Objective: The optimal simultaneous separation process of three phenylethanoid glycosides (acteoside, isoacteoside, and torenoside B) from Monochasmasavatieri were established using of macrophage resins and dynamic axial column (DAC). Methods: The adsorption/desorption experiments and dynamic separation experiments were performed on eight types of resins (AB-8, D 101, HPD 100, LSA-10, LX-11, LX-17, LX-38, and XDA-6) to find the optimal resin. Then the optimal separation parameters were investigated on the chosen resin. The total phenylethanoid glycosides obtained from the large-scale experiment were further seperated to get acteoside, isoacteoside, and torenoside B, respectively using of DAC system. Results: Among these candidate resins, LX-17 was chosen to further obtain the optimal parameters: The optimal feeding concentration of raw materials was 1.8 g/mL; The optimal adsorption time was 150 min; The optimal gradient elute conditions were ethanol/water (0/100, 4 BV; 15/85, 4 BV; 60/40, 5 BV; 90/10, 2 BV). The large-scale experiments were amplified to 10 folds on the basis of optimal parameters to obtain total phenylethanoid glycosides. Acteoside, isoacteoside, and torenoside B were simultaneously obtained from total phenylethanoidglycosides using DAC system. Conclusion: LX-17 and DAC system can be used for the purification of phenylethanoid glycosides, which will have a goodfuture for the application in industry.

Objective To explore the diagnostic value of conventional ultrasound(US) and contrast enhanced ultrasound(CEUS) in predicting extrathyroidal extension of papillary thyroid cancer(PTC).Methods Eighty-five PTCs in 75 patients were selected for thyroid surgery underwent ultrasound and contrast-enhanced ultrasound.The degrees of contact between PTCs and capsule were observed by US and CEUS respectively(0,0-25％,25％-50％,≥50％),and the diagnostic efficiency in different degree of contact (＞0 ％,≥25 ％,≥50％) as preoperative diagnostic criteria were analyzed.The diagnostic efficiency between US and CEUS in predicting extrathyroidal extension of PTC were compared.Results Of the 85 PTCs,extrathyroidal extension was presented in 57 (67.06％) based on pathologic results.When the degree of contact (＞ 0 ％,＜ 25 ％,25 ％-50 ％,≥ 50 ％) was gradually increased,the incidence of extrathyroidal extension of the thyroid cancer was also gradually risen (P ＜0.001).Comparing the sensitivity,accuracy,odds ratio,and Az value of three groups(＞0％,≥25％,≥50％),it showed that the general diagnostic efficiency between two groups(＞0％,≥25％) was similar by US and CEUS.However,the sensitivity and accuracy of ＞0％ contact with the adjacent capsule were markedly higher than those of the other two groups(P ＜0.001).Selecting ＞0％ contact with the adjacent capsule as preoperative criteria,the Az value of CEUS was markedly higher than that of US (Z =2.208,P =0.027).Conclusions The preoperative imaging feature of more than 0％ contact with the adjacent capsule is more sensitive and accurate degree in predicting extrathyroidal extension of PTC.Compared with US,CEUS may serve as a better useful tool to predict extrathyroidal extension of PTC.

OBJECTIVEThe effect of advanced glycation end products (AGEs) on the osteogenic differentiation of humanperiodontal ligament stem cells(hPDLSCs) was discussed. Changes in the Wnt signaling pathway during glycation were also determined.

METHODSIn vitro tissue explanting method was primarily applied. Limiting diluted clone was cultured to obtain hPDLSCs in vitro. The subjects were divided into two groups: the healthy group (N-hPDLSCs) and the AGEs-stimulating group (A-hPDLSCs). Osteoblast mineralization was induced in the experimental groups. The following processes were performed: alizarin red staining; alkaline phosphatase (ALP) staining; real time polymerase chain reaction (real time PCR) for detecting osteogenic genes and Wnt classical pathway-related factors, DKK-1 and β-catenin; Western blot analysis. Bone protein and β-catenin were correlated in the nuclear expression.

