AIM To observe the change of Ca 2+ /calmodulin dependent protein kinaseⅡ (CaMKⅡ) signal pathway in opioids dependent NG108-15 cells and the relationships between Ca 2+ /calmodulin dependent protein kinaseⅡ (CaMKⅡ) signal pathway and cAMP accumulation. METHODS NG108-15 cells were used as an in vitro model system. Competitive protein binding assay and radioimmunoassay were used to examine the intracellular cAMP accumulation. Calmodulin activity was assayed by PDE method. CaMKⅡ activity was assayed by ?- 32 P incorporation of syntide-2. mRNA expression of mDOR was measured by RT-PCR. RESULTS DPDPE long-term treatment also increased cAMP accumulation, and resulted in opioids dependence in NG108-15 cells; DPDPE long-term treatment increased calmodulin activity and CaMKⅡ activity in NG108-15 cells. Specific calmodulin antagonist W-7 was found to inhibit significantly the elevation of calmodulin and CaMKⅡ activity which resulted from DPDPE long-term treatment, and CaMKⅡ inhibitor KN-62 also inhibited elevation of CaMKⅡ activity by DPDPE long-term treatment. DPDPE long-term treatment increased cAMP accumulation, when W-7 or KN-62 was added at the same time, cAMP accumulation decreased. When naloxone was added to NG108-15 cells which were long-term treated by DPDPE, calmodulin and CaMKⅡactivity increased more. mRNA level of mDOR did not change significantly in opioids dependent NG108-15 cells. CONCLUSION Ca 2+ /CaMKⅡ signal pathway was involved in the mechanisms of opioids dependence through regulating cAMP level when DPDPE is long-term administered to NG108-15 cells.

Objective To investigate the clinical curative effect of oral and acupoint application of traditional chinese medicine combined with Azithromycin for the children with Mycoplasma Pneumonia. Methods A total of 160 children with mycoplasma pneumonia were divided into two groups according to digital random table method, with 80 cases in each group. The patients of the two groups were given conventional treatment to relieve cough and reduce phlegm; On the basic treatment of conventional treatment, the control group were treated with azithromycin, wile the treatment group were treated with oral traditional chinese medicine Tinghuang runfei decotion and acupointion application of Tinglizi, Baijiezi, Shengnanxing, Dahuang, and Bingpian. The treatment of both groups last 3 weeks. The time of defervescence, the disappearance time of cough and pulmonaryrales of the two groups were compared, and the serum levels of inter leukin 8 (IL-8), tumor necrosis factor-a (TNF-a), thrombin regulatory proteins (TM), D-dimer were compared before and after treatment. The total effect rate and the incidence of adverse events were compared.Results The defervesce time (3.79 ± 1.68 dvs. 4.88 ± 1.61 d,t=3.846), disappearance time of cough (8.21 ± 2.42 dvs. 10.35 ± 2.60 d,t=5.389) and disappearance time of pulmonary rales (6.21 ± 1.89 dvs. 7.78 ± 2.08 d,t=4.997) in the treatment group were significantly less than those in the control group (P<0.01). The levels of IL-8 (9.98 ± 4.26 ng/Lvs.14.18 ± 4.82 ng/L, t=5.840), TNF-a (20.78 ± 5.93ng/Lvs. 26.07 ± 6.42 ng/L,t=5.414), TM (9.63 ±  2.88μg/Lvs. 13.08 ± 3.37μg/L,t=6.961), D-dimer levels (0.09 ± 0.04 ng/Lvs. 0.15 ± 0.06 ng/L,t=7.442) the treatment group were significantly better than those in the control group (P<0.01). The total effect rate in the treatment gruop was significantly higher than that of the control group (97.5%vs. 88.8%;χ2=4.783,P=0.029), but there was no significant difference in the incidence of adverse events between the two groups (16.3%vs. 10.0%;χ2=1.370,P=0.272).Conclusions Oral and acupointion of traditional chinese medicine Tinghuang runfei decotion combined with azithromycin could relieve the symptoms, improve the clincal effect and show its safety for the children with mycoplasma pneumonia.

