Phage display technology describes a genetic engineering technique in which a library of peptide or protein variants is expressed on the outside of a phage virion,while the genetic material encodes each variant resides on the inside and maintains the relatively independent spatial structure and biological activity. Studies have shown that the phage display technology can be used to screen tumor homing peptides,target trans-porting anti-substances,develop vaccines,research diagnostic regents and image in blood vessels.Thus phage display technology is a powerful method to overcome the challenge of tumor disease.

Objective:To screen the peptide binding to human bladder carcinoma cells specifically by using phage display technology in vivo.Methods: Nude mice were inoculated with bladder carcinoma cells BIU87 for establishing tumor-bearing mice model.The Ph.D.-C7CTM Peptide Library was injected intravenously via tail vein.Then we screened Phage containing exogenous peptides binding to bladder transitional carcinoma cells specifically.The phage peptide homed to the tumor tissues was obtained after 3 rounds screening in vivo.The phage clones affinity to BIU87 were identified by immunohistochemistry and ELISA.The positive peptide was synthetized by chemical methods after sequencing the positive monoclonal phage DNA.The tumor cell specificity of target peptide was identified by confocal laser scanning microscope and flow cytometry.Results:After 3 rounds screening in vivo,enrichment rate of phage was 4.334×102 times.Immunohistochemistry results showed that the dyeing of the tumor tissue had a rising trend following each round of phage screening,while liver had a lot of non-specific binding phage because the phages were metabolized through liver and kid-ney.The 30 phage clones were identified by ELISA and 10 clones had a strong affinity on BIU87 among 24 positive clones.Three amino acid sequences of positive phage clones were obtained.The highest rate of repeat sequences CSSPIGRHC(8/10) named NYZL1 and the FITC-C6-NYZL1 peptide was synthesized.Our results showed that it could bind to bladder carcinoma cells BIU87 specifically.Conclusion:We obtained the small molecular peptide NYZL1 binding to human bladder carcinoma specifically by means of phage display in vivo,which provide a theoretical basis for bladder carcinoma early diagnosis and targeted therapy.

Objective To investigate the characters and targeted ability of FITC-CSNRDARRC molecular probe in labeling orthotopic transplantation tumor of bladder cancer in vivo.Methods From July 2013 to June 2014,the stability and characters of FITC-CSNRDARRC molecular probe were detected by spectrophotometer and molecular imaging and the optimum concentration and imaging time window were determined.30 BALB-C nude mice were randomly divided into experimental group (n =20) and control group(n =10).In control group,5 of them (group A) were ligated bilateral ureter,others(group B) were not.We established orthotropic transplanted bladder tumor (BIU-87) model by operation.And 0.2 ml probes (220 μmol/L) was then injected intravenously in all mice after 2 weeks.We obtained images and analyzed average gray value of the heart,lung,liver,spleen,bilateral kidney and orthotropic transplantation bladder tumor by using optical probe molecule fluorescence imaging system after 30 min,1 h,2 h,4 h and 12 h,respectively.Results After injected the FITC-CSNRDARRC molecular probes intravenously at 220 μmol/L,the fluorescence signal of tumor tissue strengthened gradually.The optimal imaging time window was 4 hours after injection.The illumination and temperature had little effect on the fluorescence signal.With the time passing after injection,the intensity of florescence signal progressively increasing,which reached the peak at4 h.The average gray value of tumor tissue at 1 h,2 h,3 h,4 h,5 h,6 h,8 h,12 h were 74.22,76.2,80.11,89.38,83.29,85.1,81.22,83.01,respectively.The fluorescence signal of normal tissue weakened gradually with the passage of time.Only liver and gall bladder could notice the fluorescence signal 4 hours after injection in group A.However,the relatively strong fluorescence signal could be found in liver and gall bladder in group B.Conclusions The characters of fluorescence probe are affected by its concentration.Its optimal concentration of labeling tumor is 220 μmol/L.The optical imaging time window was about 4 h after intravenous injection.The FITC-CSNRDARRC molecular probe can specifically bound to orthotopic transplanted tumor of bladder cancer in vivo.

