Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Diagnosis, Differential ; Histiocytoma, Benign Fibrous ; metabolism ; pathology ; surgery ; Humans ; Male ; Middle Aged ; Neprilysin ; metabolism ; Receptors, Cell Surface ; metabolism ; Skin Neoplasms ; metabolism ; pathology ; surgery ; Thigh ; Vimentin ; metabolism

Objective: To establish a multiplex PCR-microarray method for detecting important enteropahogens.Methods: Uniplex and multiplex PCR were performed to obtain the best primer sets for identifying the target bacteria at species and multi-species level.Fluorescent dyes were mixed into PCR reaction to determine whether it can affect the efficiency of amplification.To improve the efficiency of microarray,a 35 pairs primer-labeling system was optimized based on the hybridization results to find the best combination to avoid false negative results.Results: Specific PCR products were all obtained using species-specific primer sets.More preferential amplification may happen when more primer pairs were added to the reaction.The hybridization results showed a positive association between the efficiency of multiplex-PCR and signal intensity.Conventional PCR yielded more products than fluorescent dyes labeled PCR.Thirty-five primers were divided into three different combinations to label target respectively,hybridization results showed a high specificity.Conclusion: Mixing fluorescent dyes into PCR may reduce the efficiency of amplification and hybridization,but may have no effect on the analysis of hybridization results.The hybridization efficiency of microarray depends on the amplification efficiency of multiplex PCR.For microarray target labeling,three primer sets could be used to avoid negative hybridization led by preferential amplification of multiplex-PCR.It indicates that the multiplex PCR-microarray method is an attractive diagnosis tool for the high-throughput identification of enteropathogenic organisms especially for multiple causative agents and epidemiological investigations.

Objective To determine the impact of fluorine and aluminum,and both action combined on the number of rat osteoclasts and bone resorption cultured in vitro and to explore its mechanisms.Methods The osteoclasts and bone marrow stromal cells (BMSCs) isolated from long bone of new born rats were cultured,respectively,in TC199 medium (containing 10％ fetal bovine serum) with fluoride,aluminum and fluoride combined with aluminum.The osteoclasts were inoculated in 96-well culture plate and ivory slice,BMSCs in 6-well culture plate,and culture medium was changed after 2 hours incubation.The cells were divided into control group,fluoride group,aluminum group and fluoride combined with aluminum group; the doses of sodium fluoride were 0,1.0 × 10-4,0,1.0 × 10-4 mol/L and the doses of aluminum chloride were 0,0,1.0 × 10-5,1.0 × 10-5 mol/L,respectively.Tartrate-resistant acid phosphatase (TRAP) staining positive cells were counted under light microscope after TRAP staining on the 5th day and the pit formed in ivory slices were measured by histomorphometry after staining with toludine blue.The expression of osteoprotegerin(OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) was detected by real-time fluorescence quantitative PCR in BMSCs after 8 h treatment.Results ① Fluoride,aluminum and the interactive effects of fluoride and aluminum all had impact on the numbers of osteoclasts (F =7.15,6.56 and 7.98,respectively,all P ＜ 0.05).The numbers of osteoclasts in fluoride group,aluminum group and fluoride combined with aluminum group[(136.9 ± 22.99),(135.4 ± 23.5),(163.0 ± 24.4) per well] were higher than that in the control group[(92.5 ± 22.1) per well,all P ＜ 0.05].② Fluoride,aluminum and the interactive effects of fluoride and aluminum all had impact on the resorption pit area on ivory slices(F =10.47,12.64,14.29,respectively,all P ＜ 0.05).The resorption pit area on ivory slices in fluoride group,aluminum group and fluoride combined with aluminum group[(0.242 ± 0.031),(0.293 ± 0.026),(0.333 ± 0.016)mm2 per slice] was higher than that in the control group [(0.088 ± 0.030)mm2 per slice,all P ＜ 0.05].③Fluoride,aluminum and the interactive effects of fluoride and aluminum all had impact on the expression ratios of RANKL/OPG in BMSCs (F =8.15,15.38,23.59,respectively,all P ＜ 0.05).The expression ratios of RANKL/OPG in BMSCs in fluoride group,aluminum group and fluoride combined with aluminum group [(193.98 ± 137.93)％,(326.11 ± 176.78)％,(599.84 ± 275.82)％] were higher than that in the control group[(100.00 ± 56.02)％,all P ＜ 0.05].Conclusions Both fluoride and aluminum can cause increase in the number of osteoclasts in vitro and promote cell differentiation and bone resorption activity,which may be related to increased expression ratio of RANKL/OPG mRNA in BMSCs.The stimulating effects of fluoride on osteoclasts differentiation and bone resorption is enhanced by aluminum.