RESULTSThe cells were osteogenically induced. ALP staining showed that the N-hPDLSCs displayed the deepest color. Alizarin red staining indicated that the A-hPDLSCs group had less calcified nodules than the N-hPDLSCs group. The real time PCR results suggested that the expression of relative osteogenic genes in A-hPDLSCs was quite low. Statistically significant differences in differentiation were found between groups (P < 0.05). The Western blot result was similar to that of real time PCR. Classical Wnt signaling pathway-related factor β-catenin was higher in A-hPDLSCs than in N-hPDLSCs. By contrast, DKK-1, which is an inhibitor in the Wnt pathway, had a significantly lower expression rate in A-hPDLSCs than in N-hPDLSCs. The Western blot result also showed that β-catenin expression in the nucleoprotein in A-hPDLSCs was notably higher than in N-hPDLSCs.

CONCLUSIONAGEs can inhibit hPDLSCs osteogenic differentiation. AGEs induce changes in the normal periodontal ligament stem cells classical Wnt pathway. Canonical Wnt pathway is reactivated because of AGEs stimulation.

Cell Differentiation ; Glycation End Products, Advanced ; Humans ; In Vitro Techniques ; Osteoblasts ; Osteogenesis ; Periodontal Ligament ; Stem Cells ; Wnt Proteins ; Wnt Signaling Pathway ; beta Catenin

OBJECTIVEThis study aims to detect microRNA-17(mir-17) expression on the osteogenic differentiation of advanced glycation end products (AGEs)-stimulated hunman periodontal ligament stem cells (HPDLSCs) and to analyze the influence of these cells on this process.

METHODSHPDLSCs were isolated using limited dilution technique. After osteogenic differentiation occurred, different time points of mir-17 expression in the experimental groups were detected by real time polymerase chain reaction (PCR). The mir-17 overexpression and inhibition were evaluated using cell transfection technique. Differences in gene expressions were detected by real time PCR; differences in protein expressions were analyzed by Western blot.

RESULTSThe mir-17 expression was reduced after osteogenic differentiation occurred at 3, 7, and 14 d compared with that in the control group (P < 0.05). The expression levels of bone sialoprotein (BSP), Runt-related transcription factor-2 (Runx-2)and alkaline phosphatase (ALP) in the experimental groups were lower than those in the mimic control group when mir-17 expression increased. In addition, the protein expression levels of Runx-2 in the experimental groups were lower than those in the control group. The expression levels of BSP, Runx-2 and ALP in the experimental groups were higher than those in the inhibitor control group when mir-17 expression decreased. Likewise, the protein expression levels of Runx-2 in the experimental groups were higher than those in the control group.

CONCLUSIONAGEs inhibit the osteogenic differentiation of HPDLSCs by affecting mir-17 expression.

Alkaline Phosphatase ; Cell Differentiation ; Glycation End Products, Advanced ; Humans ; MicroRNAs ; Osteogenesis ; Periodontal Ligament ; Stem Cells

Objective To study the enhanced patterns of papillary thyroid carcinoma (PTC)on contrast-enhanced ultrasound(CEUS),and explore the relationship between the degree of enhancement and extracapsular invasionand cervical lymph node metastasis of the tumor.Methods Seventy-three PTCs in 61 patients selected for thyroid surgery underwent conventional ultrasound andcontrast-enhanced ultrasound. The enhanced patterns were analyzed.The differences between different degree enhancement and extracapsular invasion and cervical lymph node metastasis of PTCs were compared.Results Seventy-three PTCs enhanced patterns showed that 52(71 .2%)nodules with hypoenhancement,13(1 7.8%)nodules with isoenhancement,8 (1 1 .0%)nodules with hyperenhancement.Twenty-three (44.2%,23/52 )nodules with extracapsular invasion in hypoenhancing patterns,and 1 5 (71 .4%,1 5/21 ) nodules with extracapsular invasion in isoenhancing or hyperenhancing patterns.There were significant differences between them(P <0.05).Twenty-five (48.1 %,25/52 ) nodules with cervical lymph node metastasis in hypoenhancing patterns,and 13 (61 .9%,13/21 ) nodules with cervical lymph node metastasis in isoenhancing or hyperenhancing patterns,there was no significant difference between them(P >0.05 ).Conclusions The degree of enhancement are correlate to invasiveness in PTCs,PTCs with isoenhancement or hyperenhancement patterns were more often with extracapsular invasion.

