Objective:To construct an adenovirus-mediated anti-sense RNA targeting K-ras exon 1 of SW1990 cell line and observe its effect on ceil proliferation and apoptosis after transferred into SW1990 cell line.Methods:K-ras exon 1 cDNA was cloned into shuttle vector pShuttle-CMV and the resultant plasmid was confirmed by enzyme digestion and PCR.Clones with inverted insertion were selected and co-transferred into E.coli BJ5183 with an adenoviral backbone plasmid pAdEasy-1 to produce recombinant plasmid by homologous recombination.Recombinants were then selected and transfected into 293 cell line to produce recombinant adenovirus.Recombinant adenovirus production was confirmed by PCR analysis and was amplified and purified;the virus titer was determined.Ad-LacZ was used to infect SW1990 cells and the infection efficiency was observed by X-gal staining.SW1990 cells was infected with the recombinant adenovirus and their proliferation and apoptosis were determined by MTT and annexin V/PI FCM assay.Results:A 282 bp target gene fragment was acquired by PCR;the titer of recombinant adenovirus was 7.6?10~8 pfu/ml before purification by CsCl_2 gradient centrifugation and 5.0?10~(10)pfu/ml after CsCl_2 gradient centrifugation.When the recombinant adenovirus was at 100 MOI,the infection efficiency of SW1990 cells nearly reached 100%.The transfection of recombinant adenovirus significantly inhibited SW1990 cell proliferation(P

Objective To discuss the influence of different doses budesonide aerosol inhalation on serum cytokines levels in children with severe asthma.Methods 96 children with severe asthma aged 4 to 14 years old in our hospital were chosen,and they were randomly divided into the observation group and the control group,48 cases in each group.All patients were given conventional treatment, and the control group was given 1mg/time budesonide treatment on the basis of conventional treatment (3 times a day),while the observation group was given 2mg/time budesonide treatment (3 times a day).Before and 1 week after treatment,the clinical symptoms of two groups were observed and compared,as well as the changes of IL-4,IFN-gamma,IL-10 and TNF-α.Results In the obser-vation group,wheezes,coughing,wheezy sound and rales disappearance time were (2.10 ±0.77)d,(5.45 ±1.20)d, (3.46 ±1.03)d,(5.55 ±1.35),which were significantly shorter than (2.98 ±1.02)d,(7.48 ±1.19)d,(5.43 ± 1.06)d,(7.56 ±1.67)d in the control group (t=4.77,8.32,9.23 and 8.32,all P<0.01).4 weeks after treat-ment,the total effective rate of the observation group was 89.6%,which was significantly higher than 72.9% of the control group (χ2 =4.376,P<0.05).After treatment,the IL-4,IL-10,TNF-alpha,IFN-gamma levels in the observation group were (4.06 ±1.77)pg/mL,(12.77 ±2.05)pg/mL,(4.15 ±1.11)ng/mL,(26.23 ±2.78)pg/mL, which had significant changes compared with (9.02 ±2.23)pg/mL,(10.21 ±1.30)ng/mL,(6.66 ±1.62)pg/mL, (17.33 ±2.31)pg/mL before treatment(t=12.07,24.56,16.20,17.25,all P<0.01).After treatment,the IL-4, IL-10,TNF-alpha,IFN-gamma levels in the control group were (9.11 ±2.05)pg/mL,(6.80 ±1.23)ng/mL, (9.88 ±2.20)pg/mL,(21.22 ±2.80)pg/mL,which had significant changes compared with (9.11 ±2.05)pg/mL, (10.38 ±1.37) ng/mL,(6.71 ±1.77) pg/mL,(17.30 ±2.05) pg/mL before treatment( t=5.36,13.47,7.77, 7.83,all P<0.01).But IL-4,TNF-alpha levels in the treatment group were significantly lower than those in the control group (t=7.32,11.08,all P<0.01),while IL-10 and IFN-gamma levels were significantly higher than those in the control group (t=6.65,8.80,all P<0.01).The incidence of adverse reactions of the two groups had no statistically significant difference (χ2 =0.771,P>0.771).Conclusion High doses of budesonide aerosol inhalation in the treatment of children with severe asthma has obvious clinical curative effects,which could significantly improve the patients'clinical symptoms,and also has low incidence of adverse reactions,which is worthy of clinical promotion.

