Objective To establish a mouse lethal model of influenza B virus , which will facilitate the study on the mechanism of pathogenesis , transmission of influenza B virus , development of new vaccines and drugs against influenza B virus.Methods We obtained a mouse adaptive B/Lee/1940 virus by continuously passaging it in mice for 5 cycles.The P5 virus was propagated in MDCK cells , which was used for infecting mice .The body mass and survival rate of mice were monitored during the following 14 days after infection.At the same time,the 8 gene segments (PB2, PB1, PA, HA, NA/NB, NP, M, and NS) of P0 and P5 virus were sequenced and analyzed .Results and Conclusion Virus was detected in the lungs of mice in each generation in the process of virus passaging .The body mass of mice infected with the deadly mouse adaptive virus changed dramatically .The mortality of mice was 100%, and virus was detected in mouse lungs . Sequence analysis results indicated that the amino acid mutations occurred in PB 2 and NP.A series of experiments indicated that we had established a mouse lethal model of influenza B virus .

Objective:To explore whether apoptotic ovarian cancer cells induced by chemotherapy drugs paclitaxel and cisplatin can be cross-presented by dendritic cells(DCs) and enhance immune responses.Methods:DCs were induced from peripheral blood monocytes cells by GM-CSF/IL-4 for 6 d,then they were stimulated with either apoptotic ovarian cancer HO8910 cells,frozen-thawed HO8910 cells or control cells for 4 h.Their surface markers and phagocytotic ability were detected by flow cytometry and confocal microscopic scanning assay,respectively.DCs of different groups were cultured with CD8+ T cells isolated by magnetic cell sorting,and the ability of DCs to activate CD8+ T cells was evaluated by 3H-TdR,the activity of CTL to kill tumor cells was evaluated by LDH.Production of IFN-? by CD8+ T cells was measured by ELISPOT.Results:Apoptotic ovarian cancer cells induced by chemotherapy drugs paclitaxel and cisplatin could be phagocytized by DCs,which subsequently promoted the maturation and antigen presenting ability of DCs.Apoptotic ovarian cancer cells implused DCs significantly promoted proliferation of CD8+ T cells compared with that of control cells(P

Objective:To artificially design and express a recombinant protein named as ScFv-pLLO by fusing ScFv gene of Rituximab(C2B8)and dominant antigen epitopes from listeriolysin O(LLO),and studying its anti-tumor activity.Methods:VH and VL gene sequences of C2B8 against CD20 were acquired by searching United States Patent database,and ScFv sequence was constructed by linking VL and VH with a short peptide linker.Two CD4+T cell epitopes from LLO were selected and designed to splice ScFv sequence.The recombinant gene of ScFv-pLLO was cloned into prokaryotic expression vector and purified after induction.The capacity of ScFv-pLLO target-binding to B-cell lymphomas was evaluated by flow cytometry ( FCM ) and co-immunoprecipitation ( Co-IP ) .The effects of ScFv-pLLO on B-cell lymphomas proliferation and apoptosis were detected respectively.The immunogenicity of ScFv-pLLO was assessed by lymphocyte proliferation assay.Results: ScFv-pLLO was successfully expressed.It could bind to different B-cell lymphomas cell lines and obviously inhibit the growth of Raji cells as well as inducing apoptosis.Moreover,ScFv-pLLO was able to stimulate proliferation of spleen lymphocytes of immunized mice.Conclusion: The recombinant protein ScFv-pLLO can target-bind to B-cell lymphomas,and perform inhibitory effect and induce apoptosis on Raji cells that indicate ScFv-pLLO retain the capacity of ScFv derived from monoclonal antibody against CD20.Besides, ScFv-pLLO can induce immune response.This study provides a basis for further research about the role of ScFv-pLLO on simulating tumor cell antigens as well as being tumor vaccine adjuvant.

