Background and purpose:Most breast cancer cell lines were derived from malignant pleural fluid or other tissues that metastasized from breast carcinoma.Breast carcinogenesis is under represented due to the significant distinction of biological characteristics between cancer cell lines and primary breast cancers.Few primary breast tumors spontaneously developed into immortal cell lines.Overexpression of human telomerase reverse transcriptase(hTERT) in normal epithelial cells led to immortalization.We aimed to establish immortalized cultures derived from primary breast cancer by introducing hTERT and determine the gene profile of hTERT-transfected cultures.Methods:hTERT was introduced into breast tumor cultures with a puromycin-resistant retroviral construct,pBABE-hTERT-puro.Real-Time RT-PCR was used to analyze the expression level of hTERT after transfection.The upregulated gene profile was determined with microarray techniques.Results:hTERT was stably over expressed,at least 1 400-fold in hTERT-transfected cells compared to pBABE-puro transfected cultures.These ectopic over-expressions led to immortalization of transfected cultures,which could undergo at least 40-50 passages.Microarray analysis further confirmed the over-expression of hTERT in transfected cultures and demonstrated that 22 genes were up-regulated due to over-expression of hTERT.No down-regulated genes were observed cross all cultures.Conclusions:Over-expression of hTERT led to immortalization of breast tumor primary cultures and up-regulated certain genes.

Background and purpose:The epidermal growth factor receptor (EGFR) is commonly overexpressed in a variety of solid tumors, and has important roles in cancer pathogenesis and progression. EGFR thus provides a rational target for cancer therapy. We studied siRNA-mediated inhibition of epidermal growth factor receptor expression and its biologic effects in different human cancer cell lines (A431, HeLa and SPC-A-1). Methods:Cells were transfected with chemically synthesized siRNA-EGFR. EGFR mRNA was quantified by real-time PCR and was detected by immunofluorescence staining and flow cytometry. The biologic effects on cell growth were assessed by colony-formation assay.Results:siRNA-EGFR significantly decreased mRNA level of EGFR by 73.9 %, 44.6 % and 57.7 %, protein expression of EGFR by 77.0 %, 61.3 % and 65.2 %, and reduced colony number by 27.2 %, 53.9 % and 59.1 % in A431, HeLa and SPC-A-1, respectively.Conclusions:Our data suggested that RNA interference could downregulate EGFR and inhibit colony forming ability and EGFR expression at mRNA/protein levels in human cancer cell lines with different pathological types. siRNA could be one of the promising strategies in future targeted cancer therapy.

Objective To study the relationship between stress rise of blood glucose and the PEEL prognostic index of the patients with acute myocardial infarction, and provide the basis for a quick judgement and evaluation of prognosis to nurses. Methods Taking the 6.8,7.8 and 8.8 mmol/L as demarcation point to study the PEEL prognostic index in 59 patients with acute myocardial infarction, and compare the PEEL score value on two sides of each demarcation point. Results No significant difference in PEEL evaluation score when taking 6.8 mmol/L as the demarcation point, there was significant difference when taking 7.8 mmol/L as the demarcation point, PEEL score in blood glucose > 7.8 mmol/L group was higher than that of ≤7.8mmol/L group, so was when taking 8.8 mmol/L as the demarcation point. Conclusions There was instructive signifi-cance for nurses to observe a stress rise of blood glucose timely in evaluating the prognosis of patients with a-cute myocardial infarction. Nurses should strengthen disease observation and preventive nursing of admission patients whose fasting blood glucose are above 7.8 mmol/L as well their blood glucose monitoring.

