Objective To investigate the effects of silencing Smo gene on proliferation and apoptosis of rat prima-ry chondrocyte in vitro.Methods The primary chondrocyte was obtained by mechanical-enzyme digestion and identified by Immunohistochemical cells ( ColⅡ) .The animals were divided into control group , control siRNA group and Smo siRNA 1 ~3 group.The siRNA was transfected into chondrocytes by lentivirus vector .After 72 h, the cell viability was detected by MTT, Smo expression was detected by RT-PCR and Western blot, and the apoptosis of chondrocyte was assessed by flow cytometry .Results All types of siRNA were transfected into primary chondrocyte by vectors, the Smo siRNA 1 ~3 may inhibit the expression of Smo mRNA and protein in chondrocytes, and Smo siRNA2 had the highest silencing rate ( the expressions of Smo mRNA and protein were 0.19 ±0.03 and 0.39 ±0.07 ) .The cell viability in Smo siRNA2 group was lowest ( 77.38% ±7.19%) , while the apoptosis rate of Smo siRNA2 was highest ( 21.43%±2.97%) .Conclusions Silencing Smo gene in primary chondrocytes may inhibit proliferation and promote apoptosis , Smo may have a protecting role from apop-tosis of the chondrocyte.

Objective To study the proliferation and apoptosis and investigate the expression of Indian hedgehog (Ihh),parathyroid hormone-related peptide (PTHrp),smoothened (Smo) protein and mRNA in the cultured rat primary chondrocytes exposed to different doses of NaF.Methods The third generation articular chondrocytes of neonate rat were cultured in vitro and treated with 0 (control),5,10,20 and 40 mg/L of fluoride.The proliferation activities of cells at different times (24,48 and 72 h) were tested by Thiazolyl Blue Tetrazolium Bromide (MTT).The apoptosis rate was determined by flow cytometry.The expressions of protein and mRNA of Ihh,Smo and PTHrp at 48 h were determined by Western blotting and semi-quantitative RT-PCR,respectively.Results After exposed to 5 mg/L of fluoride for 24,48 and 72 h,the proliferation rates were significantly increased [(1.17 ± 0.07)％,(1.20 ±0.06)％,(1.16 ± 0.08)％] compared with those of control group [(1.10 ± 0.08)％,(1.13 ± 0.08)％,(1.15 ± 0.08)％],but the proliferation activity at 48 and 72 h in 40 mg/L group [(0.72 ± 0.11)％,(0.68 ± 0.04)％] was significantly lower than those in control group (all P ＜ 0.05).Compared with the control group,apoptosis rate of cartilage cell in fluoride treatment group increased gradually [(1.47 ± 0.05)％,(19.87 ± 3.03)％,(25.30 ± 1.28)％,(45.73 ± 4.63)％,F =123.328,P ＜ 0.01].Western blot analysis and RT-PCR results showed that the Ihh,PTHrp,Smo mRNA and protein expression increased in the fluoride groups at 48 h (Ihh protein:0.77 ± 0.08 vs.0.98 ±-0.07,1.23 ± 0.06,1.37 ±0.07,1.34 ± 0.07;PTHrp protein:0.68 ± 0.04 vs.0.89 ± 0.05,0.83 ± 0.05,1.29 ± 0.05,1.16 ± 0.08;Smo protein:0.37 ± 0.01 vs.0.64 ± 0.06,0.67 ± 0.03,0.96 ± 0.06,0.69 ± 0.06;Ihh mRNA:0.77 ± 0.05 vs.0.98 ± 0.05,1.09 ±0.05,1.27 ± 0.03,1.46 ± 0.06;PTHrp mRNA:0.67 ± 0.07 vs.0.97 ± 0.05,1.07 ± 0.08,1.37 ± 0.05,1.45 ± 0.05;Smo mRNA:0.45 ± 0.03 vs.0.63 ±-0.04,0.71 ± 0.05,0.81 ± 0.01,1.00 ± 0.02,all P ＜ 0.05).Conclusions Low doses of fluoride can promote the proliferation of chondrocytes cultured in vitro,and high doses of fluoride can promote the apoptosis of chondrocytes cultured in vitro.The expression of Ihh signaling pathway RNAs and proteins of the cartilage cells are increased following increased levels of fluoride.The results suggest that fluorine has activated the Ihh signaling pathway in chondrocytes and promoted the proliferation and apoptosis processes which might be involved in chondrocytes injury.

