Objective To observe the influence of electroacupuncure(EA) at different therapeutic time windows on the effects of inosine on neuronal apoptosis and expression of heat-shock-proteins-70 (HSP70)after focal cerebral ischemic reperfusion injury (CIRI) in rats.Methods The model was established by ligation of the artery for 2 hour.EA was delivered to “baihui” and “dazhui” through acupuncture needles 0.5 hr,2 hr,6 hr and 12 hr respectively following MCAO.The expression level of HSP70 and the number of apoptotic cells were examined by immunohistochemical technique and TUNEL,comparing the average optical density of HSP70 and the number of apoptotic positive cells of cortex penumbra and hippocampus CA1 area in rat brain.Results Compared with the model group,the average optical density of HSP70 in every EA group was increased in apoptotic positive cells of cortex penumbra and hippocampus CA1 area 0.5 h (89.98± 6.55),(128.73 ± 8.03),2 h (90.96±6.38),(132.25±8.78),6 h (93.71±6.12),(132.58±7.04),12 (96.19±7.30),(133.57±6.19)and the number of apoptotic positive cells of cortex penumbra in every EA group was decreased 0.5 h (1.80±0.84),2 h (3.40± 1.14),6 h (5.00± 1.00),12 h (5.00±2.45).The number of apoptotic cells of hippocampus CA1 area was decreased just in the EA group of 0.5 h (1.60± 1.89).Conclusion EA could increase the expression level of HSP70 and at the same time decrease the number of apoptotic cells,EA plays an important part in protecting the neuronal cells of brain after focal cerebral ischemic reperfusion injury.The acupuncture treatment should be taken as far as early.

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.

BACKGROUND:Bone marrow derived mesenchymal stem cells (BMSCs) will be homing to the lesions after balloon injury in the inflammatory reaction process.However,the molecular mechanism of transforming growth factor-β1 (TGF-β1) to promote BMSC into smooth muscle-like cell remains unclear.OBJECTIVE:To investigate the BMSC differentiation rate under different TGF-β1 levels after acute vascular injury.DESIGN,TIME AND SETTING:A randomized controlled animal experiment and in vitro induced cell observation were performed at the SPF Laboratory Animal Center and Laboratory of Tissue Engineering,Ninth People's Hospital of Shanghai Jiao Tong University Medical School between January 2008 and May 2009.MATERIALS:A total of 24 male SD rats were randomly divided into normal control and model groups with 12 animals in each group.In addition,6 4-weak-old male SD rats were used to prepare BMSCs.METHODS:Model of acute carotid artery injury was established in model group.Serum of normal control group and model group after 1,3,7 days of injury to detect TGF-β1 level after the vascular injury by ABC-ELISA.The BMSCs after one passage were cultured at a density of (1.0-3.0)×10~5 cells/100 mm in culture dish,and divided into two groups:in routine culture group,cells were cultured in high-glucose DMEM containing 20% fetal bovine serum;in TGF-β1 group,cells were induced by different concentrations of TGF-β1 (1,3,5 and 10 μg/L) based on routine culture for 1 week.MAIN OUTCOME MEASURES:Serum TGF-β1 level after the vascular injury;,cell morphological changes by inverted phase contrast microscopy;smooth muscle α-actin expression.RESULTS:The normal serum TGF-β1 level in rat was low,but increased rapidly after 1 day of injury,reached its peak at 3 days,and declined gradually but did not return to the basic level till day 7.After 1 week of induction,BMSCs were confluent,forming peak valley appearance.Immunocytochemistry showed that compared with routine culture,the rate differentiation of smooth muscle-like cell was significantly increased in cells stimulated by TGF-β1,especially 5 and 10 μg/L TGF-β1 (P ＜ 0.01).Real-time quantitative PCR results were similar to immunocytochemistry.CONCLUSION:Serum TGF-β1 level increased after acute vascular injury and peaked at day 3.In vitro,a similar concentration of TGF-β1 could induce cultured BMSC to differentiate into smooth muscle-like cells.

