The authors developed an ABC-ELISA method using the double antibody sandwich.The sandwich is composed of labled and unlabled rabbit antihuman Neuron- Specific Enolase.The method is sensitive, specific and it's standard curve is linear interrelated. In clinical practice,it can be used to assay the concertration of NSE in serum and diagnose small cell cancer of the lung.

BACKGROUND:In sports or in daily life, damage due to sudden power, especialy due to non-physiological release,is commonly seen. For example, during basketbal, soccer, rugby, or martial arts movement, oppositional and explosive movements result in a higher incidence of ankle injuries. While conventional methods can improvesymptoms, the long-term efficacy is unsatisfactory, accompanied by a higher incidence of complications that are likely to cause secondary damage.
OBJECTIVE:To prepare a calcified biomimetic scaffold for bone tissue engineering and to observe the therapeutic effect of this scaffold onmartialarts-induced ankle injuries.
METHODS:Eighty patients withmartialarts-induced ankle injury were selected from Chengdu Sport Institute between December 2014 and December 2015. These patients were randomly assigned to control group with drug treatment and biomimetic scaffold group with calcified biomimetic scaffold implantation (n=40 per group). Acelular suspension prepared by goat cartilage was used to make cartilage tissue blocks with a calcified layer with a diameter of 8 mm in a prechiled abrasive apparatus. Then, the calcified biomimetic scaffold for bone tissue engineering was prepared using lyophilization and chemical crosslinking methods.
RESULTS AND CONCLUSION:Osteochondral tissues were partialy hyalinized on the surface with the presence of osteochondral calcified layer. The hyalinized cartilage was white in color, the calcified layer existed between normal osteochondral tissues, and the subchondral bone was considered as the cancelous bone. Then the calcified layer was stained using hematoxylin-eosin. We found that cartilage cels in the calcified layer were basicaly removed, forming “empty nests” one by one. But the structure of bone cartilage in the tissue block, the calcified layer and the subchondral bone retained wel. For pain assessment, visual analog scale scores were detected and showed no difference between two groups prior to treatment (P> 0.05), but became significantly higher in the biomimetic scaffold than the control group at 1 and 4 weeks after treatment (P< 0.05). Besides, the biomimetic scaffold exhibited better therapeutic efficacy than the drug treatment (P< 0.05). Overal, this study successfuly prepare the calcified biomimetic scaffold for bone tissue engineering that is suitablefor repair of sport-induced ankle injuries.

Livin is a novel member of IAPs (inhibitors of apoptosis protein) family, which is expressed highly in a variety of tumors but not in majority of normal tissues. This protein contains a BIR domain and a RING finger domain just like other members of IAPs. It will be of great sigificance to investigate the relationship between Livin and tumors, and it might be a potential target for anti-cancer drugs.

AIM: Antisense oligonucleotides against livin were designed with computer software. METHODS: Antisense oligonucleotides were designed according to the secondary structure of livin mRNA which was simulated with RNAdraw and Sfold. And then these oligonucleotides were transfected into Hela cells for inducing apoptosis. RESULTS: Five antisense oligonucleotides were designed using RNAdraw and Sfold, and effectively induced Hela cell apoptosis. CONCLUSION: The approach is effective and feasible to design antisense oligonucleotides by means of calculation with two kinds of software.

Objective:Identification of the antigenic determinants of hALR.Methods:Theoretic antigenic determinants of hALR was predicted by using Hopp&Wodds method and and Goldkey software package.The four polypeptides according to the amino acid sequences of the predictive linear epitopes of hALR were synthesized and were used to analyze the antigenic determinants of hALR recognized by antibodies.Results:The polypeptides corresponding to residues 6～5,68～80 and 105～112 of hALR were its antigenic determinants.Conclusion:Prediction of the protein antigenic determinants by computer program and detection of them by competitive ELISA with synthesized polypeptides is a useful method of identification of the antigenic determinants.

