BACKGROUND:Intervertebral disc degeneration is the pathological basis of degenerative spinal diseases. Studies on the influentialfactors of intervertebral disc degeneration contribute to the prevention and treatment of degenerative spinal disease.
OBJECTIVE:To observe the growth and proliferation of nucleus pulposus cels isolated by trypsin plus type II colagenase digestion in complete medium with different glucose concentrations, exploring the optimal glucose concentration for growth of nucleus pulposus cels.
METHODS:Nucleus pulposus cels isolated and cultured by trypsin plus type II colagenase digestion method were observed under an inverted microscope, and thecelnumber was counted. Morphology of nucleus pulposus cels was observed afterhematoxylin-eosinstaining and toluidine blue staining. Colagen type II immunoreactivity was detected by immunohistochemical staining combined with immunofluorescent staining.Nucleus pulposuscels were incubated in complete medium containing various glucose concentrations (0, 6.25, 12.5, 17.5, and 25 mmol/L) for 24 hours, and then cel proliferation and apoptosis were determined.
RESULTS AND CONCLUSION:The stained nucleus pulposus cels showed polygonal and short spindle, with one or two nuclei. Celularpseudopod appeared gradualy and then became slim with increased passage numbers. The isolated and cultured nucleus pulposus cels positively expressed colagen type II and aggrecanProliferative activity of nucleus pulposus cels cultured in medium with 17.5 mmol/L glucose was significantly higher than that in medium with 0 and 25 mmol/L glucose (P< 0.05 orP< 0.01). There wasno significant differencein cel apoptosis between these groups except for 0 mmol/L glucose (P<0.05). These results confirm that a large number of nucleus pulposus celscan beharvested by trypsin plustype II colagenase digestion and the optimal glucose concentration is 17.5 mmol/L.

Twenty kinds of amino acids are simplified into 3 types: hydrophobic amino acids (H), hydrophilic amino acids (P) and neutral amino acids (N). Each residue is reduced to a bead which locates in the position of the C?琢 atom. The off-lattice model is adopted and the relative entropy is used as a minimization function to predict the tertiary structure of a protein. A new contact intensity function is given to consist with protein design research based on the relative entropy. Testing on several real proteins from Protein Data Bank (PDB) shows the good results obtained with the model and method. The root mean square deviations (RMSD) of the predicted structures relative to the native structures range from 0.30 to 0.70 nm. A foundation for studying protein design using the HNP model and the relative entropy was made.

The drug resistant mutations in human immunodefieiency virus type 1 (HIV-1) are a major impediment to successful highly active antiretrovirai therapy (HAART) and new drug design. In order to understand the drug resistance mechanism of HIV-1 integrase (IN) mutually existed for multiple drug-resistant strains to the most potent IN inhibitors diketo acids (DKAs), three S-1360-resistant HIV-1 strains were selected and molecular docking and molecular dynamics (MD) simulations were performed to obtain the inhibitor binding modes. Based on the binding modes, compelling differences between the wild-type and the 3 mutants for IN have been observed. The results showed that: 1) In the mutants, the inhibitor is close to the funetional loop 3 region but far away from the DNA binding site. Different binding sites lead to the decrease in susceptibility to S-1360 in mutants compared to the wild-type IN. 2) The fluctuations in the region of residues 138～166 are important to the biological function of IN. 2 hydrogen-bonds between S-1360 with residues N155 and K159 restrict the flexibility of the region. Drug resistant mutations result in a lack of the interaction, consequently, the less susceptible to S-1360. 3) In the 3 mutant IN complexes, the benzyl ring of S-1360 is far from the viral DNA binding site, thus, S-1360 can not prevent the end of the viral DNA from exposure to human DNA. 4) After T66I mutation, the long side chain of I occupied the active pocket in the 3 mutants, consequently, the inhibitor could not move into the same binding site or have the same orientation. All the above contribute to drug resistance. These results will be useful for the rational inhibitor modify and design.