Animals ; Epithelial-Mesenchymal Transition ; Fibrosis ; Kidney ; pathology ; Kidney Tubules ; cytology ; Male ; Rats ; Rats, Sprague-Dawley ; Ureteral Obstruction ; pathology

Objective To evaluate the efficacy of tibial bone-skin flap grafts in the management of se- vere traumatic osteomyelitis complicated with bone and skin defect in leg to avoid amputation.Methods From March 1998 to Aug.2004,12 cases of the traumatic osteomyelitis complicated with bone and skin defect in leg were treated with vascularized tibial bone-skin flap graft.The longest flap was 17cm,widethest 10cm, The longest bone flap was 12cm.They were followed up for 0.6 to 5 years.Results All the tibial bone-skin flaps survived completely,2 cases of osteomyelitis recurred.The followed-up,from 0.5 to five years,showed good bone union in all cases,averageing 15 weeks.The infection was under control.The leg function and con- tour were satisfactory.Conclusion The tibial bone-skin flap has the advantages of having distinguished sign of anatomy,highly vascularized,easy to obtain,simply and flexible procedure,improving circulation,short- ens hospitalization and suitable for treatment of traumatic osteomyelitis complicated with bone and skin defect in leg.

Objective To modify the methods of operation and establishment of cerebral ischemia rat models made by thread occlusion. Method We randomly divided 120 male SD rats into a common group (n = 50), an improvement group (n = 60) and a sham-operated group (n - 10). Rats in the common and improvement groups were made into models using the common and improvement methods separately. All models were evaluated on the basis of physical sign indices and 2,3,5-triphenyltetrazolium chloride (TTC) staining. The TTC staining results were taken as gold standards. Then, we compared the achievement ratios of the two groups, and computed the sensitivity and specificity of every physical sign index based on these standards. The χ~2 or correction χ~2 test was used to compare the ratios. Results (1) The achievement ratio in the improvement group was significantly higher than that in the common group (71.67% vs. 52.00%, P = 0.034). (2) The sensitivity of evaluation for both common and improvement methods was 98.55%. However, the specificity of evaluation for the improvement method was significantly higher than that for the common method (100.00% vs. 40.00%, P =0.000). Conclusions The establishment achievement ratio and evaluation correctness of models are obviously elevated by modification of the operation and establishment methods.

Objective To evaluate the effects of the range and the frequency of the compression load on the accuracy for discerning target stiffness differences in ultrasound elastography.Methods Quantitative ultrasound elastography was achieved by integrating two compression force sensors,a laptop computer and a clinical ultrasound elastographic system.The force sensors and the ultrasound probe were assembled in a 3D printed mounting bracket for continuous monitoring of compression loads during ultrasound elastography. Both the force measurements and the elastographic maps were acquired and displayed on the laptop computer in real time.Four targets of the same diameter(10.4 mm),the same depth (3 cm) and different stiffness levels (8,14,45 and 80 kPa) were examined by a HITACHI preirus,L74M linear-array transducer.Each target was evaluated 45 times with two different method(i.e.,freehand elastography and quantitative elastography),yielding 1 80 evaluations.The data were divided into the following three groups:group Ⅰ(80 kPa vs 45,14 and 8 kPa),group Ⅱ(80,45kPa vs 14,8 kPa)and group Ⅲ(80,45 and 14 kPa vs 8 kPa).Area under ROC curves(AUC)were calculated for different stiffness levels.Results In group Ⅲ, quantitative elastography yielded an greater AUC level than that of freehand elastography(P =0.0379).In group Ⅰ and group Ⅱ,two methods yielded the similar AUC levels (P = 1 .000).However,quantitative elastography was able to discern 8 kPa and 14 kPa targets (P <0.001),while freehand elastography was hard to differentiate them(P =0.258).Conclusions In comparison with freehand elastography,quantitative ultrasound elastography is able to improve the accuracy for discerning different target stiffnesses.