Objective To explore the feasibility for the tissue dispersion quantitative analysis software of ultrasonic strain elastography in diagnosing benign or malignant of thyroid lesions.Methods Eighty-two patients with 98 lesions were examined by ultrasonic strain elastography.There were 11 parameters of elastography imaging obtained by the tissue dispersion quantitative analysis software,including average relative strain value(MEAN),standard deviation of relative strain value(SD),area ratio of low-strain region (AREA％),complexity(COMP),kurtosis(KURT),skewness (SKEW),contrast (CONT),entropy(ENT),inverse different moment (IDM),angular second moment (ASM),correlation (CORR).The receiver operating characteristic (ROC) curve was constructed if there were statistically significant differences between benign and malignant lesions,and the areas under the ROC curve were got.Results All parameters except CORR had statistically significant between the groups of benign and malignant thyroid lesions (P ＜0.05).The AREA％ and IDM were the best valuable parameters,the areas under the curve(AUC) of which were 0.965 and 0.908,respectively.Their cut-off point were 81.96％ and 0.42,the sensitivity and specificitywere 98.4％ and 89.2％,91.8％ and 86.5％,respectively.Conclusions The tissue dispersion quantitative analysis software is helpful in the evaluation of benign ormalignant of thyroid lesions,parameters of AREA％ and IDM has the highest relationship with pathology and good diagnostic value.

Background Epithelial-mesenchymal transition (EMT) is a major event in the pathogenesis of posterior capsular opacification (PCO),and the expressions of α-smooth muscle actin (α-SMA) is the marker of EMT.Previous studies showed that breviscapine plays an important role in anti-fibrosis and suppression EMT,however,the mechanism of its effect on EMT in LECs is unclear.Objective Present study was to investigate the effect of breviscapine on the expression of α-SMA and fibronectin (FN) in human LECs induced by transforming growth factor-β2 (TGF-β2).Methods Human LECs strain,HLE-B3,was cultured and passaged in DMEM containing 10％ fetal bovine serum.Different concentrations of breviscapine (6.75,12.75,25.00,50.00 and 100.00 mg/L)were added into the medium for 24,48 and 72 hours respectively,and then cell cunting kit-8 (CCK-8) was used to evaluate the half maximal inhibitory concentration (IC50) of breviscapine to HLE-B3.In addition,HLE-B3 was subcultured at the density of 1 × 106/hole and divided into 4 groups.The cells were in free-serum medium as the normal control;the cells were exposed in 10 μg/L TGF-β2 as TGF-β2 group;while in the breviscapine group,10 mg/L of breviscapine was added into the culture medium and another group was the combination of 10 mg/L breviscapine and 10 μg/L of TGF-β2 treatment.Real-time PCR and Western blot were used to detect the mRNA expressions of α-SMA and FN as well as their protein in HLE-B3 72 hours after cultured.Results The inhibitory rate of breviscapine to HLE-B3 proliferation was gradually elevated with the increase of concentration of breviscapine,showing a significant inhibition of the cell proliferation among the different groups and at various time points (F =292.851,P=0.000;F =65.037,P=0.000).IC50 of HLE-B3 at 72 hours was 22 mg/L,and therefore,in rest of experiment 10 mg/L of breviscapine was used which was 1/2 only half of the IC50.α-SMA and FN were expressed in cultured normal HLE-B3.The expressing level of α-SMA mRNA and FN mRNA in the HLE-B3 was significantly different among the normal group,TGF-β22 group,breviscapine treatment group and combination group as well as at various time points (F =105.490,P =0.000 ; F =1041.414,P =0.000).Similarly,the protein expressions of α-SM A and FN in the HLE-B3 was significantly different among the four groups and different time points (F=136.872,P=0.000;F=119.820,P=0.000).The expression levels of α-SMA and FN mRNA and their proteins in HLE-B3 were remarkably increased in the 10 μg/L TGF-β2 group compared with the normal control group (at all P=0.000),and those in the combination group were obviously declined in comparison to the TGF-β2 group (P =0.001,0.001,0.001,0.010).No significant difference was found in the expressions of α-SMA and FN in the HLE-B3 between the breviscapine group and normal control group in both transcriptional level and protein level (P =0.551,0.292,0.551,0.360).Conclusions 10 mg/L breviscapine can arrest the proliferation and EMT of human LECs.This result suggests that using breviscapine may be a potential prophylactic approach in the prevention of PCO.