Protein-protein interactions and recognition are the focal and hot themes of the life science field in the 21st century. Molecular docking approach is the effective computer modeling technology in the study of this topic. Generally, the protein-protein docking procedure is composed of four stages: searching of the binding modes of the receptor and the ligand, filtering of docked modes to eliminate the irrational docked structures, optimizing the structures, evaluating the docked modes with the refined scoring function and ranking them to obtain the near-native structures. Combining the research group's works, in terms of the international and national progress of protein-protein docking approaches, the detailed review was made about the four stages mentioned above. Additionally, the existing major questions are analyzed and the prospects of the future study are made.

The application of the protein design method based on the HNP model and the relative entropy theory is discussed for four structural classes of real proteins, and the results are compared with that of the HP model. Testing on 190 proteins shows that this method is generally effective for the different structural classes of proteins. Further studies show that the success rate of this method on regular secondary structures is higher than that on the random coil. Additionally, the success rate for different types of amino acids is also analyzed. It is found that the success rate on the hydrophilic residues is higher than those of the other two types. Furthermore, the success rate of this method on the conserved residues is higher than the non-conserved residues. The reasons resulting in the difference of the success rate on different systems were also analyzed. All analyses mentioned above make the foundation for the development and the application of this method in the future.

AIM: To investigate the mutagenicity of cadmium and zinc and the effect of zinc on the mutagenicity of cadmium. METHODS: The frequency of sister chromatid exchange (SCE) induced by cadmium, zinc and the combination of cadmium and zinc were observed in cultured human lymphocytes. RESULTS: Cadmium induced the increase in frequency of SCE in human lymphocytes from the concentration 1?10~(-8) mol/L to 1?10~(-6) mol/L, but zinc had a negative result. Zinc reduced the frequency of SCE induced by cadmium at the concentration from 1?10~(-6) mol/L to 1?10~(-4) mol/L and a relationship between dose and effect was also observed. CONCLUSION: Zinc inhibits the mutagenicity of cadmium in cultured human lymphocytes.

ObjectiveTo study changes of molecular markers of prothrombotic state:Platelet granule membrane protein ( GMP-140 ),Von Willebrand factor ( vWF:Ag),thrombomodulin (TM),Two-D dimer ( DD),antithrombin Ⅲ ( AT- Ⅲ ) in plasma and puerarin for treatment functions of acute pancreatitis (AP).MethodsIn 78 patients with AP [ severe acute pancreatitis (SAP):26 cases,mild acute pancreatitis (MAP):52 cases ],using a random number table,the patients were given puerarin treated base (n =40) and conventional treated base group (n =38 ).The two groups were given fast,continuous gastrointestinal decompression,correction of electrolyte and acid-base balance disorders,vein support,antisecretory drugs,antibiotics inhibit pancreatic secretion and inhibition of trypsin activity of drug treatment.Puerarin group:Puerarin injection 0.5 g in 5％glucose injection intravenous infusion of 500 ml,1 time a day.GMP-140 vWF:Ag,TM,DD were measured by the methods of analysis of enzyme-linked immunosorbent assay and AT-Ⅲ was measured by the methods of analysis of chromogenic substrate method preformed in all patients,plasma amylase and uric amylase were determined by the method of somogyi and after the treatment.And 22 healthy people were selected as normal controls ( NC,Group C,n =22).ResultsCompared with the Group C and MAP,the plasma GMP-140 [ ( 86.26 ± 15.28 )ng/Lvs (32.56 ± 18.17) ng/L and (58.68 ± 15.86)ng/L],vWF[(236.22 ±31.78)％vs (95.12 ±31.68)％ and (126.68 ± 17.06)％ ],TM [(65.70 ± 12.27) μg/L vs (4.26 ±0.92) μg/L and (9.80 ± 6.98) μg,/L],DD [ (0.87 ±0.04) mg/L vs (0.36 ±0.06) mg/L and (0.56 ±0.05) mg/L] were significantly elevated,however the AT-Ⅲ [ (56.13 ± 15.78) U/ml vs (98.76 ±22.68) U/ml and (80.38 ± 18.29)U/ml )was significantly decreased SAP ( P ＜ 0.01 ).There were significant differences on the levels of GMP-140 [ (31.52 ± 15.81 ) ng/L vs (59.62 ± 13.73 ) ng/L,t =- 23.283 ],vWF [ ( 93.32 ± 28.62) ％ vs ( 128.81 ±16.23)％,t=-28.205,P＜0.01 ],TM[ (4.36 ± 0.82) μg,/L vs (11.23 ± 7.62)μg/L,t =-43.419,P ＜0.001],DD[ (0.32 ±0.05) mg/L vs (0.68 ±0.04) mg/L,t =- 15.642,P ＜0.001],AT-Ⅲ ((97.68 ±21.69) U/ml vs (76.86 ± 17.92) U/m,t =14.967,P ＜ 0.01 ) between puerarin treated base group and conventional treated base group.Comparing with treated base,the group given puerarin obviously shortened the increased of plasma [ ( 81.26 ± 17.12) U/L vs ( 119.63 ± 51.87 ) U/L,t =- 7.618,P ＜ 0.001 ],uric amylase [ (416.37 ± 116.50) U/L vs (576.32 ± 126.58) U/L,t =- 36.659,P ＜ 0.001 ],the time of abdominal pain relief and therapy to spend [ ( 2.18 ± 0.76 ) d vs ( 5.26 ± 0.58 ) d,t =- 13.619,P ＜ 0.001 ].Conclusion The molecular markers of prothrombotic state:GMP-140,vWF:Ag,TM,DD,AT- Ⅲ might all play key roles in the development of AP.Puerarin can improve the pancreatic microcirculation and adjust molecular markers of prothrombotic state,and had certain treatment functions with AP.

