonide were separated from plasma by alkalinized liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Capcell Pak C18 MG column with the mobile phase consisted of acetonitrile -5 mmol x L(-1) ammonium acetate (60:40, v/v) at a flow-rate of 0.50 mL x min(-1). A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the negative ion mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 489 --> m/z 357 and m/z 493 --> m/z 413 for budesonide and the internal standard, respectively. The linear calibration curves were obtained in the concentration range of 25.0-2000 pg x mL(-1). The lower limit of quantification was 25.0 pg x mL(-1). The intra- and inter-day relative standard deviation over the entire concentration range was less than 15%. The accuracy was in the range of -8.1% to -1.7% in terms of relative error. The method was applied to a pharmacokinetic study of budesonide controlled-release capsules in Beagle dogs. Maximal budesonide plasma level was observed after (3.5 +/- 3.3) h and the Cmax was (786 +/- 498) pg x mL(-1) after a single oral administration of 9 mg budesonide capsules, Cmax was increased to (2142 +/- 1515) pg x mL(-1) after multiple oral administration (9 mg x 5 d) of budesonide capsules. This method was selective and rapid, and the sensitivity was sufficient for the purpose of the pharmacokinetic study of budesonide controlled-release formulation.
Animals ; Anti-Inflammatory Agents ; blood ; pharmacokinetics ; Area Under Curve ; Budesonide ; blood ; pharmacokinetics ; Chromatography, Liquid ; methods ; Delayed-Action Preparations ; Dogs ; Male ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry ; methods

Objective To investigate the effect of miR-106b on the apoptosis and proliferation of nasopharyngeal carcinoma (NPC ) cells. Methods We analyzed differences in miRNA expression in nasopharyngeal carcinoma and adjacent normal tissues with miRNA microarray.Taq Man miRNA detection kit and Real-time fluorescence quantitative PCR were used to detect the expressions of miR-106 and RhoC mRNA in nasopharyngeal carcinoma and adjacent tissues.The miR-106b and target gene binding sites were predicted with miRnada.The target gene was verified by double luciferase.Western blot was used to detect the expression of RhoC regulated by miR-106b.Annexin and TUNEL were used to detect the effect of miR-106b on the apoptosis of nasopharyngeal carcinoma cells;the effect of miR-106b on the proliferation of nasopharyngeal carcinoma cells was detected by MTT assay.Results miRNA microarray analysis showed that the expression of miR-106b was lower in NPC tissues than in adjacent normal tissues.The results of RT-PCR showed that the expression of miR-106b in nasopharyngeal carcinoma was decreased (P <0.05)while the expression of RhoC was increased in nasopharyngeal carcinoma (P <0.05).The expressions of miR-106b and RhoC in NPC were negatively correlated (r =-0.5866, P <0.001).The results of luciferase reporter assay showed that the activity of luciferase in miR-106b group was lower than that in empty plasmid group (P < 0.05 ).The results of Western blot showed that miR-106b could decrease the expression of RhoC in NPC tissues (P <0.05).Annexin V-PI and TUNEL showed that the apoptosis ofnasopharyngeal carcinoma cells was significantly higher in miR-106 group than in empty plasmid group (P <0.05). MTT results showed that the proliferation of nasopharyngeal carcinoma cells in miR-106b group was lower than that in empty plasmid group (P <0.05).Conclusion miR-106b may induce the apoptosis of nasopharyngeal carcinoma cells and inhibit the proliferation of nasopharyngeal carcinoma cells by down-regulating the expression of RhoC.

OBJECTIVETo construct pNTAP-MK2 eukaryotic expression plasmid and establish a HEK293 cell line stably expressing tandem affinity purification (TAP)-tagged MK2.

