s〕 Objective:To investigate the influence of repeated firings on the color of porcelain fused to metal crown(PFM) . Methods: 5 PFM samples were prepared and fired for 9 times.The color of the samples was measured following firing with CIE1976 L*a*b* color system . The color difference ?E ab *,hue angle h ab * and chroma C ab * were calculated according to the following calculations:?E ab * ={(?L*) 2 +(?a*) 2 +(?b*) 2} 1/2 , C ab * ={(a*) 2+(b*) 2} 1/2 , h ab *=tg 1 b*/ a*, L* presented brightness. Results: After repeated firing, L* value of PFM , C ab * and L* value of dentin layer and h ab * value of opaque layer increased. Conclusion: The color of PFM after repeated firing may become brighter. The color change is resulted from the increase of L* and C ab * of dentin layer.

Objective: To study the influence of repeated firings on the PFM bond strength. Methods: 25 specimens of PFM in the diameter of 13.0 mm and thickness of (1.0?0.02) mm were prepared and fired at 920 ℃ for 1, 3, 5, 7 or 9 times(5 samples for each time). The remained porcelain rate(RPR) was measured by Mackert method and used to evaluate the bond strength.Results: After 3 firings, the RPR of PFM decreased obviously, and additional firings had no obvious effect on the RPR. Conclusion: 3 times firing or more may decrease the bond strength of PFM.

BACKGROUND: Bone marrow stromal stem cells (BMSCs) are characterized by rapid amplification and wide differentiation.Thus, to establish in vitro BMSC induction models contributes to the study of tissue engineering.OBJECTIVE: To investigate the possibility of inducing the differentiation of rat BMSCs into cardiomyocytes by 5-azacitidine in vitro.OESING, TIME AND SETTING: The cytological in vitro study was performed at the Central Laboratory of First Affiliated Hospital of Liaoning Medical University from June 2005 to June 2008.MATERIALS: A total of 20 Sprague Dawley rats were supplied by the Experimental Animal Center, Liaoning Medical University.5-azacitidine (Sigma, USA) was used.MHEHODS: Following anesthesia, the rats were used to isolate the femur and tibia. BMSCs were isolated and cultured by the whole bone marrow method + adherent method. When 90% BMSCs were confluence, BMSCs were passaged. BMSCs at the third passage were incubated in a 24-well plate at 2×10~4/well, with 5,10, 15, 20 μmol/L 5-azacitidine. Simultaneously, a blank control group (without inductor) was set. Following 24 hours of induction, BMSCs were incubated in normal medium for 3 weeks.MAIN OUTCOME MEASURES: The following parameters were measured: cell appearance and growth curve, morphological changes following induction, and expression of connexin-43 and a-striated muscle actin.RESULTS: Cultured BMSCs were spindle-shape, with some cell confluence. P3 cells following incubation entered static phase at 1 and 2 days, and entered logarithmic phase at 3 days, reached a peak at 9 days, and then entered platform stage. Cell number became decreased at 12 days. Following induction of 5 μmol/L 5-azacitidine, no significant difference was found in BMSCs.Following induction of 10 μmol/L 5-azacitidine, BMSCs became long and big, extended towards a direction, with the property of myotube formation cells. Following induction of 15 umol/L 5-azacitidine, a few cells survived surrounding the 24-well plate.Following induction of 20 μmol/L 5-azacitidine, cells died. Following induction of 10 μmol/L 5-azacitidine for 3 weeks, expression of connexin-43 and a-striated muscle actin was determined in BMSCs. However, a negative expression was detected in the blank control group.CONCLUSION: BMSCs cannot differentiate into cardiomyocytes by itself. Following in vitro induction, BMSCs can differentiate into cardiomyocytes. Low-dose 5-azacitidine concentration cannot induce the differentiation, but high-dose 5-azacitidine concentration will induce death in a large number of cells. Thus, 10 μmol/L is an optimal concentration.

OBJECTIVE:To establish an HPLC method for the determination of lipoic acid in lipoic acid sustained-release tablets.METHODS:HPLC was conducted on a Hypersil ODS with acetonitrile-0.05 mol?mL-1 potassium dihydrogen phosphate(45∶55,adjust pH to 2.0)as mobile phase at a flow rate of 1.0 mL?min-1.The injection volume was 10 ?L;the detection wavelength was set at 219 nm and the column temperature was 25 ℃.RESULTS:The linear range of lipoic acid was 9.99～399.70 ?g?mL-1(r=0.999)with an average recovery rate of 99.17%(RSD=0.40%);the intra-day and inter-day RSD were all less than 2.6%.CONCLUSION:The method is simple and accurate,and suitable for the determination of the content of lipoic acid in lipoic acid sustained-release tablets.

