Objective To construct expression vectors that Renilla reniformis (Rluc) fused with neurotensin type 1 receptor (NTSR1),and to investigate the interaction between NTSR1 and other receptors,as well as intercellular signal transduction mechanism mediated by neurotensinl-R.Methods The human NTSR1 gene was amplified by PCR using the plasmid pcDNA3.1-hNTSR1 as template.The PCR product was digested,ligased with the plasmid pRluc and then be transformed into the competent cell Top10.The construct was identified by DNA sequencing.The recombinant plasmid was transiently transfected into human embryonic kidney 293 ( HEK293 )cells,and the expression of pRluc-hNTSR1-pcDNA3.1 was detected by confocal microscopy and Western blot.Results The fragment of 1257 bp was amplified by PCR,and the DNA sequences were identical with the gene in GenBank ( NM_002531 ).Western blot showed a band about 90kDa.Confocal microscopy showed that NTSR1 was expressed on the plasma membrane.Conclusion The pRluc-hNTSR1-pcDNA3.1 eukaryotic expression vector is successfully constructed,and the expression vector can be used to investigate the interaction between NTSR1 and other receptors,as well as intercellular signal transduction mechanism mediated by neurotensinl-R,which will provide new target for drug development.

OBJECTIVE: To analyze the use situation and trend of cardiovascular and cerebrovascular drugs during 2007～2009 in a military sanatorium, and provide reference for the rational use of cardiovascular and cerebrovascular drugs and its essential medicines. METHODS: Cardiovascular and cerebrovascular drugs in the rehabilitation center during 2007～2009 were analyzed statistically in the respect of category, consumption sum and DDDs. RESULTS: The consumption sum and DDDs of cardiovascular and cerebrovascular drugs were increasing in the past three years. And its essential drugs account for 94.8%.The most widely used drugs were cerebrovascular drug, brain metabolism drug, nootropics and antihypertensive drugs. Oxiracetam and Xueshuantong were widely used cerebrovascular drug, brain metabolism drug and nootropics. Amlodipine were widely used antihypertensive drugs in the clinic. CONCLUSION: Cardiovascular and cerebrovascular drugs and its essential drugs were characterized with complex classification, numerous categories, extensive application and potential market demand, which require rational use of drug should be strengthened.

Objective: To evaluate the mechanical properties of Rene95 alloy component made by laser engineered net shaping (LENS). Methods: The component was fabricated from Rene95 alloy powder by LENS, then the mechanical properties of the component, Rene95 alloy casting and dental Ni-Cr alloy casting were studied. Results: The tensile strength(MPa), yield strength(MPa), elongations(%) and the microhardness(Hv) of Rene 95 alloy component made by LENS were 1 354.27?63.44, 1 175.29?34.32, 16.53?5.85, and 464.33?10.95, those of Rene95 alloy casting 1 430.28?55.73, 1 145.52?29.78, 10.31?4.25 and 457.56? 11.38, those of dental Ni-Cr alloy casting 612.05?18.46, 426.06?12.70, 6.21?1.21 and 260.14? 6.08, respectively.Conclusion: The mechanical properties of Rene95 alloy component made by LENS were same as those of Rene95 alloy casting, better than dental Ni-Cr alloy casting.

Objective:To evaluate the anti-corrosion property of Ti-Zr alloy fabricated by laser solid forming technic in artificial saliva.Method:The corrosive behavior of Ti-Zr alloy fabricated by laser solid forming was studied by potentiadynamic polarization technique in artificial saliva at 37 ℃(pH=6.8).Ti-Zr alloy formed by casting was applied as the control.With the polarization curves given by the electrochemical corrosive measure system and the picture of corrosive plane given by SEM, the corrosive property of the Ti-Zr alloy were analyzed.Results:Ti-Zr alloy fabricated by laser solid forming showed more relatively positive corrosion potential, smaller corrosion current density and lower passive potential, and did not show any evidence of corrosive phenomena.Conclusion:Ti-Zr alloy fabricated by laser solid forming has good corrosive resistance in oral circumstances.

objective: To study the effects of laser engineered net shaping(LENS) in the manufacture of dental prosthesis. Methods: An experiment LENS system, including a 5 kW continuous wave CO 2 laser, a four-axes numerical control working table and powder feeding system, was built up, by which, a rectangular LENS component was made from Rene95 alloy powder. The microstructure of the component was observed with scanning electron microscope and ESA analysis. Results: The size of the component was 120 mm?15 mm?15 mm , and the wall of the component was perpendicular to the base. The microstructure of the component was made up of fine columnar dendrite with the length of 10～20 ?m, 10 times less than that of the cast made from the same material. The main elements of the powder distributed homogeneously,the negative effect of segregation could be limited and the properties of the component could be improved.Conclusion: The microstructure of components made by LENS is much better than that of those made by casting from the same material.

Objective To construct the expression vector of early growth response-1 promoter-macrophage inflammatory protein 3? (pEgr-1-MIP-3?, pEM) to study MIP-3? expression in transfected Lewis lung cancer cells. Methods MIP-3? cDNA was inserted into plasmid with Egr-1 promoter by directional cloning. The vectors were transfected into Lewis lung cancer cells with liposomes. The expression of MIP-3? in transfected Lewis lung cancer cells induced by electron ray irradiation at different doses were confirmed by RT-PCR and Western blotting. Results The recombinant vector was sequenced, and the result indicated the correct insertion in the expression vector. After electron ray irradiation at different doses, MIP-3? expression in the transfected Lewis lung cancer cells increased significantly. Conclusion Electron ray can enhance the expression of MIP-3? in Lewis lung cancer cells. This has laid the experimental foundation for the studies of pEM for radio-genetic therapy in vivo.