Objective To explore the value of detection of systemic and local oxidation antioxidation level of ascites in the differential diagnosis of benign and malignant ascites.Methods Thirty-five patients with malignant ascites hospitalized in the Yidu Central Hospital of Weifang from December 2012 to November 2013 as the malignant ascites, and 35 cases of benign ascites as the control group.The ascites of malignant ascites group and plasma total antioxidant capacity (T-AOC) level of two group were detected by ferric ion reduction method.Plasma malonaldehyde (MDA) level of two group were detected by thiobarbituric acid content assay.Peripheral blood mononuclear cell (PBMCs) and tumor associated lymphocytes (TALS) in ascites were isolated by Ficoll density gradient centrifugation,the DNA damage of PBMCs and TALS were detected by single cell gel electrophoresis (SCGE) , expressed in the tailing rate.The value in the diagnosis of benign and malignant ascites was judged according to the analysis of above indexes.Results (1) The difference analysis : the levels of T-AOC in plasma in malignant ascites group was (9.26 ± 1.88)mM, significantly lower than that in benign ascites group((11.26± 1.78) mM, t =16.520,P =0.000);T-AOC levels of malignant ascites group ascites was (6.59± 1.38) mM, significantly lower than the plasma levels of T-AOC ((9.22± 1.86) mM, t =13.869, P =0.000);the MDA level of malignant ascites group was (6.26± 1.83) nM, significantly higher than that of serum benign ascites((3.26±1.12) nM,t=18.267,P=0.000);tailing rate of PBMCS in malignant ascites group was (17.9 ± 6.7) ％, significantly higher than that in benign ascites group ((9.6 ± 5.3) ％, P＜ 0.01);tailing rate of TALS in malignant ascites group was (442.6± 10.8) ％, significantly higher than that of PBMCS ((17.2±6.1)％ ,P＜0.01).(2)The correlation analysis: there was a negative correlation between plasma T-AOC and MDA in malignant ascites group (r =-0.518, P ＜ 0.01), tailing rate of T-AOC and TALS ascites (r =-0.566,P＜0.01) ,tailing rate of plasma T-AOC and PBMCS(t =-0.472,P＜0.01);there was a positive correlation between the tailing rate of plasma MDA and PBMCS (r =0.476, P ＜ 0.05).Conclusion In the malignant ascites group, there is oxidative stress in the whole body and the ascites, and the local oxidative stress in the ascites is more obvious.The sensitivity and accuracy of the diagnosis of malignant ascites can be increases by detected by the detection of the local oxidation resistance of the whole body and the ascites.