Objective To determine the effects of fluoride on osteoclasts's quantity and bone resorption function in vitro and its mechanisms. Methods The osteoclasts and bone marrow stromal cells(BMSCs) isolated from long bone of new born rats were cultured respectively in TC199 medium (containing 10% fetal bovine serum) with fluoride. The osteoclasts were inoculated in 96-well culture plate and ivory slice, BMSCs were inoculated in 6- well culture plate, respectively, medium were changed after 2 hours incubation. They were divided into control group, low-dose fluoride, medium-dose fluoride and high-dose fluoride groups, the doses of sodium fluoride were 0,2.5 × 10-5,5.0 × 10-5,10.0 × 10-5 mol/L, respectively. Tartrate-resistant acid phosphatase(TRAP) staining positive cells were counted under light microscope after TRAP staining on the 2nd and the 5th day and the pit formed in ivory slices were measured by histomorphometry after staining with toludine blue. The expression of receptor activator of NK-κβ ligand(RANKL) and osteoprotegerin(OPC) was detected by real-time fluorescence quantitative (337.5 ± 70.5), (447.5 ± 43.4), (472.9 ± 34.8), (475.3 ± 24.3)/well in the control group, the low-dose, mediumdose and high-dose fluoride groups, respectively. The differences were statistically significant between these groups and the control group (all P < 0.05). After in vitro culture for 5 days, the numbers of osteoclasts were (92.5 ± 22.1), (123.0 ± 26.4), (135.5 ± 22.2), (136.9 ± 23.0) per well in the control group, the low-dose, medium-dose and high-dose fluoride groups, respectively. The differences were statistically significant between these groups and the (0.088 ± 0.030), (0.100 ± 0.018), (0.152 ± 0.015), (0.242 ± 0.031 )mm2 per piece in the control group, the lowdose, medium-dose and high-dose fluoride groups, respectively. The values of medium-dose and high-dose fluoride BMSCs in the control group, the low-dose, medium-dose and high-dose fluoride groups were 100.00 ± 56.02, 144.95 ± 97.21,223.25 ± 184.48,193.98 ± 137.93, respectively. The values of medium-dose and high-dose fluoride groups were significantly higher than that of control group (all P < 0.05). Conclusions Fluoride can cause increase in the number of osteoclasts in vitro and promote their cell differentiation and bone resorption activity, which may be related to increased expression ratio of RANKL/OPG mRNA in BMSCs.

Objective To explore the influence of fluorine on mRNA and protein expression of the insulin-like growth factor-1 (IGF-1) and its receptor of rat osteoblasts.Methods Osteoblasts were isolated from rat bone by enzyme digestion.Different fluorine concentration [0 (control),10-7,10-6,10-5,10-4,10-3 mol/L] was add to the second generation osteoblasts.The IGF-1 in the culture medium was determined by radioimmunoassay (RIA) at different fluorine concentration and different time (24,48 h).The expression of IGF-1 receptor was measured by the method of fluorescent quantitation PCR and the expression of protein IGF-1 receptor was measured by Western blotting.Results ①With increased dose of fluoride exposure,IGF-1 concentration in the osteoblastic culture medium increased first and then decreased at 24,48 h,respectively.Compared to the control group [(38.83 ± 3.48)ng/L],IGF-1 concentration of the 24 h 10-6 mol/L group[(65.45 ± 4.84)ng/L] was higher,and the difference was statistically significant(P ＜ 0.05).The same result was also shown in the 48 h 10-5 mol/L group [(59.14 ± 1.53)ng/L] to its corresponding control group [(33.79 ± 1.84)ng/L,P ＜ 0.05].②The mRNA expression of IGF-1 receptor of the 24,48 h 10-5 mol/L groups (0.0055 ± 0.0004,0.0262 ± 0.0040) was significantly higher than their corresponding control groups (0.0022 ± 0.0001,0.0073 ± 0.0008,all P ＜ 0.05).③With increased dose of fluoride exposure,the protein expression of IGF-1 receptor increased first and then decreased ;the expression of 24 h 10-5 mol/L group (1.39 ± 0.16) was compared with the corresponding control group (0.86 ±0.12),and the difference was statistically significant (P ＜ 0.05) ; the expression of 48 h every fluorine group was also compared with the corresponding control group,and the difference was not statistically significant(all P＞ 0.05).Conclusions Fluorine can affect the mRNA and protein expression of osteoblastic IGF-1 and its receptor.It indicates that IGFS signal transduction pathways play an important role in fluorine regulation of bone metabolism.