As for the ? opioid receptor, there are some disputes about the domains involved in the selective recognition of ligands, the relative spatial orientation of the amino acids within the TM affect the affinities of selective ligands. Regulation of opioid receptor activities does not appear to involve in their ability to promote the association of GTP onto the G proteins and the subsequent dissociation of heterotrimers. It is not very clear if phosphorylation correlates with agonist induced receptor desensitization. The celluar processing of ? opioid receptors requires the formation of multiple protein complexes, it is clear that interactions of ubiquitous transcriptional factors determine gene transcription.

BACKGROUND:Generaly, the stent surface modification, especialy seeding cels, may accelerate or cause stent endothelium, and cause restenosis for prevention of in-stent thrombosis.
OBJECTIVE: To develop the optimal conditions for vascular stents coated with bone marrow mesenchymal stem cels.
METHODS: Cerebrovascular stent was co-cultured with passage 3 bone marrow mesenchymal stem cels from rats at 1×106, 1×107, 1×108, and 1×109/L. Cels on the stents were examined with transmission electron
microscopy after 48 hours. A total of 160 male Sprague-Dawley rats were enroled, among which, 20 rats were as normal control group, and the remaining 140 were used for producing models of ischemic stroke that were
randomly sub-divided into seven groups at 8 weeks after modeling: stainless steel stent implanted group, polymer stent group, and different concentrations of cellstent composite groups. After 8 weeks of implantation, the
expression of vascular endothelial growth factor in these cels was examined by western blot assay. Rat platelet activation in different groups was determined by flow cytometry.
RESULTS AND CONCLUSION:Implanted stem cels were able to grow adherently on the stainless steel stent wal. When the planting cellconcentration was 1×107 cels/L, the cels and organeles were morphologicaly
normal and covered the stent surface wel. These coated cels also expressed vascular endothelial growth factor, suggesting that they functioned as endothelial cels, and they also significantly lowered platelet activation. When
co-cultured with 1×107/L bone marrow mesenchymal stem cels, the stent was covered wel with endothelial-like cels and had significant lower platelet activationin vivo.

Objective To develop a new designed Kirschner wires guider for patellar fracture,and to evaluate its clinic outcomes.Methods From October 2010 to November 2015,totally 72 patients with patellar fracture were detected for this study.Divided them into 3 groups, named as the second-generation guider group,the first-generation guider group and the control group.And then evaluated the clinic outcomes in treatment of patellar fracture of the 3 groups.Results The surgery time,accuracy of placement needle position of the second-generation guider group were better than the other two groups with significant statistical differences (P <0.01).And the clinic outcomes of the first-generation guider group were better than the control group,and the differences were statistically significant (P <0.05).Conclusion The second-generation Kirschner wires guider is reasonable in design and convenient for use.Moreover,it can improve the accuracy and quality of surgery,reduce the operation time,and enhance the clinical effects.

Objective To explore an effective way for medical students and young surgeons learning operative approach .Methods A total of 50 medical students and young surgeons in our hospital were detected for this study .They were randomly divided into the BOOK class (group A) and the 3D computer class (group B) to learn the course of anthropotomy.After learning each section, they were examined to-gether on fresh corpse , and the examined results of the two groups were compared and assessed .Results In the general surgery examina-tions, there was no difference in the examined results with no statistically significant difference (P>0.05).But in the orthopedics examina-tions, the difference of intra-group comparison both in group A and group B were of statistical significance (P<0.05), and the results of group B were better than group A with statistically significant difference (P<0.05).Conclusion For the general surgical approach learn-ing, both of the two methods could achieve good effect .For the orthopedics surgical approach learning ,it can receive better results through computer software ,but it still cannot achieve the ideal effect ,which indicates that the teaching through fresh corpse could not be replaced yet .