OBJECTIVE: To evaluate the effect of aerobic exercise on genome expression in human skeletal muscle. METHODS: Six healthy sedentary elderly men aged (66+9) years were selected from military cadre retirement centers and after exercise. Testing indices included height, weight, vital capacity, step index and maximal oxygen uptake. Needle biopsies were obtained from the skeletal muscle before and after the last training. Total RNA extracted from the samples was hybridized to Affymetrix U 133A platform, the gene expression datum was analyzed.RESULTS: Aerobic exercise was shown to improve cardiorespiratory function and reduce body fat of elder subjects. It could alter the genome expression in human skeletal muscle, the number of genes that passed filtering criteria was 725. The most differently expressed genes (n=20) were investigated in this study, in which there were 3 upregulated and 17 downregulated. According to gene function annotations, the differential genes were classified into 8 categories which concerned cellular component and biological process, Kyoto Encyclopedia of Genes and Genomes (KEGG) searching showed 4 genes' metabolism pathway. CONCLUSION: Systematic aerobic exercise upregulates expression of enzyme genes concerning tricarboxylic acid cycle, and downregulates expression of genes conceming muscle protein synthesis and sphingolipid. It is suggested that aerobic exercise is good to protect human nerves' integrity, exerts positive action on anti-aging and accelerate the aerobic metabolism of lipid materials in vivo.

Objective To construct bispecific antibody BsAb1/17 against both IL-1β and IL-17A,express and purify the biologically active BsAbl/17 protein in prokaryotic system for further studies and applications. Methods VH1VL17-CL and VL1VH17-CH1 gene segments were constructed by overlap-PCR.Restriction enzyme sites Nco Ⅰ and BamH Ⅰ were designed at the 5'and 3' end primers respectively. The products of overlap-PCR were ligated to the Nco Ⅰ/BamH Ⅰ -prepared pET-27b vector. The recombinant plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 ( petB ) were transformed into E. coliRosetta separately. The expressing products were analyzed by SDS-PAGE and Western blot. Neutralization activity of the bispecific antibody for blocking the induction of IL-18 expression by IL-1β in human T cells was determined by real-time PCR. Neutralization activity of the bispecific antibody for blocking the induction of IL-6 expression by IL-17A in HeLa cells was determined by ELISA assay. Results The structure of the plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 (petB)was confirmed by DNA sequencing. After induction, the fusion proteins were expressed mainly as inclusion bodies. The purity of the both proteins exceeded 90%. SDS-PAGE analysis suggests the relative molecular mass of both products expressed by petA and petB were approximately 38× 103, which is in accordance with the theoretical value. The results of Western blot and ELISA test demonstrated that BsAb1/17 molecule had binding ability to both IL-1β and IL-17A. The BsAb1/17 could block IL-1β to stimulate human T cell to express IL-18 and block IL-17A to stimulate HeLa cell to express IL-6. Conclusion We successfully constructed a novel bispecific antibody BsAb1/17 against both IL-1 β and IL-17A, and expressed biologically active BsAb1/17 protein in prokaryotic system.

Objective:To transfect the non-viral vector polyethylenimine (PEI)mediated miR-2861 mimic into the MC3T3-E1 cell line,and to explore the transfection efficiency of PEI/miR-2861 complex and its effects on the proliferation and osteogenesis differentiation in pre-osteoblasts. Methods:The proper amount of PEI was blended with miR-2861 mimic and negative control (NC)separately in a ratio of N∶P=10∶1,and they were divided into experiment group and NC group. The NC/PEI complex acted as the NC group was used to eliminate the interference of osteogenesis from the addition of double-stranded RNA mimic.MTT assay was used to determine the optimal concentration of PEI/miR-2861 mimic complex.The fluorescence imaging technique and bulge-loop RT-PCR were used to detect the transfection efficiency and mRNA expression of miRNA-2861 in the cells with different concentrations (10,30, 50,and 100 nmol · L-1 ), separately.The osteogenesis ability of MC3T3 cells was identified with RT-PCR and Alizarin red staining with the selected concentration of PEI/miR-2861 by transient transfection.Results:Compared with blank control group,the proliferation rates of MC3T3 cells in 100 nmol·L-1 PEI/miR-2861 group was decreased significantly at 72 h (P < 0. 05 ). With the increasing of transfected concentration the transfection efficiency of miRNA/PEI complex was increased gradually.The results of Alizarin red staining and quantitative analysis showed that calcium deposits were more and bigger in experiment group after induced for 21 d,while both in blank control group and NC group they were less.Conclusion:The miRNA-2861 mimic can be effectively transfected into the MC3T3-E1 cell line and expresses with a high level,which is mediated by PEI as the gene vector.miR-2861 mimic has a certain ability of promoting osteogenesis differentiation of MC3T3-E1 cells.