Objective To observe changes in bone mass and symptom duration in patients with lumbar disc herniation(LDH) and explore the relationship between osteoporosis and clinical features of LDH. Methods 83 LDH patients undergoing surgery were enrolled in the study from November 2008 to September 2009. Before surgery, dual-energy X-ray absorptiometry was used to detect bone mineral density of lumbar spine and hip, and calculate T score. The patients were divided into three groups according to the T score: group A (normal bone mass group, n=27), group B (osteopenia group, n=31) and group C (osteoporosis group, n=25). The differences in the duration of symptoms and pathological types were compared between the groups. The relationship between BMI and lumbar spine T-score was explored. Results There were no significant differences in the pathological types among the three groups. The symptom duration in group C was significantly shorter than in group A (P < 0.05). There was no correlation between BMI and lumbar spine T-score (r=0.20, P=0.070). There was positive correlation between BMI and Hip T-score (r=0.263, P=0.016). Conclusion Osteoporosis may affect the symptom duration of LDH patients. We should attach great importance to patients with osteoporosis and LDH.

Objective To compare safety and feasibility using radial versus femoral access during cardiac catheterization of patients who had previously undergone coronary artery bypass graft ( CABG) surgery. Methods We retrospectively evaluated 116 consecutive patients who underwent graft intervention via the transradial (TRA group, n = 46) or transfemoral approach (TFA group, n = 70), and observed the baseline clinical characteristics, angiography characteristics and complications between the 2 groups. Results The baseline clinical characteristics between the 2 groups were similar ( all P > 0. 05) . No significant difference was observed in angiography characteristics and procedural parameters including operation time, radiation exposure and puncture time between the 2 groups (all P > 0. 05). There was no significant difference in major adverse cardiac events during hospitalization. PCI to graft vessels were all successful and procedural success rates were similar between the 2 groups (P = 0. 669). Vascular access site complications were significantly lower ( P = 0. 03) in the TRA group. No access site complication was recorded in the TRA group. 7 cases (10. 0% ) with complications were recorded in the TFA group including 1 case of major bleeding (1. 4% ), 3 cases of minor bleeding (4. 3% ), 2 cases of local hematorna (2. 9% ) and 1 case of A-V fistula formation. Conclusions In contrast to the transfemoral route, the rate of major vascular complications was negligible using the transradial approach.

Objective To explore the feasibility of one-stop examination with Revolution CT for axial perfusion of normal pancreas.Methods Thirteen patients who received axial perfusion scan by one-stop examination with Revolution CT were analyzed as perfusion group.Two radiologists measured pancreatic CT perfusion (CTP) parameters independently and selected optimal phase for CTA and three phases of enhanced images.The effect dose (ED) was calculated.Eighteen patients who underwent abdominal enhanced CT and CTA with spiral scan were included as control group.Patients in both groups had no pancreatic disorders.The interobserver variation of CTP parameters was estimated.Two independent radiologists separated the superior pancreaticoduodenal artery (SPDA) image into 5 points according to image quality,and the consistency was assessed.The subjective points of SPDA image quality of two groups was compared.CT value,images noise,CNR and SNR of SPDA on CTA images and those of pancreas on three phases enhanced scan images between two groups were compared.Results ICC values of all CTP parameters were higher than 0.75.The ED of perfusion protocol was (24.52±-0.01)mSv.The subjective image scores of SPDA on CTA images in both groups were both 5,the consistency was good (Kappa=0.629,0.769).The CT value,CNR,and SNR of SPDA on CTA images of CTP group were higher than those of control group (all P＜0.05).The CT value,CNR,and SNR of pancreas of CTP group were higher than those of control group in venous phase and balanced phase (all P＜0.05).Conclusion The pancreatic CT one-stop examination can be performed by Revolution CT scanner with maximum detector width with acceptable radiation dose,from which pancreatic CT perfusion data,enhanced images with high quality and better CTA images can be extracted.

