Objective To investigate the clinicopathological features and risk factors of lymph node metastasis of gastrointestinal neuroendocrine neoplasms (GI-NENs).Methods The retrospective case-control study was conducted.The clinicopathological data of 467 patients with GI-NENs who were admitted to the Fourth Hospital of Hebei Medical University from January 2006 to December 2015 were collected.Observation indicators:(1) occurrence sites and pathological classification of GI-NENs;(2) pathological characteristics of surgical specimens of GI-NENs;(3) univariate analysis and multivariate analysis affecting lymph node metastasis of GI-NENs:sex,age,tumor location,tumor diameter,pathological classification,pathological stage and tumor invasive depth.The univariate analysis and multivariate analysis were respectively done using the chi-square test and Logistic regression model.Results (1) Occurrence sites and pathological classification of GI-NENs:of 467 patients with GI-NENs,tumors of 304,15,7,14 and 127 patients were located at stomach,duodenum,small intestine,colon and rectum,respectively.Tumor diameter was 0.3-12.0 cm,with an average diameter of 2.2 cm.Of 467 patients with GI-NENs,G1 and G2 of neuroendocrine tumors (NETs),G3 of neumendocfine carcinomas (NECs) and mixed adenoneuroendocfine carcinomas (MANECs) were respectively detected in 209,64,146 and 48 patients.Lymph node metastasis rate of GI-NENs was 31.48％ (147/467).(2) Pathological characteristics of surgical specimens of GI-NENs:NETs were high-differentiated NENs.Ceils of NETs were solid and nest-,trabeculum-and tubular-shaped,and consisted of small or medium cells,with moderate amount or massive cytoplasms,round or oval nucleus,particle-shaped chromatin,unobvious nucleolus and positive endocrine markers.There were abundant of small blood vessels and surrounding fibrous stroma in peripheral tumor cell nests.NECs were low-differentiated NENs and included small cell carcinoma and large cell NEC.Cells of small cell carcinoma were small round or oval and looked similar to lymphocytes,with few amount cytoplasms,fine granularshaped or hyperchromatic nucleus and common mitosis figures.Cells of large cell NEC were large and greater than 3 lymphocytes,arrayed in organoid-or chrysanthemum-shape,with massive cytoplasms,coarse particle-shaped chromatin,obvious nucleus,clear mitosis figures and large laminar-shaped necrosis.There were different positive expressions of endocrine markers between small cell carcinoma and large cell NEC.MANECs had the characteristics of glandular cavity formation of traditional adenocarcinoma and NENs.Results of immunohistochemical staining in 467 patients showed that Ki-67 of 467 patients was positive;CD56 in 379 of 428 with CD56 test was positive;synaptophysin (Syn) in 416 of 422 with Syn test was positive;cytokeratin (CK) in 354 of 396 with CK test was positive;chromogranin (CgA)in 264 of 388 with CgA test was positive;neuron specific enolase (NSE) in 287 of 346 with NSE test was positive.(3) Univariate analysis and multivariate analysis affecting lymph node metastasis of GI-NENs:results of univariate analysis showed that sex,tumor location,tumor diameter,pathological classification,pathological satge and tumor invasive depth were related factors affecting lymph node metastasis of patients with GI-NENs (X2 =20.654,18.182,26.788,184.709,163.738,195.391,P＜ 0.05).Results of multivariate analysis showed that pathological classification and pathological stage were independent influenced factors affecting lymph node metastasis of patients with GI-NENs (HR =2.129,7.171,95％ confidence interval:1.273-3.561,-2.327-22.098,P＜0.05).Conclusions GI-NENs are mostly located on the stomach and rectum.Results of immunohistochenical staining could help diagnosis of GI-NENs.Pathological classification and pathological stage are independent influenced factors affecting lymph node metastasis of patients with GI-NENs.