Objective:To investigate the prevalence,and the factors that influence depressive symptoms among chronic respiratory disease patients in tertiary hospital.Methods:A total of 1713 outpatients and inpatients with chronic respiratory disease were selected from 8 tertiary hospitals in Jiangsu Province from July to September,2014 and screened according to the Hospital Anxiety Depression Scale-D (HADS-D).A questionnaire developed by this research group,was used to collect demographic and clinical information.Logistic regression was used to identify factors that were associated with depressive symptoms.Results:The overall rate of depressive symptoms was 46.0％.Multiple logistic analysis showed that spinsterhood (OR ＝ 0.45),higher education level (middle school /high school/technical school OR ＝0.65;college degree or aboveOR ＝0.28),BMI ≥24 (OR ＝0.71) were associated with decreased risk of depressive symptoms (P ＜ 0.05).B MI ＜ 18.5 (OR ＝ 1.52,),average income of family ≥10000 RMB (OR ＝ 1.37-1.96),limited daily activities (OR ＝ 1.72),poorer sleep quality (OR ＝ 1.45),and negative life events (OR ＝ 1.62) were associated with increased risk of depressive symptoms (P ＜ 0.05).Conclusion:The prevalence of depressive symptoms among chronic respiratory disease patients in tertiary hospitals in Jiangsu Province was higher.Marital status,education level,income,BMI,limited daily activities,subjective sleep quality,negative life events may be the related factors of depressive symptoms of chronic respiratory diseases patients.

Objective To efficiently builds up and expand breast cancer cells from cancer tissue and to identify their biological properties , provide abundant materials for research and personalized medicine .Methods Feeder cell layer and ROCK inhibitor Y-27632 were employed to faciliate the breast cancer cells;CCK-8 was used to determine the proliferation of the breast cancer cells; Cell cycle distribution was analyzed by flow cytometry; Histochemistry ( FH) assay to show the expression level of CK .The mRNA expression of HER-2, ER, PR and the breast cancer stem cell associated molecules (such as CD44, CD24, etc.) were detected by RT-PCR;STR assay was used for identifying verification of the cells .Results The use of feeder cells and Y-27632 facilitates rapid expand of the original breast cancer cells , and the cells have kept the original features of the tumor .Conclusions To use the method could obtain a large number of cells within a short time , which can promptly be used for the research of per-sonalized medicine .

Objective:To study the dental and skeletal changes after orthodontic treatment for malocclusion patients with rapid maxil-lary and mandibular expansion companied with fixed appliance.Methods:36 patients underwent the treatment with rapid maxillary and mandibular expansion companied with straight wire appliance.Pre-and post-treatment dental casts and lateral cephalometric radiographs were measured and compared.Results:After treatment,the maxillary and mandibular arch width perimeters increased(P <0.05).The anterior arch depths decreased(P <0.05).No significant differences were found in vertical skeletal variables(P >0.05).U1-SN and U1-NA decreased,L1-MP and L1-NB increased(P <0.05).Conclusion:Dental crowding can be solved effectively and occlusion re-lationship can be kept well with rapid maxillary and mandibular expansion companied with straight wire appliance without influence on the vertical skeletal relationship.

Objective To provide supporting data for the quality control and evaluation of Nitraria Spp .from Xinjiang region by analyzing the content of isorhamnetin-3-rutinoside in its leaves .Methods HPLC method was used for the determina-tion of isorhamnetin-3-rutinoside in leaves of Nitraria Spp .Results There is a good linear relationship in the range of 0 .007813~1 mg/ml .The average recovery rate is 100 .13% (RSD=3 .02% ) .Conclusion There were large interspecific and intraspecific differences of isorhamnetin-3-rutinoside content in leaves of Nitraria Spp from Xinjiang region .A simple method was identified to give accurate and reproducible results .This method can be used for quantitative analysis of isorhamnetin-3-ru-tinoside content in leaves of N itraria Spp .