Objective To test the biomechanical properties and evaluate the feasibility of the new developed external fixator system,so as to provide reliable theoretical basis for further clinical research.Methods The tibia of 10 fresh male cadaver were made into fracture model.In order to fix the fracture of tibia,experimental group were treated with the new developed external fixator system and the control group were treated with the Hoffman 2 external fixator which was used in clinic commonly.Then axial compression and horizontal torsion test were made on tibia fracture model.The test results were recorded and statistically analyzed.Results There was no statistical difference on the aspect of the ability to resist axial compression and horizontal torsion between the experimental group and the control group(P >0.05). The new developed external fixator system could meet the criterion in clinical application.Conclusion The new developed external fixation system can meet the requirement of biomechanics on fixing the fracture of bone,and it can greatly simplify and diversify the clinical applica-tion of external fixation device.

Objective To develop a newly designed guide and evaluate its clinic application prospect in treatment of patella fracture. Methods In our mechanical experiment, we carried out the tensile test of the two generation drilling guides by biomechanics machine and then analyzed the difference in their deformation. In the corpse experiment , we transversed fractures at the equator of the patella obtained from 10 fresh cadaver speci-mens. They were divided into 2 groups randomly: a test group and a control group. The second-generation of the aiming guide was used for the test group but not for the control group during the experiment. Then we evaluated its clinic application in the treatment of patella fracture. Results In the mechanical experiment, the displace-ment of the first generation guide was significantly larger than that of the newly one. The accuracy in the place-ment of k-wires by the second generation guide was significantly higher than by the first generation guide (P <0.05). Conclusions The drilling guide is reasonable in design and convenient in use. Moreover, it can improve the accuracy and quality of insertion of tension-band wiring of patellar fractures. The second-generation one is better and thus it is worth clinically spreading.

Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR),and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2).Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study.The PDR patients were divided into non-injection group (30 patients,32 eyes) and injection group (27 patients,28 eyes).The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery.The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy.Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls.The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression ofNotch1,Dll4 and VEGFR2.In the meantime,the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted.Results The immunohistochemical staining revealed that there were positive expression ofNotch1,Dll4 and VEGFR2 in all PDR membranes,regardless of the injection of the ranibizumab.The levels ofNotch1,Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45,6.01,4.08;P=0.030,0.008,0.023).In injection group,the number of endothelial cells in the membranes reduced (17.17 ± 2.48) compared with that of the non-injection group (41.50± 5.57).There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05).RT-PCR showed that the differences of the mRNA expression ofNotch1,Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50,12.50,12.02;P＜0.05).The expression ofNotch1,Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients.In the PDR group,the expression of Notch1,Dll4 and VEGFR2 of non-injection group was higher than that of injection group.Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83),VEGFR2 and Notch1 (r=0.81),Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05).Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group,and it is positively correlated with the expression of the VEGFR2.Notchl and Dll4 play a regulatory rule in the neovascularization in PDR,the acting way may be correlated with VEGFR2.

AIM: To quantify magnesium isoglycyrrhizinate(MGL) and glycyrrhetic acid in the plasma of dog by develop a simple, rapid, sensitive high-performance liquid chromatography (HPLC-UV) method. METHODS: HPLC-UV methods with wavelength 252 nm were used for the quantitation of MGL and GA in plasma. The concentration of MGL and GA were assayed on a Kromasil ODS-1 C18 column with the column temperature 25 ℃. The mobile phase was a gradient system with 0.1 % diethylamine in water (pH 4.60 ) and acetonitrile at a flow rate of 1.0 ml?min~ -1 . RESULTS: There was a good linear response range of 0.2 -2.5 mg?L~ -1 and 2.5 -100 mg?L~ -1 , while the limit of quantification was 0.2 mg?ml~ -1 . The relative recovery rate of MGL was 94.3 %-101.9 %,GA 96.4 %-101.9 % (n=5);the absolute recovery rate was MGL 78.7 %-87.0 %, GA 77.5 %-87.7 % (n=5). The intra-day and inter-day variations were all less than 15% (n=5). The plasma samples preserved in refrigerator is stable at -20 ℃ for 14 days, freeze thawing 3 times and at room temperature for 10 h without degradation. CONCLUSION: This method is shown to be specific, sensitive, reliable and suitable for the quantitative determination of magnesium isoglycyrrhizinate following oral administration in biological sample.