A 50?year?old woman presented with intermittent dull pain in the forehead and mild dizziness occasionally after her forehead was subjected to a mild bump accidentally 20 days prior to the presentation, and was diagnosed with angioneurotic headache in a local hospital. After the treatment with oral sibelium tablets, the condition wasn′t relieved obviously. Computed tomography (CT) scan showed multiple localized bone destruction and low?density area in the frontal and bilateral parietal bones with adjacent soft tissue swelling. Magnetic resonance imaging(MRI)revealed equal T1 signals and slightly long T2 signals for multiple nodules in the frontal and bilateral parietal bones, high signals on diffusion?weighted imaging (DWI), obvious enhancement on contrast?enhanced MRI, and linear enhancement in adjacent meninges. Whole?body bone scintigraphy showed multiple increased radionuclide uptake in the skull. Laboratory examination demonstrated that specific antibodies to Treponema pallidum (Tp) were positive, and the serum rapid plasma reagin(RPR)titer was 1∶128. Cerebrospinal fluid(CSF)examination showed normal CSF pressure, nucleated cell counts(8 × 106/L)and glucose level(4.0 mmol/L), slightly high chloride flux(129.1 mmol/L), high protein level(0.9 g/L), high CSF?RPR titer of 1∶16 and presence of specific antibodies to Tp. Histopathological examination revealed hyperemia of adjacent tissues in the cranial osteolytic area, hyperplasia of interstitial fibrous tissue, endothelial cell swelling, and infiltration of inflammatory cells mainly containing plasma cells. The treatment regimen for neurosyphilis was given, and headache was relieved after 1 week of treatment, basically disappeared after 2 weeks, and completely disappeared after 4 weeks, and no similar headache occurred thereafter. Finally, the patient was diagnosed with acquired syphilitic skull osteomyelitis complicated by syphilitic meningitis.

Objective:To design the small ubiquitin modification-fibroblast growth factor receptor 4 (SUMO-FGFR4) fusion gene and construct the expression vector pET22b-SUMO-FGFR4, to optimize the expression conditions. Methods:The SUMO-FGFR4 fusion gene was obtained by Overlap PCR and was connected to pET22b;the recombinant expression vector pET22b-SUMO-FGFR4 was obtained. The influence of lactose concentration, induction time,induction temperature,induction point and adding mode of lactose in the expression levels was observed,and the best induction condition was determined; then the solubility of recombinant protein was analyzed.Results:The SUMO-FGFR4 fusion protein was highly expressed,the molecular weight of the fusion protein was about 40 000 and it could bind with FGFR4 specific antibody.When the lactose concentration was 1.0 g·L-1 ,the induction time was 3 h,the induction temperature was 37℃,the value of A (600)was 0.8,the expression level was highest;but adding mode of lactose had no remarkable effect on the protein expression.The expression level of recombinant protein induced by lactose was higher than IPTG.SUMO-FGFR4 protein existed in a form of inclusion body.Conclusion:The SUMO-FGFR4 fusion protein is expressed successfully in this study while lactose is used as inducer and the best expression conditions are confirmed.

Objective To analyze the genetic characteristics and the evolution of the influenza A (H3N2) virus strains circulating in Jiangsu province between 2013 and 2014.Methods This study analyzed thirty-one representative strains of influenza A (H3N2) virus, which were isolated in different regions of Jiangsu province and during different time periods from 2013 to 2014.Results Genetic distances in nucleic acid and amino acid between a strain used for vaccine production (A/Texas/50/2012) and the 31 strains were 0.010 5 and 0.012 4.Similarities between them in nucleic acid and amino acid sequences were 97.9%-99.6% and 97.2%-99.3%.Phylogenetic analysis showed that the hemagglutinin (HA) genes of the 31 strains were divided into three different groups.Three strains isolated in 2013 and three strains isolated in 2014 belonged to Group 1 and Group 2, respectively, while the others belonged to Group 3.Three positive selection sites (237, 366 and 367) in HA protein were observed by REL model.Compared with the strain used for vaccine production, the 31 strains were characterized by amino acid substitutions (N128A/T and P198S/A) in HA protein and all of the mutations located in B-cell epitopes.The total number of mutation sites reached 24.Compared with the A/Texas/50/2012 strain, seven strains presented the glycosylation site 126NWT, and three strains showed disappeared glycosylation sites of 45NSS and 144NNS.Evaluation of vaccine efficacy for A(H3N2) virus strains showed that the vaccine efficacy was not very well.Conclusion The HA gene of A(H3N2) virus had undergone a greater variation and the vaccine efficacy was not very well in Jiangsu province during 2013 to 2014, which made the influenza A(H3N2) virus become the circulating strain.

Objective To select the herba anti- human papillomavirus (HPV) active fraction from herba Arnebia. Methods The fractions from herba Arnebia. were separated with systematic solvents including petroleum arieal part of benzin, n- butanol , ethanol and distilled water, and their effects on HPV- DNA were evaluated by fluorescence quantitative polymerase chain reaction (FQ- PCR) technique. Results Only the water- extract of.this drug showed in- vitro inhibitory effect on HPV- DNA and its minimum effective concentration is 0.08g/mL. Conclusion Herba Arnebia. has in- vitro inhibitory effect on HPV- DNA and the active components exists in the water- extract.