Aim To investigate the roles of intracellu-lar reactive oxygen species ( ROS ) and Nrf2 pathway in shikonin-induced A549 cell apoptosis. Methods The cytotoxicity was analyzed by MTT assay. The ap-optosis of A549 cells was analyzed by both cellular morphological and biochemical methods. The relative changes of the redox marks ( ROS/GSH) were studied by fluorescence assay in the shikonin-treated A549 cells in accompany with the changes of the intracellular redox homeostasis by GSH/GSSG ratio. ROS inhibitor was also employed in the treatment to find the role of ROS in shikonin-induced A549 cell apoptosis. Real-time PCR analysis and ELISA assay were performed as well to determine the role of Nrf2 pathway in the shiko-nin-induced A549 cell apoptosis. Results The IC50 of shikonin on A549 cells was 3. 2 mg·L-1 . The cellu-lar redox homeostasis shifted toward oxidation signifi-cantly in shikonin treatment in a time-dependent man-ner. The expression of the Nrf2 pathway related genes was up-regulated by shikonin ( 3 . 2 mg · L-1 , 8 h ) . The expression of the anti-apoptotic genes was down-regulated , and proapoptotic genes were up-regulated by shikonin (3. 2 mg·L-1, 24h). Futhermore, the inhi-bition of intracellular ROS alleviated the cytotoxicity of shikonin in A549 cells. Conclusion The critical role of shikonin-induced redox imblance in A549 cell, coped with the secondary produced ROS and Nrf2 path-way antioxidants, result in A549 cell apoptosis.

G-protein-coupled receptor kinases (GRKs) are a family of serine/threonine protein kinases. The investigators pay much attention to the roles of GRKs in the signal transduction through G-protein-coupled receptors (GPCRs) with arrestin ever since a long time ago. Due to the physiological and pathological observations with the methods of deletion or overexpression, GRKs are considered as new drug targets. The kinases play a role in the pathogenesis of hypertension and cell migration through GPCRs and Hedgehog signaling pathways. As the development of research techniques, especially bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), the special mechanism of GRKs for GPCRs is more evident. In this review, we discuss the recent achievement in the roles of GRKs signaling and the related newest research techniques.

BACKGROUND: Apelin/APJ system has a wide range of physiological functions,but its intracellular signal transduction,in particular,apelin receptor desensitization,internalization,resensitization degradation,have still no consistent opinion.OBJECTIVE: To construct eukaryotic expression vector expressing human apelin receptor(APJ)tagged to red fluorescent protein(pDsRED-express-C1),and to determine the expression in human embryo kidney 293 cells.METHODS: The plasmid pcDNA3.1-hAPJ was used as a template for PCR amplification of human APJ.Following PCR amplification the PCR product were removed and enzymatic digestion with EcoR I and BamH I.Same enzymes were used to cut vector pDsRED-express-C1.The digestive product was ligated by conventional methods of connection,then transfected into Competent E.coli TOP10.Single clones were picked plasmid extraction,followed by restriction enzyme digestion and finally DNA sequencing.The recombinant plasmid with correct sequencing was transfected into human embryonic kidney cells,PI staining,followed by the observation under a confocal microscope.RESULTS AND CONCLUSION: PCR amplified a 1.2-kb fragment,which was consistent with the expected size of the human APJ.The pDsRed-hAPJ recombinant plasmid was cut into two fragments,one corresponded to the pDsRED-express-C1 vector size,and the other fragment corresponded to APJ target fragment.Confocal microscopy analysis showed that,APJ was expressed mainly in the membrane of human embryo kidney 293 cells.The pDeRed-hAPJ eukaryotic plasmid expression vector was successfully constructed and effective expression of this fusion protein is achieved,which might be instrumental in the study of displacement and intracellular localization of human APJ.

Objective To investigate the influence of acpuncture on free calcium in rat brain cells after focal cerebral ischemia reperfusion.Methods 145 adult male SD rats were randomly divided into control group,simple ischemia reperfusion group and acupuncture with ischemia reperfusion group.The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established by the modified Longa occlusion method. ①The part of free calcium in rat brain cells,focal cevebral ischemia model of rats were made by thread locking up the blood vessel for 15 min.30 min later after reperfusion, the Baihui and Shuigou Point in Du meridian were acupunctured electrically 30 min.After 3h, 6h and 12h, the rat was killed and its brain cells were made into single cell suspension,marked by Fluo-3/AM.The fluorescence optical density was recorded by laser confocal microscopy.②The part of nerve functional reconstruction, focal cevebral ischemia model of rats were made by thread locking up the blood vessel for 12 hours.30 min later after reperfusion, the Baihui and Shuigou Point in Du meridian were acupunctured electrically 30 min.After 7 d, 14 d,30 d,60 d and 90 d, the rat was forced to detect it's strength of the dog.Results ①Free calcium in rats of acupuncture therapy group(6h:10.96±1.18;2h:20.9±4.37) was significantly less than that in control group in 6 h and 12 h after reperfusion (6 h: 16.87 ± 3.56,12 h: 34.10 ±1.06)(P<0.05).②The dog in rats of acupuncture therapy group was significantly more than that in control group in 7 d, 14 d after reperfusion (P＜ 0.05 ).No difference of the dog was detected in 30 d ,60 d and 90 d after reperfusion between the two groups.Conclusion Acupunture could decreases the concentration of free calcium and the expression of Caspase-3 mRNA in rat brain cells after focal cerebral ischemia reperfusion, and it can facilitate the recovery of nerve function.