Objective To analyze the corneal flap thickness in laser in situ keratomileusis(LASIK) using Moria M2 microkeratome and to identify the related factors. Methods Sixty patients with LASIK were divided into two groups: M2 90 group,using the Moria M2 90 microkeratome,n=30;M2 110 group,using the Moria M2 110 microkeratome,n=30.All were performed on both eyes with the right one treated first.Subtraction pachymetry was used to measure corneal flap thickness which was analyzed statistically with the data including age,preoperative corneal diameter,curvature,corneal thickness and refraction. Results In the 30 patients of M2 90 group,the mean corneal flap thickness of right eye and left eyes were(128.03?12.03)?m(105~156 ?m) and(123.40?12.38) ?m(92~147 ?m),respectively,and the corneal flap thickness were statistically different between the right and left eyes(P

Objective To investigate the dynamic changes of osteopontin (OPN) in the guinea pig model of cholesterol gallstone disease.Methods Forty-four guinea pigs were randomly assigned to cholesterol gallstone group and control group.The animals were sequentially killed on Day 7,14,28,42,56 and 70 after operation.The expressions of OPN mRNA in gallbladder and liver tissues were detected by real-time PCR.The changes of OPN,mucin,α1-acid glycoprotein (AAP) and apolipoprotein A1 (APOA1) in bile and blood plasma were detected by ELISA kits.Results The expression of OPN mRNA in gallbladder and liver tissues increased gradually and reached the peak in the 6th week,and then decreased.The increased expression of OPN in bile in the gallstone group started from the 1st week and reached the peak in the 6th week (P ＜ 0.05),which gradually decreased to the baseline in the 10th week (P ＞ 0.05).The expressions of OPN in bile and blood demonstrated similar trends,while the peak time in blood samples was much earlier (4th week).The changes of APOA1 in bile and blood were similar to OPN,although there was no advanced peak value in blood.The levels of mucin and AAP in bile and blood increased after operation,and were kept at high level throughout the study.Conclusions OPN is involved in the whole process of cholesterol gallstone formation,which may be associated with other nucleation-active factors.

Objective To discuss the blood substitutes during the perioperative period for hemophilic children with surgical disease as well as the coping strategies to its postoperative complications.Methods A retrospective study between 2003 and 2013 from one our centre identified a total of 41 perations performed in haemophiliac patients.33 patients were diagnosed with haemophilia A,among whom 10 were severe cases,13 moderate cases and 8 mild cases.8 patients were diagnosed with haemophilia B,among whom 2 were severe cases and 6 were mild cases.Different kinds of operation were required for each case.Results According to the monitoring of the coagulation tests PT and APTT before and after the operation respectively,after the use of the blood substitutes such as FV Ⅲ,PPSB and fresh frozen plasma,38 patients underwent surgical treatment successfully and had full recovery without any surgical complications,1 patient was dead,1 suffered intraperitoneal hemorrhage and 1 had delayed wound healing.Conclusion Full preparation and thorough plan before the operations,combined with appropriate blood substitutes can effectively reduce postoperative bleeding for hemophilic children.

Objective To establish a Wuzhishan minipig model of atherosclerosis(AS) induced by high fat/cholesterol diet,and observe the changes of expression of lipoprotein associated phospholipase A2(Lp-PLA2) in plasma and plaques.Methods 10 Wuzhishan minipigs were randomly divided into 2 groups:The normal control(Ctr,n=4) group was fed with normal diet,and AS model(n=6) group fed with high fat/cholesterol diet for 24 weeks.After the modeling for 24 weeks,the changes of total cholesterol(TC),low density lipoprotein(LDL-C),high density lipoprotein(HDL-C),triglyceride(TG),C-reactive protein(CRP),Lp-PLA2 activity and composition were detected.The changes of vascular lipid deposition and plaques were assessed by pathology using oil red O staining and HE staining,respectively,and immunohistochemical staining for IL-6 protein expression.Moreover,the expression of Lp-PLA2 mRNA determined by RT-PCR and protein by Western blot were observed in the abdominal aortic tissues.Results Compared with the control group,the body weight,body mass index(BMI),TC,LDL-C,HDL-C,CRP,Lp-PLA2 activity and composition and aortic lipid deposition were significantly increased,and AS plaque formation was observed in the AS model group(P<0.05,P<0.01).The expression of Lp-PLA2mRNA and protein and IL-6 protein in abdominal aortic tissues were also significantly increased(P<0.05).Conclusions Long-term high fat/cholesterol diet feeding for 24 weeks can induce atherosclerosis in Wuzhishan minipigs,and Lp-PLA2 plays a key role in the vascular inflammation and plaque formation.