Objective To explore the feasibility of axial whole-liver one-stop examination with Revolution CT.Methods Totally 19 patients were underwent upper-abdominal enhanced examination with Revolution CT and acquired whole-liver CT perfusion (CTP),vein phase and balanced phase enhanced images.Two observers recorded the rank and peak CT value corresponding to time-density curve (TDC) of abdominal aorta and portal vein respectively on the CTP images.The perfusion parameters including blood flow (BF),blood volume (BV),hepatic arterial fraction (HAF),mean transit time (MTT),time to peak (TP) of left and right liver lobe were measured.The images of hepatic artery CTA and portal vein CTV were reconstructed and the arterial phase enhanced images were extracted using the images corresponding to abdominal aorta and portal vein peak TDC.And the radiation dose of CT perfusion and one-stop examination were recorded.The differences between perfusion parameters of left and right liver lobe were compared and the consistency of two observers were analyzed.Results The differences between BV and MTT of left and right liver lobe were statistical significance (both P＜0.05).The subjective scores of hepatic artery CTA,portal vein CTV and arterial phase images were greater than 1 point.The two observers were in great consistence (Kappa＞0.6).The effective radiation dose in perfusion phase and one-stop examination were 14.47 mSv and 21.29 mSv.Conclusion With low radiation dose,Revolution CT axial wholeliver perfusion one-stop examination can provide multiple quantitative parameters of liver CTP and clear hepatic artery CTA,portal vein CTV and 3 phase enhanced scan images,which has broadly prospective in clinical application.

The global regulatory gene, nsdA, negatively regulates antibiotics production in Streptomyces coelicolor. Southern blot experiment, using an nsdA fragment of S. coelicolor as probe, indicated that nsdA gene existed in many Streptomyces. Primers were designed based on the published sequences of S. coelicolor and S. avermitilis. PCR amplification and sequencing showed that nsdA in Streptomyces was conservative and that of S. lividans ZX64 has a 100% identity in the nucleotide sequence comparing with that of S. coelicolor A3 (2). The nsdA disrupted mutant of S. lividans was constructed named as WQ2. WQ2 was able to produce actinorhodin but the wild-type strain ZX64 did not, which has a silent gene cluster contributing to the biosynthesis of actinorhodin. However, the ability was lost when another copy of the wild nsdA gene was introduced into WQ2. All the results above indicate that nsdA homologous gene is wildly existent and conserved in Streptomyces. And it plays a role in negatively regulating the actinorhodin synthesis in S. lividans and disruption of it can activate the silent gene cluster.
Anti-Bacterial Agents ; biosynthesis ; Blotting, Southern ; Genes, Bacterial ; physiology ; Genes, Regulator ; physiology ; Multigene Family ; Streptomyces lividans ; genetics

BACKGROUNDAsthma is a chronic inflammatory disease characterized by reversible bronchial constriction, pulmonary inflammation and airway remodeling. Current standard therapies for asthma provide symptomatic control, but fail to target the underlying disease pathology. Furthermore, no therapeutic agent is effective in preventing airway remodeling. A substantial amount of evidence suggests that statins have anti-inflammatory properties and immunomodulatory activity. In this study, we investigated the effect of rosuvastatin on airway inflammation and its inhibitory mechanism in mucus hypersecretion in a murine model of chronic asthma.

METHODSBALB/c mice were sensitized and challenged by ovalbumin to induce asthma. The recruitment of inflammatory cells into bronchoalveolar lavage fluid (BALF) and the lung tissues were measured by Diff-Quik staining and hematoxylin and eosin (H&E) staining. ELISA was used for measuring the levels of IL-4, IL-5, IL-13 and TNF-α in BALF. Periodic acid-Schiff (PAS) staining was used for mucus secretion. Gamma-aminobutyric acid type A receptor (GABAAR) β2 expression was measured by means of immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSRosuvastatin reduced the number of total inflammatory cells, lymphocytes, macrophages, neutrophils, and eosinophils recruited into BALF, the levels of IL-4, IL-5, IL-13 and TNF-α in BALF, along with the histological mucus index (HMI) and GABAAR β2 expression. Changes occurred in a dose-dependent manner.