Objective To evaluate the feasibility of active breathing control (ABC) in conformal radiotherapy (CRT) for patients with non-small cell lung cancer (NSCLC). Methods From Feb 2005 to Mar 2008, 29 patients with inoperable NSCLC (stage Ⅱ-Ⅳ) were evaluated. For each patient, two series of CT scans were obtained with free breathing (FB) and ABC system during simulation, respectively. Then two confonnal radiotherapy (CRT) plans were finished based on the two sets of reconstructed images. The pattern of post-inspiratory breath-hold was triggered at 80% of the peak of inspiration curve. The margin of clinical target volume (CTV) to planning target volume (PTV) was 0. 6 cm for lesions of the superior lobe, and 1.0 cm for the lesions of middle and inferior lobes. Three to five coplanar fields were performed in conformal radiotherapy. The gross tumor volume (GTV), CTV, PTV, volume of the bilateral lungs (Volume_(lung)), V_(20) and mean lung dose (MLD) of two plans were evaluated by dose-volume histogram (DVH). The World Health Organization criteria and National Cancer Institute Common Toxicity Criteria 3.0 (NCI-CTC3.0) scale were used to assess the immediate response and acute side-effect, respectively. Results Significant differences of GTV, CTV, FIN, Volum_(lung), V_(20) and MDL were observed between the two plans (36. 35 cm~3 vs. 31.40 cm~3, t = 9. 70, P <0. 001 ;82. 33 cm~3 vs. 70. 83 cm~3, t = 8. 19, P < 0. 001 ; 230. 73 cm~3 vs. 197.59 cm~3 ,t=5.72,P <0. 001 ;21.66% vs. 18. 76% ,t = 11.16,P <0. 001 ;1329. 07 Gy vs. 1143. 14 Gy, t = 13. 24, P < 0. 001). With ABC, all patients completed their treatment successfully except one patient for financial problems. The median radiation dose to the GTV was 64 Gy (60 -64 Gy). The overall immediate response rate was 64% (18/28). According to the NCI-CTC 3.0, grade 1 and 2 acute radiation-related toxicities occurred in 68% (19/28) and 18% (5/28) of patients for esophagitis, 82% (23/28) and 7% (2/28) for pneumonitis, respectively. Grade 1, 2 and 3 bone marrow suppression occurred in 57% (16/28), 25% (7/28) and 14% (4/28) of patients, respectively. Grade 1 and 2 acute cardiac injuries occurred in 86% (24/28) and 14% (4/28) of patients. Conclusions During CRT for patients with NSCLC, the use of ABC can decrease the radiation dose and acute complications of normal tissues.

Aim To investigate the expression and im-plication of HIF-1α, ROCK-2 , FoxM1 in PC12 cell in-jury induced by lead acetate. Methods PC12 cells were treated with lead acetate at the doses of 100 , 200 and 400 μmol·L-1 . The cell viability was determined by MTT reduction assay and LDH assay, the intracellu-lar production of oxygen species was measured by as-sessing SOD and MDA levels, cell apoptosis was deter-mined by Hoechst 33342 staining, the expressions of HIF-1α, ROCK-2 , FoxM1 , Bcl-2 and Bax were deter-mined by immunoblotting analysis. Results Lead ac-etate induced cell injury in PC12 cells in a dose-de-pendent manner, and it potentiated oxygen radical pro-duction and cell apoptosis. In addition, lead acetate enhanced HIF-1α and ROCK-2 expressions, increased Bax/Bcl-2 ratio and decreased FoxM1 expression. Conclusion Lead acetate can induce PC12 cell apop-tosis, which may be related with the expressions of HIF-1α, ROCK-2 and FoxM1 . Cellular oxidative stress may contribute to the injury as well.

OBJECTIVETo compare the influence of cardiac-pulmonary function on clinical acute respiratory failure patients using Proportional assist ventilation (PAV), Pressure support ventilation (PSV) and intermittent positive pressure ventilation (IPPV). Here, we also describe some our experience with the clinical use of PAV.

METHODSUsing the IPPV mode in ten acute respiratory failure patients, calculate Elastance (Ers) and Resistance (Rrs), then change to PSV, set inspiratory positive airway pressure (IPAP) according to IPPV, so that tidal volume (V(T)) is the same as that of IPPV. We then changed the mode into PAV and set the assist ratio according to PSV, so that V(T) and Ppeak were the same as that of PSV. Then we observed the changes of respiratory mechanics, blood gas levels and hemodynamics during ventilation.