Objective To investigate the impact of enhanced recovery after surgery (ERAS) program on postoperative recovery in patients undergoing laparoscopic colorectal resection. Methods Eighty-four patients undergoing laparoscopic colorectal resection from March 201 5 to June 201 6 (55 males,29 females,aged 36-78 years,ASA physical status Ⅰ or Ⅱ),were randomly divid-ed into two groups (n = 38 each).Patients in group E were received epidural block combined with general anesthesia,and a series of perfect ERAS strategies,such as strengthen preoperative educa-tion, maintaining perioperative normothermia, perioperative goal-directed fluid therapy, intraoperative and postoperative analgesia.While the patients in group C received routine anesthetic management.The volume of fluid,the nasopharyngeal temperature,the time of recovery of bouel sound,first anal exhaust,eating fluid food,ambulation and remove of the catheter were recorded in two groups.Furthermore,time of PACU after surgery,the total days of hospitalization and total hos-pital costs were recorded.Results The volume of fluid [(1 328 ± 64)ml vs.(2 463 ± 135 )ml]in group E were significantly lower than group C (P <0.05),the nasopharyngeal temperature [(36.2± 0.2)℃ vs.(35.1±0.5)℃]was significantly higher in group E (P <0.05).Compared with group C,the time of recovery of bowel sound [(33.4 ± 12.5 )h vs.(42.8 ± 14.3 )h],first anal exhaust [(43.6±13.9)h vs.(60.7±1 5.4)h],eating fluid food [(26.8±4.1)h vs.(67.4±13.5)h],first ambulation [(7.4±1.6)h vs.(26.5±3.8)h]and remove of the catheter [(29.2±6.1)h vs.(5 1.8 ±7.6) h ], time of PACU [(26.4 ± 8.5 ) min vs.(37.2 ± 1 1.6 ) min ], the total days of hospitalization [(7.5±0.9)d vs.(9.7±1.2)d]were significantly shorter (P <0.05),and hospital costs [(2.1±0.6)ten thousand yuan vs.(2.6±0.8)ten thousand yuan]were significantly decreased (P <0.05).The incidence of adverse reactions such as nausea and vomiting (2.4% vs.21.4%),pru-ritus (7.1% vs.23.8%),agitation (4.8% vs.26.2%)and chills (0% vs.1 9.0%)were significantly lower in group E (P <0.05).Conclusion ERAS program applied to patients undergoing laparoscopic colorectal resection can reduce the intraoperative sufentanil consumption,avoid the occurrence of postoperative hypothermia, accelerate recovery of gastrointestinal function, which can obviously reduce the hospitalization costs and shorten the hospitalization time.

With the rapid development of industrial biotechnology, breakthrough in enzymology and biocatalysis has been made in recent years, especially in areas of stability and activity of enzyme in nonaqueous media, screening, construction and modification of solvent-tolerant biocatalysts, as well as the development of green solvent with excellent biological and environmental compatibility. Recent trend and future focus include: in silico virtual screening and construction of solvent-tolerant biocatalysts based on bioinformatic technology, modification and construction of native solvent-tolerant biocatalysts, the development of environmental friendly green solvent such as ionic liquids.
Animals ; Biocatalysis ; Biotechnology ; trends ; Enzymes ; chemistry ; genetics ; Humans ; Solvents

AIM:To construct myocardium-specific nucleolin ( Ncl) transgenic mice and to provide an animal model for the studies of the myocardial protection of nucleolin .METHODS:To create nucleolin transgenic mice , a myo-cardium-specific expression plasmid of nucleolin ( Alpha-MyHC clone 26-Ncl) was constructed .The gene type of transgenic mice was identified by PCR and the nucleolin protein level was tested by Western blotting .The myocardium morphology , heart weight index (HWI) and left ventricular pressure maximum rise rate were observed in nucleolin transgenic (TG) mice and wild-type ( WT) mice.RESULTS:We gained 4 transgenic mice (51, 52, 56 and 86 lines, only 52 line and 86 line were eugonic) by PCR.Western blotting analysis showed the expression of nucleolin up-regulated specifically in the myocardium .However , the myocardium morphology , HWI and left ventricular pressure maximum rise rate in the nucleolin transgenic mice were similar to those in the wild-type mice.CONCLUSION:We constructed myocardium-specific nucleo-lin transgenic mice successfully .