CONCLUSIONSBased on its ability to reduce the inflammatory response and mucus hypersecretion by regulating GABAAR activity in a murine model of chronic asthma, rosuvastatin may be a useful therapeutic agent for treatment of asthma.

Animals ; Asthma ; drug therapy ; metabolism ; Chronic Disease ; Disease Models, Animal ; Female ; Fluorobenzenes ; pharmacology ; therapeutic use ; GABA-A Receptor Antagonists ; pharmacology ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Lung ; chemistry ; Mice ; Mice, Inbred BALB C ; Mucus ; secretion ; Pyrimidines ; pharmacology ; therapeutic use ; Receptors, GABA-A ; analysis ; Rosuvastatin Calcium ; Sulfonamides ; pharmacology ; therapeutic use

OBJECTIVETo investigate the changes in plasma levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) and in peripheral CD34(+) cells in patients with acute myocardial infarction (AMI), and explore their role in AMI.

METHODSEnzyme-linked immunoassay (ELISA) was employed for measuring the levels of VEGF and SDF-1 in AMI patients on days 1, 3, 7, 10, and 14 of onset and in normal control subjects. The absolute counts of CD34(+) in the peripheral blood were measured on days 1, 7, and 14 by flow cytometry in AMI patients, with their myocardial enzyme and troponin I detected and electrocardiography (ECG) and echocardiography (UCG) recorded.

RESULTSPeripheral CD34(+) cells obviously increased on day 7 after AMI onset (2.35-/+0.72/microl vs 1.48-/+0.49/micro, P<0.05). VEGF levels were significantly higher in AMI patients than in the control subjects, reaching the peak level and on day 14 (197.56-/+39.87 vs 53.79-/+18.12 pg/ml, P<0.01). SDF-1 level obviously decreased on day 1 after AMI onset (1683.12-/+224.79 vs 2178.67-/+265.34 pg/ml, P<0.01), followed by gradually increased to the control level. Obvious correlation was noted between the level of VEGF on day 7 and the peak level of peripheral CD34(+) cells, and the peak plasma VEGF level was obviously associated with the peak serum CK-MB and troponin I levels.

CONCLUSIONThe stem cells are mobilized into the peripheral blood in the event of AMI. Obviously increased VEGF level following AMI may persist for at least 2 weeks, whereas SDF-1 level undergoes temporary decrement after AMI. The dynamic changes of VEGF and SDF-1 can be related to the mobilization and homing of the stem cells to the injured myocardium.

Adult ; Antigens, CD34 ; blood ; Chemokine CXCL12 ; blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Humans ; Male ; Myocardial Infarction ; blood ; Time Factors ; Vascular Endothelial Growth Factor A ; blood

pHZ1080, an E. coli-Streptomyces shuttle expression vector was constructed in order to explore the utilization of lambda phage regulated expression elements in Streptomyces. A 2.7 kb polyketide synthase (PKS) gene from Streptomyces sp. FR-008 was inserted into downstream of lambda phage promoter (PR) to give the shuttle plasmid, pHZ1067. The PKS protein was expressed in Streptomyces lividans carrying pHZ1067 in a heat-dependent manner, as it did in E. coli. The PKS protein expressed in both hosts with same molecular weight was detected by SDS-PAGE and Western-blot. The successful heat-induced expression of PKS suggested that pHZ1080 was useful and convenient for heat-induced expression of heterologous genes in both E. coli and Streptomyces.
Amino Acid Sequence ; Base Sequence ; Blotting, Western ; Cloning, Molecular ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Multienzyme Complexes ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Streptomyces ; enzymology ; genetics ; Temperature