Objective To evaluate the b value of diffusion-weighted(DW)MRI in distinguishing between benign and malignant breast lesions.Methods Three diffusion-weighted sequences were implemented with 500,1000 and 2000 s/mm~2 b values respectively on 95 breast lesions in 83 patients.All lesions were confirmed by pathology.The apparent diffusion coefficient(ADC)values and signal intensity (SI)were recorded and compared in different lesions(breast cancer,benign lesion,cyst and normal beast tissue)with the same b value and the same lesions with the different b values.Results(1)The mean ADC value and SI of breast cancer were 1.375?0.378 and 839.713?360.493 respectively with b= 500 s/mm~2,1.176?0.311 and 459.314?229.609 with b=1000 s/mm~2,0.824?0.198 and 243.825? 110.616 with b=2000 s/mm~2.The differences in the mean ADC value were significant between two type lesions(cancer and benign lesion,cancer and cyst,cancer and normal breast tissue)with b values of 500 s/mm~2 and 1000 s/mm~2.But the significant differenee was only seen between cancer and benign lesions when b value was 2000 s/mm~2.(2)The one-side upper limits of 95% confidence interval of mean ADCs were adopted as the point to separate the malignant from the benign lesions,the sensitivity was 70.92%, 70.73% and 69.77%,the specificity was 77.19%,75.70% and 54.76%,the accuracy was 77.12%, 74.32% and 62.35% respectively with b values of 500 s/mm~2,1000 s/mm~2 and 2000 s/mm~2.The areas under ROC eurves were Az_(500)=0.775?0.046(P0.05).Conclusion DWI MRI is useful for the differential diagnosis of breast lesions with b values of 500 s/mm~2 and 1000 s/mm~2.

Objective To investigate the morphological features of normal lumbar dorsal root ganglia using a three-dimensional(3D)coronal MR imaging.Methods One hundred and fifteen volunteers were included.Ages ranged from 15 to 75 years,with a mean of 40 years.Coronal 3D fast field echo(FFE) with water selective excitation(Proset)MR examination of 1150 dorsal root gangha were underwent at nerve root levels from L1 to L5.The source coronal images were further reconstructed into a series of rotational alignment coronal images with an interval angel of 12 degree using maximum intensity projection(MIP) technique.All DRGs of bilateral spinal nerve from L1 to L5 were morphologically analyzed on the original and MIP images including qualitative evaluation of the location,signal intensity,architecture and quantitative dimensional measurement.Results There were 225,225,219,210 and 160 foraminal ganglia from L1 to L5 level,respectively.The incidence of intraspinal ganglia from L3 to L5 gradually increased with a maximum at L5 level of 29.1%(X~2=188.371,P

Objective To assess the clinical values of MR angiography(MRA)in the detection of deep vein thrombosis of the lower limbs.Methods Two-dimensional time of flight(2D TOF)MRA was performed in thirty patients who were suspected of having deep vein thrombosis in the lower limbs.The findings of MRA were compared to that of digital subtraction angiography(DSA).Results twenty-five cases showed deep vein thrombosis in the lower limbs,the MRA findings included venous filling defect (14 cases),occlusions and interruptions of veins(8 cases),venous recanalizations(3 cases),collateral veins(25 cases).Taking the results of DSA as a golden standard,MRA detected all of the affected cases with only one case as the false positive.Conclusion 2D TOF MRA is a method of choice in the diagnosis of deep vein thrombosis of the lower limbs.

Objective To study the exact function of human gene 5 transactivated by pre-S1 protein of hepatitis B virus(PS1TP5)by investigating the gene expression of PS1TP5 in yeast cells. Methods Reverse transcription-polymerase chain reaction(RT-PCR)was performed to amplify the gene of PS1TP5 using the mRNA of HepG2 cells as template and the gene was cloned into pGEM-T vector.The gene of PS1TP5 was cut from pGEM-T-PS1TP5 vector and cloned into yeast expressive plasmid pGBKT7,then pGBKT7-PS1TP5 was transformed into yeast cell AH109.The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western hybridization.Results PS1TP5 gene was successfully amplified and identified by DNA sequencing.The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109.The results of SDS-PAGE and Western assay showed that the relative molecular weight of the expressed product was about 36 950,and PS1TP5 protein existed in yeast cells.Conclusion The findings suggest that PS1TP5 can be successfully expressed in yeast cell.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer with a very poor prognosis.In order to guide better clinical management of ICC patients,the American Joint Committee on Cancer (AJCC) cancer staging manual (7th edition) have established a unique TNM staging scheme for separating ICC from hepatocellular carcinoma (HCC) for the first time,and reflected a difference between risk factor of ICC and HCC.This TNM staging system for ICC has been most recently updated by the AJCC cancer staging manual (8th edition),in which T staging has been redefined without gross features,and lymph node metastasis (N1) in N staging has been grouped as stage Ⅲ B,but not stage Ⅳ as required by the 7th edition of AJCC cancer staging manual.In addition,region lymphatic and distant metastases have been clearly redefined by the AJCC cancer staging manual (8th edition) that also requires recovering at least 6 lymph nodes for the N staging scheme.The apparent advantages of the AJCC cancer staging manual (8th edition) for ICC pathologic staging may better stratify the prognosis of ICC patients and provide an improved guidance in clinical practice.