Objective: To investigate the expressions of programmed death ligand 1（PD-L1）in triple-negative breast cancer (TNBC) and its correlation with angiogenesis. Methods: 120 cases of TNBC patients who underwent surgery in the Fourth Hospital of Hebei Medical University from March 1, 2011 to June 1, 2012 were collected. The tumor tissues of patients were surgically resected and confirmed by pathology. PD-L1 protein expression in TNBC tissues of 120 patients was detected by tissue microarray combined with immunohistochemistry, and its relationship with various clinical indicators was analyzed. Blood vessels and lymphatic vessels were labeled withCD34andD2-40todetectmicrovesseldensity(MVD)andlymphaticvesseldensity(LVD)inTNBC.Results:Thepositiveexpression rate of PD-L1 in the tumor cells and interstitial infiltrating lymphocytes fromTNBC was 56.7% (68/120); No correlation was found between PD-L1 protein expression and the gender, age, histological grade, clinical stage, or tumor size of patients with TNBC (P>0.05), but related to the lymph node metastasis (P<0.05) and vascular thrombus (P<0.05). TNBC with high PD-L1 expression exhibited high incidence of lymph node metastasis and formation of vascular thrombus, and the expression of PD-L1 was positively correlated with MVD (r=0.500, P=0.02) as well as LVD (r=0.662, P=0.01). Log-Rank test showed that the survival time of TNBC patients with positive PD-L1 protein expression was significantly shorter than that of patients with negative expression (P<0.05). Cox multivariate analysis suggested that PD-L1 protein expression could be an independent prognostic factor for TNBC overall survival. Conclusion: PD-L1 plays an important role in TNBC angiogenesis and lymphangiogenesis, and is closely related to TNBC invasion and metastasis; blocking PD1/PD-L1 signal pathway is expected to be an effective new strategy for TNBC treatment.

Objective:To construct GJB2 gene mutaitons common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutaitons in GJB2 gene. Method: Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutaiton methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA cloning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutaions were inserted into pEG-FP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were trans-fected with the recombinant DNA samples by the liposome complex method. Results The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence singals were distributed uniformly in cytoplasm. Conclusion; GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.

Objective To evaluate effects of tea polyphenols on the mRNA and nucleoprotein expression of Nrf2/Bach1 in human skin fibroblasts (HSFs).Methods Some HSFs were incubated with tea polyphenols at different concentrations of 0,2.5,5,10,20 and 40 mg/L for 24 hours.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate the proliferative activity of HSFs to screen the optimal concentration of tea polyphenols.Then,some other HSFs were treated with tea polyphenols at this optimal concentration for 24 hours.Real-time quantitative PCR (RT-qPCR) was performed to determine mRNA expression of Nrf2 and Bach1,Western blot analysis to measure nuclear expression of Nrf2 and Bach1 proteins,and immunofluorescence assay to determine the distribution of Nrf2 and Bach1 protein in the cell nucleus.Results MTT assay showed that 5 mg/L tea polyphenols had no obvious effects on the proliferation of HSFs,so 5 mg/L was chosen as the optimal concentration of tea polyphenols for subsequent experiments.HSFs cultured without tea polyphenols served as control group.After the treatment,the 5-mg/L tea polyphenol group showed significantly decreased mRNA and nuclear protein expression of Bach 1 (mRNA:0.629 ± 0.077 vs.0.940 ± 0.033,t =6.397,P ＜ 0.05;protein:1.424 ± 0.171 vs.16.966 ± 1.702,t =15.730,P ＜ 0.05),but significantly increased mRNA and nuclear protein expression of Nrf2 (mRNA:1.467 ± 0.076 vs.0.977 ± 0.091,t =7.133,P ＜ 0.05;protein:6.929 ± 0.121 vs.3.537 ± 0.126,t =33.636,P ＜ 0.05) compared with the control group.Immunofluorescence assay showed increased accumulation of Nrf2 protein,but decreased accumulation of Bach1 protein in the nucleus.Conclusion Tea polyphenols can promote the mRNA and nuclear protein expression as well as nuclear distribution of Nrf2,but suppress the mRNA and nuclear protein expression as well as nuclear distribution of Bach 1,finally exerting antioxidative effects.