RESULTSCompared with PSV and IPPV, peak pressure (Ppeak) of PAV was markedly lower while V(T) was similar; work of breathing of patient (WOBp), and work of breathing of ventilation (WOBv) were also lower; center vein pressure (CVP) and pulmonary capillary wedge pressure (PCWP) of PAV were markedly lower than that of IPPV while V(T) were similar. Compared with PSV, V(T), mean blood pressure (mBP) and cardiac output (CO) of PAV were higher. Mean pulmonary artery pressure (mPAP) and WOBp of PAV were lower while Ppeak was similar; the differences in WOBp were notable.

CONCLUSIONSFor clinical acute respiratory failure patients, compared with PSV and IPPV, PAV has lower airway pressure, less WOBp and less influence on hemodynamics.

Acute Disease ; Adult ; Aged ; Aged, 80 and over ; Cardiac Output ; physiology ; Female ; Hemodynamics ; physiology ; Humans ; Male ; Middle Aged ; Pulmonary Ventilation ; physiology ; Pulmonary Wedge Pressure ; physiology ; Respiration, Artificial ; methods ; Respiratory Insufficiency ; physiopathology ; therapy ; Ventilators, Mechanical

Objective To investigate the specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B (PorB) fused with B subunit of Escherichia coli heat-labile enterotoxin B (LTB) in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic vector pcDNA3.1(-). The recombinants were identified by PCR, enzyme digestion and DNA sequencing.The vectors were transfected into Hela cells, and expressed proteins were checked by cytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium (MTT) colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls (P＜0.01 ; P＜0.05). Moreover, the sIgA level in pcDNA3.1 (-)/ltB-porB group was higher than that in peDNA3, 1(-)/porB group (P＝0. 002). The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in the supernatants and stimulation index (SI) of spleen lymphocyte culture in pcDNA3, 1(-)/porB group were (170.04±23.89) pg/mL, (114.68±14.27) pg/mL and 1. 68±0.19, respectively; and those in pcDNA3, 1(-)/ltB-porB group were (161.42±27.50) pg/mL, (124.16±19.04) pg/mL and 1.73±0.28, respectively; which were both higher than those in pcDNA3.1(-)/ phosphate buffered saliae (PBS) group (P＜0. 01; P＜0.05) and pcDNA3.1 (-)/ltB group (all P＜0.05), while there was no significant difference between pcDNA3.1 (-)/ltB-porB group and pcDNA3. 1 (-)/porB group (0. 998, 0. 696, 0. 994; all P＞0.05). Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 (- )/porB and pcDNA3.1 ( -)/ltBporB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.

OBJEVTIVETo study the effect of spvB/spvC gene on Salmonella virulence and the Host immune.

METHODSSTM.211, STM.211-Delta;spvB, STM.211-Delta;spvC, STM.211-Delta;spvB.spvC and PBS were infected with 0.2 mL 10(5) CFU corresponding strain respectively by intraperitoneal. We observed the mental status, movement, diarrhea, weight, pelage changed hair of the infected mouse. Then the level of IL-10, IL-12, IFN-γ were detected by ELISA. Finally, we observe the pathological changes of liver and spleen with the general view and the microscope.

RESULTSInfection symptoms of STM.211, STM.211-Delta;spvB and STM.211-Delta;spvC were significantly worse than PBS group, but there was no significant difference between STM.211-spvB.spvC group and PBS group. The secretion of IFN-γ and IL-12 of STM.211, STM.211-Delta;spvB, STM.211-Delta;spvC group were significantly lower than those in the STM.211-Delta;spvB.spvC group (P<0.05), but IL-10 secretion was significantly higher than STM.211-Delta;spvB.spvC group (P<0.05). There were no statistical significance among the STM.211, STM.211-Delta;SpvB, STM.211-Delta;spvC groups (P>0.05).

CONCLUSIONSSalmonella virulence can be affected obviously by spvB combined with spvC gene, but not by spvB or spvC. spvB/spvC gene can inhibit the TH1 cytokines (IFN-γ and IL-12) secretion but promote the TH2 cytokines (IL-10) expression, leading immune response trend to TH2 shift. It shows that spvB/spvC gene can help the bacteria evade the host immune defenses, leading to aggravation of infection.