Objective: To assess intravoxel incoherent motion(IVIM) in evaluating and predicting response to neoadjuvant chemotherapy(NAC) in esophageal squamous cell carcinoma(ESCC). Methods: Forty-seven patients with ESCC diagnosed by pathological findings on biopsy from September 2015 to March 2017 were prospectively collected. All patients were examined before and after NAC using routine MRI scan and IVIM. The standard apparent diffusion coefficient (ADC_standard), diffusion coefficient (D), perfusion coefficient (D^*) and perfusion score (f) were measured. The patients were divided into complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD) according to the 1.1 version of the response evaluation criteria in solid tumors (RECIST). Thirty-one patients underwent surgery after NAC, and the patients were divided into TRG 0-3 according to tumor regression grade (TRG). The differences of parameter values before and after NAC between different groups were analyzed using Student's t test (normal distribution) and Wilcoxon rank sum tests (non-normal distribution). The parameters with statistical significance were evaluated by the receiver operating characteristics (ROC) curves. Results: The ADC_standard values before and after NAC were (1.97±0.51) ×10^-3, (2.42±0.52)×10^-3 mm²/s. The D values before and after NAC were (1.30±0.30)×10^-3, (1.63±0.35)×10^-3 mm²/s. The ADC_standard and D values after NAC were significantly higher than those before NAC, and the differences were statistically significant (t=-6.35, -5.25 respectively, both P<0.01). There were no statistical differences on D^* and f between before and after NAC (P>0.05). The patients were divided into PR group (29 cases) and SD group (18 cases) after NAC, without CR and PD patients. The ADC_standard, D and f values of PR group were significantly lower than those of the SD group before NAC (t=-3.11, -2.53 and -2.10 respectively, all P<0.05). The ADC_standard, D, D^* and f values after NAC revealed no significant difference between PR and SD groups. Thirty-one patients received operation after NAC, which were divided into TRG 2 group (14 cases) and TRG 3 group (17 cases) according to TRG standard, without TRG 0 and TRG 1 patients. All the parameter values before NAC revealed no significant difference between TRG 2 and TRG 3 groups. The D values after NAC in TRG 2 and TRG 3 groups were (1.81±0.31)×10^-3, (1.46±0.39)×10^-3 mm²/s respectively, and the significant difference was found between two groups (t=2.76, P<0.05). The efficiency of efficacy evaluation for NAC was the highest at D value of 1.68×10^-3 mm²/s, with sensitivity and specificity being 85.7% and 70.6%, respectively. Conclusion IVIM can be used as a new imaging method to evaluate and predict the efficacy of NAC for ESCC, among which the parameter D was the most valuable.