BACKGROUND:The oxygen free radicals and apoptosis play an important role in limb ischemia/reperfusion injury, so we can al eviate limb ischemia/reperfusion injury by inhibiting the production of oxygen free radicals and apoptosis.
OBJECTIVE:To discuss the application and effect of edaravone on limb ischemia/reperfusion injury in rats.
METHODS:Of the 30 female Sprague-Dawley rats, 20 rats were randomly selected to make models of limb ischemia/reperfusion injury by ligating the root of right lower limb with a self-made bal oon cuff at 40 kPa pressure to block blood flow for 4 hours and reperfusing. After success model establishment, they were randomly assigned to two groups. In the edaravone perfusion group, edaravone 3 mg/kg was injected via the left femoral vein at 5 minutes before reperfusion. In the model group and normal group (the remaining 10 rats), an equal volume of physiological saline was given at the same time point. At 24 hours after reperfusion, the right anterior tibial muscle of each group was removed and these ultrastructural changes were observed by transmission electron microscope. Bcl-2 mRNA and Bax mRNA of rat anterior tibial muscle of each group were semiquantitatively detected with the RT-PCR and the ratio of bcl-2/bax was calculated.
RESULTS AND CONCLUSION:(1)Electron microscope results:compared with the model group, the muscle fibers were neater;the M line and the N line were clearer;the swel ing of mitochondria was al eviated;the numbers of mitochondria and mitochondrial crista were also increased in the edaravone perfusion group. (2)RT-PCR results:At 24 hours after reperfusion, the relative expression of bcl-2 mRNA and the ratio of bcl-2 mRNA to bax mRNA in right anterior tibial muscle were lower in the model group compared with the edaravone perfusion group (P<0.05). However, relative expression of bax mRNA was greater in the model group than that in the edaravone perfusion group, which were both higher than the normal group (P<0.05). Results indicated that the free radical scavenger edaravone relieved limb ischemia/reperfusion injury by improving the mitochondrial ultrastructure and promoting expression of bcl-2 mRNA and inhibiting expression of bax mRNA, and could provide a new choice for the treatment of limb ischemia/reperfusion injury.

This study investigates the metabolic regulatory effects of exogenous lactate on obesity mice induced by high-fat diet.We established obesity and metabolic disorder C57 mice model using a synthetic high-fat forage containing 60% fat.Some mice were fed with high-fat diet for 4 weeks to establish the model, being given 500 mg/(kg?d) lactate with ip for 4 weeks at the same time; the others were fed with high-fat diet for 8 weeks to establish the model, being given 500 mg/(kg?d) lactate 4 weeks after 4 weeks of modeling.During the trial period, the change of body weight and food intake, as well as serum glucose, lactate, triglycerides, insulin, and liver glycogen levels of both groups of mice were measured.Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were used to assess glucose metabolism and insulin resistance in the body.At the end of the experiment, adipose tissue was dissected for weighing and histopathological examination.The expression of lipid synthesis and lipolysis genes in adipose tissue was detected by real-time PCR.The results showed that: (1) in the 4-week preventive medication trial, lactate had no significant effect on the body weight of normal and high-fat diet (HFD) mice, yet it increased the subcutaneous fat/visceral fat weight ratio; lactate could significantly reduce fasting blood glucose and liver glycogen levels in HFD mice while increasing blood lactate levels, significantly improving impaired glucose tolerance in HFD mice; lactate could improve the size and arrangement of adipocytes in the HFD group while significantly down-regulating the expression of fatty acid synthesis and lipolysis genes in adipose tissue; (2) in the 8-week treatment, both routes of lactate administration could partially reduce body weight in HFD group mice and reduce food intake, with the improvement trend for fat weight; both routes of lactate administration could significantly reduce fasting blood glucose levels in HFD mice, while significantly improving glucose and insulin tolerance, with some improvement of fasting insulin levels and insulin resistance index; both routes of lactate administration showed different degrees of improvement effect on adipocyte morphology in obese mice while significantly down-regulating lipolysis gene expression in adipose tissue.Therefore, for high-fat diet-induced obese mice with metabolic imbalance, exogenous lactate can stimulate glucose metabolism, inhibit adipose tissue lipolysis, and prevent adipocyte hypertrophy, thereby improving glucose tolerance and insulin sensitivity and reducing sugar-lipid metabolic disorder.