Animals ; Cytokines ; immunology ; Interleukin-12 ; Mice ; Salmonella ; genetics ; pathogenicity ; Salmonella Infections ; immunology ; Virulence ; Virulence Factors ; genetics

Objective To evaluate the correlation between cerebral blood flow perfusion and memory impairment in patients with severe stenosis of vertebral basilar artery (VBA).Methods 62 cases of patients with VBA stenosis diagnosed by digital subtraction angiography(DSA) in Beijing Tiantan Hospital from September 2016 to March 2017 were enrolled.Mental State Examination (MMSE),Clinical Memory Scale (CMS) test and CT perfusion(CTP) was performed.All patients were divided into memory normal group(n=24,including 1 excellent case,6 above normal cases,and 14 normal cases) and memory impairment group(n =38,including 18 below normal cases,12 periphery cases,8 impaired cases) according to CMS.The ratios of side-to-side period were compared between bilateral mesial temporal lobe and anterior circulation area.The relative time to peak (rTTP),relative mean transit time(rMTY),relative cerebral blood flow(rCBF) and relative cerebral blood volume (rCBV) were calculate.Results The incidence of CTP decompensation in the medial temporal lobe was higher than that in the patients with memory impairment(P＜0.05).The difference of rTTP and rMTT value between the two groups in the bilateral medial temporal lobes was statistically significant (rTFP:(1.131 ±0.037),(1.437±0.139),t=10.520,P＜ 0.05);rMTT:(1.081 ±0.059),(1.281 ±0.174),t=5.423,P＜0.05).Conclusion The patients with VBA severe stenosis are more likely to get memory impairment due to cerebral hypoperfusion.

BACKGROUNDLung cancers are classified as squamous cell carcinoma (SQ), adenocarcinoma (AC) and small cell lung carcinoma (SCLC). SQ is the major subtype of lung cancer. Currently, there are no targeted therapies for SQ due to lack of understanding its driving oncogenes. In this study, we validated an SQ specific biomarker hsa-miR-205 in Chinese patients with lung cancer and screened its candidate target genes for further functional studies to enrich knowledge in SQ target therapies.

METHODSQuantitative reverse-transcription PCR (quantitative RT-PCR) was performed on 197 macro-dissected (cancerous cells >75%) surgical lung tissues (45 SQ, 44 AC, 54 SCLC and 54 adjacent normal tissues) to validate the expression profiles of miR-205. Furthermore, the targets of this microRNA were predicted through the gateway miRecords and mapped to lung cancer-associated pathways using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Then quantitative RT-PCR was performed on an independent cohort of 44 snap-frozen surgical lung tissues to concurrently assess the expression profiles of miR-205 and its 52 putative targeted genes.

RESULTSMicroRNA-205 yielded high diagnostic accuracy in discriminating SQ from AC with an area under the curve (AUC) of 0.985, and discriminating SQ from SCLC with an AUC of 0.978 in formalin-fixed paraffin-embedded (FFPE) surgical lung tissues. Predicted targets of miR-205 were associated with 52 key members of lung cancer signaling pathways. Ten target genes (ACSL1, AXIN2, CACNA2D2, FOXO3, PPP1R3A, PRKAG3, RUNX1, SMAD4, STK3 and TBL1XR1) were significantly down-regulated in SQ and had a strong negative correlation with miR-205, while one target gene (CDH3) was up-regulated in SQ and exhibited a strong positive correlation with miR-205.

CONCLUSIONSWe confirmed the high diagnostic accuracy of miR-205 in discriminating SQ from AC and SCLC in Chinese patients. Moreover, we identified 11 significant target genes of miR-205 which could be used for further functional studies as the basis for the development of SQ targeted therapies.

Adenocarcinoma ; genetics ; Aged ; Carcinoma, Squamous Cell ; genetics ; Female ; Humans ; Lung Neoplasms ; genetics ; Male ; MicroRNAs ; genetics ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Small Cell Lung Carcinoma ; genetics