OBJECTIVETo explore the effect of combination therapy of tetramethylpyrazine (TMP) with methotrexate (MTX) on collagen induced arthritis (CIA) rats.

METHODSTotally 55 male SD rats were stratified by body weight. Nine of them were randomly recruited as the normal control group. The rest 46 were immunized with type II bovine collagen (C II) for establishing rheumatoid arthritis (RA) model. Forty successfully modeled rats were randomly divided into 4 groups according to swollen toe degree, i.e., the CIA group, the TMP group, the MTX group, and the TMP plus MTX group, 10 in each group. Rats in the MTX group were administered with MTX (1. 2 mg/kg) , once per week for 4 continuous weeks. Those in the TMP group were administered with 40 mg/kg TMP, once per day for 10 continuous days, and then discontinued for 7 successive days, and continued for another 10 successive days. Rats in the TMP plus MTX group were administered with a mixture of equal dose MTX and TMP, and when MTX was discontinue, TMP was administered according to the way in the TMP group. Equal volume of saline solution was given to rats in the normal control group and the CIA group. Clinical parameters including ankle width (mediolateral diameter) and hindpaw swelling were measured at day 0, 4, 11, 18, and 26 after treatment. Rats were sacrificed 28 days after treatment, their knee joints and ankle joints were collected for pathological analyses. Serum levels of IL-1β, IL-6, and IL-17A were detected by ELISA. Changes of fibrinogen (FIB) and platelet aggregation rate (PAg) were detected.

RESULTSCompared with the normal control group, the ankle width and hindpaw swelling increased significantly (P < 0.01), contents of FIB and PAg increased obviously (P < 0.05, P < 0.01), serum levels of IL-1β, IL-6, and IL-17 increased remarkably (P <0. 01) in the CIA group. Obvious cell proliferation, inflammatory cell infiltration, hyperemia and edema of synovial tissues could be seen. Pannus formed and immerged in cartilages, resulting in necrosis. Compared with the model group, changes of ankle width and hindpaw swelling were all alleviated in each medicated group (P <0. 05, P <0. 01). Of them, the effect was superior in the MTX group to that of the TMP group and the MTX plus TMP group (P < 0.05, P < 0.01). Contents of FIB, serum levels of IL-1β and IL-6 decreased significantly in the MTX group (P < 0.05). Contents of FIB, serum levels of IL-1β and IL-6 decreased significantly in the TMP group and the MTX plus TMP group (P < 0.05). Besides, serum levels of FIB and IL-6 were obviously lower in the MTX plus TMP group than in the TMP group and the MTX group (P < 0.01). Levels of PAg and IL-17A were more significantly lowered in the TMP group than in the MTX plus TMP group and the MTX group. Pathological changes could be alleviated in each medicated group, with the optimal effect obtained in the MTX plus TMP group.

CONCLUSIONCombination of TMP with MTX could significantly ameliorate inflammatory reactions and FIB contents of CIA rats.