Objective To observe the effect and toxicity of oxaliplatin plus 5-Fu and CF (FOLFOX) vs irinteean plus 5-Fu and CF (FOLFIRI) in patients with advanced/metastatic colorectal cancer. Methods 67 patients with histologicaly confirmed advanced/metastatic colorectal cancer were non-randomized to enter the study. Patients for FOLFOX: oxaliplatin 85 mg/m2 iv 2 h d1.CF 200 mg/m2 iv 2 h followed by 5-Fu 250 mg iv bolus and 5-Fu 600 mg/m2 iv 22 h d1,2 were given, every 2 weeks as one cycle. FOLFIRI: irinotecan 150 mg/m2 iv d1. CF, 5-Fu do so. Efficacy was evaluated at 4 cycles. Results For 39 patients to FOLFOX and 37 patients to FOLFRI, the objective response rate (CR+PR) was 41.0 % vs 35.1%. The median time to progression was 5.2 months vs. 5.8 months in the FOLFOX and FOLFIRI arm. The median survival time was 13.2 months vs. 14.0 months in the FOLFOX and FOLFIRI arm respectively. The clinical benefit rate was 71.8 % vs 78.4 % in the FOLFOX and FOLFIRI ann respectively. There was no significantly differences between two arms (P>0.05). The most frequently observed toxicity reaction was hematological toxicity nausea/vomiting and neurn-sensory toxicity in FOLFOX arm, and hematological toxicity and diarrhea in FOLFIRI arm. FOLFIRI arm had a remarkably higher incidence rate of grade 3 diarrhea than FOLFOX arm(P<0.025). Conclusion FOLFOX and FOLFIRI arm provid high effective and well tolerable treatment for advanced/ metastatic colorectal cancer.

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.

OBJECTIVE:To analyze fatty oil and volatile oil in seed of Metaplexis japonica. METHODS:Fatty oil and vola-tile oil in seed of M. japonica were analyzed by GC-MS:HP-5MS quartz capillary column,high purity nitrogen as carrier gas, flow rate of 1 mL/min,injector temperature of 220 ℃,primary column temperature of 120 ℃(temperature programmed),column pressure of 80 kPa,split sampling,split ratio of 20:1,sample size of 1 μL. Mass condition:electron bombardment ion source, electron energy of 70 eV,interface temperature of 250 ℃,mass scanning range of m/z 50-550,scanning interval of 1.0 s. The dif-ference of volatile components in seed of M. japonica before and after processing was analyzed by HSGC-MS:HP-5MS quartz cap-illary column,high purity nitrogen as carrier gas,flow rate of 1 mL/min,headspace heating temperature of 90 ℃,heating time of 30 min,primary column temperature of 80 ℃(temperature programmed),column pressure of 80 kPa,split sampling,split ratio of 20:1,sample size of 1 μL. Mass condition:electron bombardment ion source,electron energy of 70 eV,interface temperature of 210 ℃,mass scanning range of m/z 50-550,scanning interval of 1.0 s. RESULTS:A totall of 30 components were identified in fatty oil,among which relative contents of linoleic acid,oleic acid,palmitic acid were in high level;54 components were identi-fied in volatile oil,main components were terpenes,among which relative contents of cananga oil diene,thujopsene,dehydro aro-madendrene were in high level. Terpinen-4-ol was found and dihydrocarveol increased 100% after frying,compared with before fry-ing. CONCLUSIONS:The study basically confirm main component of fatty oil and volatile oil in seed of M. japonica;there is dif-ference of volatile components in seed of M. japonica before and after frying.