Objective To observe the effect of transcutanous electrical acupoint stimulation(TEAS)on the exercise ability of rats.Methods After stereotaxis surgery,twenty male adult SD rats wera randomly divided into control group (n=10)and TEAS group(n=10).Two days after surgery.the rats were trained to adapt the treadmill mnning and TEAS(continuous wave,2Hz,5mA)was applied to right Zusanli (ST 36) for 30min,once a day for 6 days.Basic microdialysis samples of extracellular serotonin(5-HT)in rats'dorsal raphe nucleus(DRN)were collected 7 days after surgery.During experiment,rats were running on treadmill (10m/min for 10min,then gradually increased to 24m/min for 1 hour,0% grade).Microdialysis samples were collected continuously until 5 hours after treadmill running.The frequency of brush-stimulation during the running was recorded.Meanwhile,plasma amino acid content was examined 5 hours after exercise.Results The frequencies of brush-stimulation during the treadmill running in control group and TEAS group were 2.04+0.46 and 0.40+0.080 times per minute,respectively (P<0.05).There were no significant differences in plasma total-TRP(T-TPP)and branched chain amino acid(BCAA)contents between two groups(P>0.05).However,plasma free-TRP(f-TRP)content,f-TRP/T-TRP ratio and f-TRP/BCAA ratio decreased significantly(P<0.05) in TEAS group as compared with the control group.Extracellular 5-HT levels in DRN in control group soon after exercise were higher than their base levels(P<0.05),and decreased in sequential two hours,and then increased significantly 3 hours and 4 hours after exercise as compared with that 2 hours after exercise(P<0.05).There was a slow decline in the levels of extracellular 5-HT in TEAS group at each time-point after exercise,and significant lower than that in control group within 5 hours after exercise(P<0.05).Conclusion TEAS atZusanli(ST36)can effectively improve exercised ability by reducing peripheral free-TRP level and transporting TRP into the brain,and thus decreasing the synthesis of 5-HT in the brain.

Objective To study the influence of optimization of parenteral nutrition strategy on the head circumference and brain volume in very low birth weight infants.Method Very low birth weight infants admitted to NICU of University of Hong Kong-Shen Zhen Hospital from January 2014 to June 2016 were assigned to optimized group and conventional group according to early nutritional strategies.Early parenteral nutrition intakes were increased in infants assigned to optimizated group.Nutrition intakes and parenteral nutrition related complications within 28 days after birth were compared between groups.All participants underwent brain MRI at corrected gestational age (CGA) 36 weeks.Head circumference and brain volume measured by MRI were also compared between groups.Result A total of 40 preterm infants were recruited,with 20 infants in each group.There were no significant differences in the gestational age,birth weight,brain injury and intrauterine growth retardation rate between the two groups (P ＞ 0.05).The average daily total calories and protein intake of optimization group during the first 4 weeks were significantly higher than those of conventional group,respectively [(101.5 ± 3.1) kcal/ (kg · d) vs.(96.1 ± 3.2)kcal/(kg·d),(3.07±0.16) g/(kg·d) vs.(2.90±0.11) g/(kg· d),P＜ 0.05].Theaverage daily calorie intake and protein intake of optimization group was increased by 4.7％ and 5.5％,compared with those of conventional group.Compared with the conventional group,head circumference and total brain volume of optimized group at CGA 36 weeks was improved by 3.3％ and 4.1％,and the differences were both statistically significant (P ＜ 0.05).Cerebral cortex gray matter volume of optimized group was significantly higher than that of conventional group [(102.4 ± 4.9) ml vs.(96.4 ± 4.6) ml,P ＜ 0.05].There was no significant difference in brain white matter,deep gray matter and cerebrospinal fluid volume between the two groups (P ＞ 0.05).Conclusion The optimization of parenteral nutrition within the framework of active nutrition strategy of preterm infants can further improve the early nutritional intake of preterm infants,leading to the increase of the head circumference and the gray matter volume of the cerebral cortex.