Animals ; Arthritis, Experimental ; Arthritis, Rheumatoid ; Cattle ; Collagen Type II ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Hemorheology ; Interleukin-17 ; Interleukin-1beta ; Interleukin-6 ; Male ; Methotrexate ; therapeutic use ; Pyrazines ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane

Aim To establish a co-incubation system in cardiac fibroblasts of SD neonatal rats and spleen CD4+ CD25 + regulatory T lymphocytes (Tregs) of normal adult SD rats,and to investigate the effects of eplerenone(EPL) on the interaction of two cells and the relationship with the Kvl.3 channel on Tregs cell membrane.Methods The spleen Tregs of normal adult SD rats were sorted by immunomagnetic bead sorting,and the myocardial fibroblasts of SD rats were isolated by differential adherence method.The experiment was conducted in the following groups:CFs,CFs + Tregs,CFs + Tregs + EPL,Tregs.The proliferation of CFs was detected by CCK-8 method.The expression levels of type Ⅰ collagen,type m collagen and matrix metalloproteinase 2 (MMP-2) secreted by CFs were detected by ELISA.The mRNA expression levels of Kv1.3,KCa3.1 on Tregs cell membrane and intracellular CRAC channel were detected by RT-qPCR technique.Tregs cell membrane Kvl.3 channel protein expression levels were determined by In-Cell Western blot.Results After 48 h incubation of the co-culture system,the cell proliferation was stable.CFs proliferation was marked(P ＜0.01),which could be inhibited by EPL(P ＜0.01).The type Ⅰ,type Ⅲ collagen and MMP-2 secreted by CFs increased (P ＜ 0.01).The expression levels of Kv1.3,KCa3.1 and CRAC channel mRNA in Tregs increased by 6.95,1.99 and 1.53 fold (CFs + Tregs vs Tregs,P ＜0.01),EPL decreased the mRNA level of each channel (CFs +Tregs + EPLvs CFs + Tregs,P＜0.01),and the decrease of Kv1.3 channel was the most significant (P ＜ 0.01).The Kv1.3 channel protein of Tregs increased by 67.9％ (CFs + Tregs vs Tregs,P ＜0.01),which could be inhibited by EPL(P ＜ 0.01).Conclusions Tregs cultured with CFs after 48 h can significantly promote the proliferation of CFs,and EPL can down-regulate the Kv1.3 channel expression on the Tregs membrane and inhibit the activation/proliferation of Tregs,indirectly inhibiting myocardial fibrosis.

OBJECTIVETo provide molecular evidences for its breeding by studying the genetic relationship among varieties of Rehmannia glutinosa.

METHODNineteen varieties were detected by Randomly Amplified Polymorphic DNA(RAPD) markers.

RESULTThe 20 selected primers produced 163 bands, among which 114(69.9%) were polymorphic. A DNA molecular dendrogram was established based on Hierarchical cluster analysis of 163 DNA bands amplified by 20 primers, which divided the 19 varieties into four groups: Group Beijing, Group 85-5, Group Guolimao and the other Group.

CONCLUSION8 varieties of Group Beijing have a close genetic relationship, and so have varieties of Group 85-5, which provides information for Rehmannia glutinosa's breeding.

DNA, Plant ; genetics ; Plant Leaves ; genetics ; Plants, Medicinal ; genetics ; Polymorphism, Genetic ; Random Amplified Polymorphic DNA Technique ; Rehmannia ; genetics

OBJECTIVEIn this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB) in modulating adhesion molecules expressions.

METHODSNeutrophils and BEAS-2B cells (one human bronchial epithelial cell line) were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction (real-time qPCR). Phosphorylated p38 MAPK and inhibitor κB were analyzed by Western blot.

RESULTSIn co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly (P<0.05). Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor κB in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin (P<0.05).

CONCLUSIONSCoculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF-κB pathways, and exhibiting its anti-inflflammation activity.

Animals ; Base Sequence ; Bronchi ; cytology ; enzymology ; metabolism ; Cattle ; Cell Adhesion Molecules ; metabolism ; Cell Line ; Coculture Techniques ; DNA Primers ; Down-Regulation ; drug effects ; Epithelial Cells ; enzymology ; metabolism ; Isoflavones ; pharmacology ; NF-kappa B ; metabolism ; Neutrophils ; enzymology ; metabolism ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; p38 Mitogen-Activated Protein Kinases ; metabolism