A serum method for evaluating therapeutic efficacy of acute leukemia has been built by magnetic beads-based biological mass spectrometric technology. After analysis of quality control sample, the reproduci-)bility) of within-run assay and between-run assay was obtained and consistent with the previous reports. This result) manifested that the method used for serum peptides analysis was stable and reliable. Serum samples from 30 patients with primary acute leukemia and those with complete remission after treatment were extracted by metal-chelated magnetic beads and detected by MALDI-TOF-MS. Peaks at the m/z range 1000-10000 were analyzed by FlexAnalysis 2.4 software and Biosun_MS software. Approximately 27 differential peaks that were of statistical significance(p<0.05) were obtained. Ten peaks were up-regulated) and 17 peaks were down-regulated) after complete remission, respectively. The diagnostic model based on these differential peaks achieved) 100% sensitivity and 90% specificity. The results indicated that the model based on magnetic beads extraction and MALDI-TOF-MS detection acquires high sensitivity and specificity. Further sequence identification of differential) peaks is useful for evaluation of therapeutic efficacy and mechanism research on acute leukemia.)

The capsid protein (VP60) gene of RHDV was subcloned into the Pichia expressin vector pPICZ B to express the VP60 protein intracellularly. The recombinant plasmid was initially transformed into a E. coli strain TOP10 F'. After verification of the construct by sequencing, the recombinant plasmid was linearized by Sac I in the 5' AOX1 region and then transformed into Pichia pastoris strain GS115 using the Pichia EasyComp Kit. After selecting and verifing for the insertion of VP60 gene in the genome, two clones of Pichia transformants were select for expression test. The recombinant clones were first inoculate with BMGY in baffled flask at 28-30 degrees C in a shaking incubator (250-300 r/min) until culture reaches an OD600 = 2-6, then resuspend the cell pellet to an OD6oo of 1.0 in BMMY medium to induce expression for 5 days by methanol at a concentration of 0.5% in a 1 liter baffled flask covered with 2 layers of sterile gauze. Collect the cell pellets and break it by acid-washed 0.5 mm glass beads. The expression of recombinant Pichia strains was detected by SDS-PAGE and Western analysis with a polyclonal serum which showed a specific protein band of 60kD. Theses results indicates that the recombinant VP60 produced in Pichia was antigenically similar to the viral polypeptide. Electron microscopic observation of the recombinant Pichia-derived protein revealed the presence of virus-like particles similar in size and appearance to native virus capsids. In the haemagglutination test, the recombinant VLPs, like the native RHDV, also agglutinated human blood type O erythrocytes and could be inhibited by the anti-RHDV polyclonal serum.
Animals ; Capsid ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Hemorrhagic Disease Virus, Rabbit ; genetics ; Pichia ; genetics ; metabolism ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Viral Structural Proteins ; biosynthesis ; genetics ; isolation & purification

Background The molecular biological mechanisms of diabetic retinopathy(DR)is unclear up to now.Researches have proved that endoplasmic reticulum stress(ER Stress)-associated factors are elevated in peripheral blood in patients with diabetic retinopathy.and P58 IPK can inhibit those factors.So the relationship between P58 IPK and DR is worth to investigate. Objective The aim of this study was to detect the dynamic expression of P58 IPK in the retina of diabetic rats. Methods The diabetic animal models were established in 18 clean male SD rats by intraperitoneal injection of stilptozotiein(STZ)at a dose of 60 mg/kg.The rats were sacrificed in 1,3,6 months after injection.The expression change of P58 IPK mRNA in the rats retina was detected by quantitative real-time RT-PCR.Other 6 matched normal rats were used as control groups.This experiment followed the Regulations for the Administration of Affair Concerning experimental Animals by State Science and Technology Commission. Results The rats showed more drinking,more food and more urine after STZ injection with the blood glucose level≥ 16.5 mmol/L.The success rate of diabetic models was 100%.The A value of P58 IPK mRNA/β-actin in rat retina was 0.800±0.005 and 0.975±0.008 after injection of STZ.and that of control rats was 0.725±0.006,showing statistically significant difference between them(t=22.589,t=62.784,P＜0.05).In 6 months after injection of STZ,the expression of P58 IPK mRNA in experimental diabetic rat retina was evidently lower than the eontrol rats(0.671±0.004 versus 0.725±0.006,t=-17.984,P＜0.05).Conclusion P58 IPK has a close relation to the pathogenesis of DR,and it plays a retarding role for DR.