Traditional Chinese medicines (TCM) is one of the most important sources of new drugs.The rapid development of modern science and technology has brought new opportunities for TCM.Admittedly,new academic theory is getting into a golden period of innovation.Key technologies that enbody TCM features and adapt to modern drug-screening requirements are urgently needed.After five years' endeavor,the authors' group has made great progress in the new theories and methodologies for the discovery of bioactive compounds from TCM.In this review,a total of five key technologies:library-bioactivity-structure integration,biological and chemical fishing technology,ligand-and receptor-based virtual screening,profile-bioactivity relationship and the technology for discovering bioactive equivalent combinatorial components (BECCs),were introduced.In the text,several valuable demonstrations over the TCM-based drug discovery were provided,for uncovering the scientific basis of TCM and accelerating the process of TCM modernization.

Objective: To isolate small molecular polypeptides which bind to KDR specifically using C7 and 12 peptide libraries. Methods: KDR/IgGFc was coated directly on plates, C7 and 12 peptide libraries are then applied, followed by vigorous washings in washing buffers to remove most non-specifically bound phage and to select specific phage particals by VEGF suspension and elution in acid buffers. The positive phage clones were detected by ELISA, cell-ELISA and competitive binding ELISA. Results: After three washes, 12 positive phage clones were selected and sequenced. Conclusion: A conserved peptide motif was not found in these sequences. The peptide binding to KDR specifically may be helpful for lead drug to improve selectivity and decrease the side effect of anti cancer drug in clinical treatment.

Objective To study the effect of metformin on the growth of megakaryocytic leukemia cell line Dami and to explore the molecular mechanisms of the inhibitory effect of metformin on the proliferation of Dami. Methods The Dami cells were cultured and divided into control and 1,2,4,8,16 and 32 mmol·L-1 metformin groups.Then MTT test was performed to detect the inhitory rate of proliferation of Dami cells after treated with different concentrations of metformin. Flow cytometry was used to examine the distribution of cell cycle, and Western blotting was carried out to analyze the expressions of Cdc2 and CylinB1 and the phosphorylation of Cdc2. Results The MTT results showed that compared with control group,the inhibitory rates of proliferation of the Dami cells in 32 mmol·L-1 metformin groups at 0,24,48,72 and 96 h (35.1%±2.3%,49.7%±5.1%, 78.85±0.9%,79.1%± 3.0%%,and 85.2%± 3.2%)were significantly increased(P<0.01),Furthermore, after metformin treatment for 72 h,the inhibitory rates of proliferation of the Dami cells in 1,2,4,8,16 and 32 mmol·L-1 metformin groups were (33.8 ± 0.3)%,(51.9 ± 0.2)%,(59.4 ± 1.6)%,(65.5 ± 2.0)%, (75.5±0.9)%,and (79.1±3.0)%,respectively. Metformin inhibited the growth of Dami cells in a time-and dose-dependent manner. The flow cytometry results results revealed that compared with control group, the percentages of Dami cells in G2/M phase in 1,2 and 4 mmol·L-1 metformin groups were increased from (26.0± 0.5)% to (38.5 ± 1.5 )%, (48.4 ± 1.1 )%, and (58.2 ± 2.7 )%;there was significant difference in the percentages of Dami cells in G2/M phase between control group and 4 mmol·L-1 metformin group (P<0.01). Western blotting analysis showed that compared with control group, the expressions of Cdc2 and CyclinB were evidently reduced, the phosophorylation of Cdc2 at Tyr1 5 was up-regulated, and the phosphorylation at Thr1 6 1 was down-regulated.Conclusion Metformin can inhibit the growth of Dami cells and induce G2/M arrest,and its mechanism may be related to inhibiting the activation of Cdc2/CyclinB1 complex.