Objective: To investigate the effect of extensive retraction clefts (RC, >20% of tumor volume) on prognosis in invasive breast carcinoma of no specific type (IBC-NST). Methods: A total of 2 184 cases of IBC-NST diagnosed at the Fourth Hospital of Hebei Medical University from January 2006 to December 2008 were collected. All the cases were diagnosed according to the latest guideline and standard. After excluding cases of shrinkage due to tissue fixation, 483 cases with RC were identified, and the clinical and pathological features were retrospectively analyzed. Results: Among the 483 cases, the mean tumor size was 2.0 cm (range 0.8 to 4.8 cm). Two hundred and thirty-two cases were moderately differentiated (48.0%), 97 were well differentiated (20.1%), 154 were poorly differentiated (31.9%); 382 (79.1%) cases were of stages Ⅰ and Ⅱ. A total of 177 cases (36.7%) had lymphatic invasion; nodal metastasis were found in 202 cases (41.8%). Extensive RC was found in 237 of 483 cases (49.1%). Follow-up information was available in 407 patients, and 46 died of breast cancer with survival time from 37 to 103 months. Multivariate analysis of extensive RC showed that tumor size, histological grade and nodal metastasis were risk factors of patients with IBC-NST (P<0.05). Lymphatic invasion and nodal metastasis were risk factors for extensive RCs in patients with IBC-NST (P<0.05). There was a high probability of lymph node metastasis in patients of extensive RC without lymphatic invasion, and the difference was statistically significant (P<0.05). Conclusions Both lymphatic invasion and nodal metastasis are risk factors of extensive RC. The presence of extensive RC in IBC-NST patients is correlated with poor outcome. Tumors with lymphatic invasion are more likely to show extensive RC.

Objective:To investigate the effect of pterostilbene on the growth, apoptosis and autophagy of a human papillomavirus type 16 (HPV-16) -immortalized cervical epithelial cell line H8.Methods:H8 cells were treated with pterostilbene at different concentrations of 0 (control group) , 25, 50, 75, 100 μmol/L for 24 and 48 hours. Cell counting kit-8 (CCK8) assay was performed to evaluate the cellular proliferative activity, flow cytometry was conducted to detect apoptosis and cell cycle, monodansylcadaverine (MDC) staining and fluorescence microscopy were performed to detect autophagy, and Western blot analysis was conducted to determine the expression of the cell cycle-related protein cyclinD1, apoptosis-related proteins caspase-3 and caspase-9, autophagy-related proteins Beclin1, microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ/Ⅰ, ATG5 and P62, as well as HPV oncoproteins E6 and E7. Statistical analysis was carried out by using one-way analysis of variance, repeated measures analysis of variance and least significant difference- t test. Results:After 48-hour treatment with pterostilbene at different concentrations of 0, 25, 50, 75, 100 μmol/L, the relative cellular proliferation rate significantly differed among the groups (100.00% ± 1.56%, 99.02% ± 4.97%, 93.59% ± 2.01%, 81.28% ± 4.90%, 69.17% ± 7.56%, respectively; F = 77.22, P < 0.05) , and gradually decreased along with the increase in the concentration of pterostilbene; compared with the control group, the pterostilbene groups all showed significantly decreased cellular proliferation rate (all P < 0.05) . After 24-hour treatment with pterostilbene, the proportions of H8 cells at G1, G2 and S phases significantly differed among the above groups ( F = 7 845.00, 51.14, 266.50, respectively, all P < 0.05) ; compared with the control group, the pterostilbene groups showed significantly increased proportions of H8 cells at G1 and G2 phases (all P < 0.05) , but significantly decreased proportions of H8 cells at S phase ( P < 0.05) . After 48-hour treatment with pterostilbene, the apoptosis rate was significantly higher in the 25-, 50-, 75- and 100-μmol/L pterostilbene groups (14.66% ± 0.22%, 13.50% ± 0.49%, 14.56% ± 0.19%, 15.30% ± 0.76%, respectively) than in the control group (11.58% ± 0.50%, all P < 0.05) . After 24-hour treatment with pterostilbene, MDC staining showed only a small number of H8 cells with bright dot-like fluorescence in the control group, but increased number of autophagosome-positive H8 cells with bright dot-like fluorescence in the pterostilbene groups. Western blot analysis revealed that there were significant differences in the protein expression of cyclin D1, caspase-3, caspase-9, Beclin1, LC3-Ⅱ/Ⅰ, ATG5, P62, E6 and E7 among the control and pterostilbene groups after 24- and 48-hour treatment with pterostilbene (all P < 0.05) . The treatment with pterostilbene could down-regulate the expression of cyclin D1, E6 and E7, and up-regulate the expression of caspase-3, caspase-9, Beclin1, LC3-Ⅱ/Ⅰ, ATG5 and P62, with significant differences between the control group and most pterostilbene groups in expression of the above proteins (all P < 0.05) . Conclusion:Pterostilbene can inhibit the proliferation of H8 cells, promote their apoptosis and autophagy, and down-regulate the expression of oncogenes E6 and E7.