Objective To explore the predictive value of intravoxel incoherent motion (IVIM) imaging for the pathologic response to neoadjuvant chemotherapy in locally advanced esophageal squamous cell carcinoma (ESCC).Methods The prospective study was conducted.The clinicopathological data of 33 patients with locally advanced ESCC who were admitted to Affiliated Hospital of Zhengzhou University from September 2015 to October 2017 were collected.Patients received magnetic resonance imaging (MRI) and IVIM imaging examination before and after neoadjuvant chemotherapy.Two radiologists read the imaging together,manually delineated the region of interest in the diffusion-weighted imaging,and the apparent diffusion coefficient (ADC),diffusion coefficient (D),perfusion coefficient (D*),and perfusion score of the tumor (f) were automatically measured.Patients underwent neoadjuvant chemotherapy with paclitaxel plus cisplatin,and underwent radical surgery for esophageal cancer after 2 cycles of chemotherapy.Observation indicators:(1) comparison of IVIM imaging parameters before and after neoadjuvant chemotherapy in patients with ESCC;(2) comparison of change value and change rate of IVIM imaging parameters before and after neoadjuvant chemotherapy in patients with different tumor regression grade (TRG);(3) predictive efficacy of change value and change rate of IVIM imaging parameters before and after neoadjuvant chemotherapy for TRG.Measurement data with normal distribution were presented as Mean±SD,and comparison before and after neoadjuvant chemotherapy was done using the paired t test,and comparison between different TRG patients was done using the t test.Measurement data with skewed distribution were presented as M(P25,P75),and comparison before and after neoadjuvant chemotherapy and between different TRG patients were done using the Wilcoxon rank sum test.The receiver operating characteristic (ROC) curve was used to evaluate predictive value of IVIM imaging parameters.Results Thirty-three patients were screened for eligibility,including 26 males and 7 females,aged from 44 to 74 years,with an average age of 60 years.All the 33 patients were diagnosed as ESCC by pathological examination.(1) Comparison of IVIM parameters before and after neoadjuvant chemotherapy in patients with ESCC:33 patients with ESCC showed a significant difference in the ADC,D,and f value after neoadjuvant chemotherapy [ADC:(1.95±0.56) × 10-3 mm2/s vs.(2.54±0.50) × 10-3 mm2/s,t=-6.98;D:(1.26×10-3 mm2/s (0.81×10-3 mm2/s,2.44×10-3 mm2/s) vs.1.68×10-3 mm2/s (0.83×10-3 mm2/s,2.27×10-3 mm2/s),Z=-3.96;f:0.33％±0.14％ vs.0.42％±0.15％,t=-3.13,P＜ 0.05].(2) Comparison of change value and change rate of IVIM imaging parameters before and after neoadjuvant chemotherapy in different TRG patients:of 33 patients,15 were in TRG 2 and 18 were in TRG 3.The ADC change value,ADC change rate,D change value,D change rate were (0.85±0.52)× 10-3 mm2/s,52.91％± 32.51％,0.64× 10-3 mm2/s (0.05× 10-3 mm2/s,1.41 × 10-3 mm2/s),48.20％ (3.03％,16.95％) of TRG 2 patients,and (0.38±0.35)×10-3 mm2/s,21.94％±19.08％,0.26×10-3 mm2/s (-1.43×10-3 mm2/s,0.81× 10-3 mm2/s),20.18％ (-58.61％,77.14％) of TRG 3 patients,respectively,with significant differences between two groups (t=3.09,3.41,Z=-3.04,-2.93,P＜0.05).(3) Predictive efficacy of change value and change rate of IVIM imaging parameters before and after neoadjuvant chemotherapy for TRG:ROC curve analysis showed that ADC change value exhibited an area under curve (AUC) of 0.798,a sensitivity of 66.7％ and a specificity of 94.4％ in predicting TRG,when 0.86× 10-3 mm2/s was used as the cut-off value.With 43.3％ as the cut-off value,ADC change rate had an AUC of 0.793,a sensitivity of 66.7％ and a specificity of 88.9％ in predicting TRG.With 0.35× 10-3 mm2/s as the cut-off value,D change value had an AUC of 0.809,a sensitivity of 73.3％ and a specificity of 77.8％ in predicting TRG.With 25.9％ as the cut-off value,D change rate had an AUC of 0.800,a sensitivity of 80.0％ and a specificity of 72.2％ in predicting TRG.Conclusions The change value and change rate of ADC and D values before and after neoadjuvant chemotherapy are potential predictors of pathologic response in ESCC.The significantly increased ADC and D values after neoadjuvant chemotherapy are prone to good pathologic response.The change value and change rate of D values show a better predictive value for pathologic response to neoadjuvant chemotherapy in ESCC compared with those of ADC values.

Objective: To establish human cancer cell strains with stable Cas9 expression, and to validate the gene editing activity of Cas9 for simple gene editing in future study. Methods: Fifteen cancer cell lines of different tissue origins were infected with pLv-EF1α-Cas9-Flag-Neo or pLv-EF1α-Cas9-Flag-Puro by lentivirus and clone selection was employed to screen Cas9 stably expressed cancer cell lines. Afterward designed guide RNA vectors targeting TSC22 gene were transiently transfected into 3 of cell lines, and subsequently the gene editing activity of Cas9 was evaluated by genomic PCR, sequencing and Western blot. Results: Sixty-nine human cancer cell strains with stable Cas9 expression from different cancers were established, and by transient transfection with designed guide RNA, long fragment deletion was detected in TSC22 gene. Conclusions Sixty-nine human cancer cell strains are successfully established with stable expression of Cas9 protein and gene editing activity. These cell strains may be employed in large-scale drug screening, screening of new drug targets and gene function investigation.