METHODSThe MK2-encoding region was subcloned into the vector pNTAP to construct the recombinant plasmid pNTAP-MK2, which was subsequently transformed into DH5 alpha.E.coli. After identification by PCR, digestion with restriction endonuclease and sequencing, the recombinant expression plasmid was transfected into HEK293 cells via liposome, and the cell line with stable expression of exogenous TAP tag-MK2 gene was selected by antibiotic G418. The expression and localization of the fusion protein TAP tag-MK2 were detected by Western blotting and immunofluorescence assay.

RESULTSThe results of PCR, restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pNTAP-MK2. Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection with G418 selection. Immunofluorescence assay identified the expression product TAP tag-MK2 mainly in the cell nuclei.

CONCLUSIONThe eukaryotic expression vector pNTAP-MK2 has been successfully constructed, and in the established cell line with stable expression of TAP tag-MK2, TAP tag does not influence the localization of exogenous MK2.

Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Plasmids ; Protein-Serine-Threonine Kinases ; genetics

OBJECTIVETo acquire sufficient PDGF-BB protein and provide the basis for the further studies of its role on the fracture healing and trauma reconstruction and its clinical applications.

METHODSConstructed the prokaryotic expression vector pQE-PDGF-B with the gene rearrangement technique, and the monomeric form of recombinant PDGF-B expressed in E. coli M15.

RESULTSPDGF-B mature peptide gene was inserted into prokaryotic expression vector pQE30, which was confirmed by PCR, enzyme digestion and sequencing identification; the expressed products of pQE-PDGF-B in E. coli showed a single protein on SDS-PAGE, and their expression level was about 15% of the total bacterial protein. The molecular weight of the purified PDGF-B protein was about 15 KDs on SDS-PAGE.

CONCLUSIONSThe construction of recombinant plasmid and preparation of the monomeric protein of PDGF-B provides a solid foundation for further studying the function of PDGF-BB and producing biologically PDGF-BB protein.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; In Vitro Techniques ; Platelet-Derived Growth Factor ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-sis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

To investigate the clinical value of 1H magnetic resonance spectroscopy (1H MRS) in the evaluation of high intensity focused ultrasound (HIFU) ablation for primary liver cancer. Routine magnetic resonance sequences, contrast-enhanced magnetic resonance imaging and respiratory-triggered single voxel point resolved spectroscopy sequence (PRESS) were performed on 24 patients with primary liver cancer before and after HIFU ablation. A respiratory-triggered axial T2 weighted imaging (T2WI) was used as localizer for PRESS. Spectroscopy data was transmitted to a personal computer and was post-processed with a custom software (Saker, provided by Ning Jing, an engineer in GE Healthcare). It would be considered "technical success" if the baselines of spectra were stable and main metabolites were without overlapping and could be identified. Integral areas of choline (Cho) peak at 3.2 parts per million (ppm) and lipid (Lip) peak at 1.3 ppm were measured, and the choline to lipid (Cho/Lip) ratios were calculated. The differences of areas of Cho, Lip peak and Cho/Lip ratios before and after HIFU ablation were compared by using paired samples t test, and a P value of less than 0.05 was considered statistically significant. The technical success rate of 1H-MRS was 87.50% (42/48). Integral areas of Cho peak and Lip peak of 20 patients with satisfied spectra were measured, and the Cho/Lip ratios were calculated. The Integral area of Cho peak decreased from 34 597+/-6 802 before HIFU ablation to 6 372+/-2 466 after HIFU ablation (t = 18.02, P less than 0.01). The Integral area of Lip peak increased from 147 948+/-16 317 before HIFU ablation to 149 069+/-16 345 after HIFU ablation (t = -15.11, P less than 0.01). The Cho/Lip ratio decreased from 0.23+/-0.03 before HIFU ablation to 0.04+/-0.02 after HIFU ablation (t = 25.32, P less than 0.01). 1H-MRS could provide information of metabolites changes of primary liver cancer after HIFU ablation and could be used as a complementary sequence to other magnetic resonance sequences to evaluate all around primary liver cancer after HIFU ablation.