Objective To evaluate the treatment effect of compound Chinese medicine on skeletal fluorosis in rats by Micro-CT.Methods Eighty-eight Wistar rats which had been weaned for two weeks were divided into four groups according to body weight [(91.1 ± 10.0)g] by the method of random number table:control group(16 mts),middle fluorine(MF)group(24 rats),high fluorine(HF) group(24 rats),and high fluoride and low calcium low protein (HF-LC-LP) group (24 rats).The amounts of fluorine of MF,HF and HF-LC-LP groups were 50,100 and 100 mg/kg,respectively.The contents of calcium and protein in HF-LC-LP group were half of MF and HF groups.Six months after treatment with fluoride,eight rats of each group were put to death with femoral artery bleeding.The rest 16 rats of each fluorosis group were divided into two groups,one was the control group and the other was fed with both fluorine and the compound Chinese medicine which simulated the actual situation of fluorosis area.Each rat of the treatment group was given the medicine 194 mg/100 g for six days every week.Daily urine samples were collected when the medicine had been used for 0,30 and 60 days.All the rats were put to death with femoral artery bleeding after the medicine had beengiven for 90 days,and limbs bones were dissected.Urine fluoride was tested by the method of fluoride ion selective electrode ; bone fluoride was tested by the method of high temperature ashing-fluoride ion selective electrode; bone mineral density(BMD),tissue mineral density(TMD),structure model index (SMI),trabecular thickness (Tb.Th),trabecular separation (Tb.Sp),anisotropy (a1/a3),trabecular connection density(Conn.D),the volume ratio of trabecular and bone tissue,the ratio of bone surface area and volume(BS/BV),and trabecular number(Tb.N) were detected by Micro-CT technology.Results The level of urinary fluoride of high fluoride and low calcium low protein treatment group [(11.01 ± 3.67)mg/L] was lower than that of its control group [(34.32 ± 9.50)mg/L,t =3.13,P ＜ 0.05] when rats were remedied with the compound Chinese medicine for 60 days.The level of bone fluoride of high fluoride treatment group[(275.38 ± 171.65)mg/kg] was lower than that of its control group[(701.67 ± 178.16)mg/kg,t =5.42,P ＜ 0.05] when rats were remedied withy the compound Chinese medicine for 90 days; bone fluoride of high fluoride and low calcium low protein treatment group[(313.26 ± 124.51)mg/kg] was lower than that of its control group[(794.66 ± 261.35)mg/kg,t =3.25,P ＜ 0.05].The differences of Tb.Th,Tb.Sp,a1/a3,Conn.D,BV/TV,BS/BV and Tb.N among groups were statistically significant(F =2.785,2.681,3.039,27.231,2.595,2.854,5.050,all P ＜ 0.05).Tb.Th[(0.04 ±0.01)mm] and Tb.Sp[(0.03 ± 0.01)mm] of middle fluorine treatment group were higher than those of their control groups[(0.02 ± 0.00),(0.02 ± 0.00)mm,all P＜ 0.05]; al/a3,Corm.D,BV/TV and Tb.N[(0.77 ±0.61),(510.91 ± 304.99)mm-3,(0.42 ± 0.06) and (13.58 ± 2.48)mm-1] were lower than those of their control groups[(1.11 ± 0.01),(2 403.69 ± 124.02)mm-3,(0.46 ± 0.03) and (18.12 ± 0.69)mm-1,all P ＜ 0.05].BV/TV(0.44 ± 0.04) of high fluoride treatment group were lower than those of their control groups(0.49 ± 0.00,P ＜ 0.05) ; Tb.Th[(0.04 ± 0.01) mm] was higher than that of its control group [(0.03 ± 0.00)mm,P ＜ 0.05].Conclusion The compound Chinese medicine may has therapeutic effect on rat skeletal fluorosis.

Objective To obtain highly expressing cell lines by inserting the glutamine synthetase (GS) screening system and replacing the promoter of the vector.Methods The mutation of the point BamHⅠwas induced to build a new vector pIRES2-EGFP.The marker gene GS was inserted by AseⅠ and NheⅠ, and the promoter hCMV was replaced by PacⅠand NheⅠ.The new vector pHGS1.0 and the vector pIRES2-enhanced screen fluorescein protein( EGFP)-B were inserted by the recombinant protein TEM8 ( 1-227 )-VEGFR1 domain2-IgG2 ( TV-IgG2 ) gene to analyze the advantages of the expression.Results The glutamine synsthetase is successfully inserted, the human cytomegalovirus replaced, and recombinant protein is increased 5-fold by human immunoglobulin quantification kit.Conclusion The GS system is a highly protein expressing system.