Anti-angiogenesis mechanism plays a vital role in tumor targeting immunotherapy. Based on the amino acid sequence of an anti-VEGFR-2 scFv-Fc fusion antibody (AK404R-Fc), this article is aimed to generate an anti-VEGFR-2 human IgG1-like full length antibody (Mab-04). Firstly, the light chain (L-chain) and heavy chain (H-chain) were obtained by overlap PCR and then linked to eukaryotic expression vector pcDNA3.1, separately. The recombinant plasmids (pcDNA3.1-L-chain and pcDNA3.1-H-chain) were then co-transfected into CHO-k cells using liposome transient transfection. Subsequently, Mab-04 antibody was expressed and purified by Protein A affinity chromatography. Western blotting was applied to identify the expression of Mab-04 and its affinity was detected by ELISA assay. DNA sequencing revealed the successful construction of recombinant plasmids and Western blotting assay proved the successful expression of full-length antibody (1 microg x mL(-1)). Finally, ELISA assay illustrated that the binding of the antibody to its antigen was in a concentration-dependent manner (IC50: 50 nmol x L(-1)). These outcomes above indicated that Mab-04 was successfully expressed and assembled, which laid the foundation for further preparation and antineoplastic activity study.

Objective:To comparatively analyze the efficacy of S-1 plus cisplatin as first-line chemotherapy between advanced AFP posi-tive and AFP negative gastric cancer. Methods:A total of 89 eligible patients with advanced gastric cancer from January 2010 to June 2013 were enrolled in this retrospective study. The cases were divided into AFP positive group (n=18) and AFP negative group (n=71) based on serum AFP level before treatment. Both groups received S-1 plus cisplatin as first-line treatment. Results:Significant differ-ences were observed in remission rate (66.7% versus 32.4%, P=0.028) and radical surgical resection rate (44.5% versus 18.3%, P=0.029) after chemotherapy between AFP positive group and AFP negative group. In the two groups, neither progression-free survival nor overall survival (OS) showed any significance (P>0.05) in patients without surgery after chemotherapy. However, the disease-free survival of the two groups with radical surgical resection after chemotherapy was significantly different (median:27.0 months versus 15.6 months, P=0.034). The OS of the AFP positive group was evidently longer than that of the AFP negative group (median:16.0 months versus 11.0 months, P=0.005). Conclusion:As first-line chemotherapy, S-1 plus cisplatin was more effective for the advanced AFP positive gastric cancer and prolonged overall survival.

Purpose To study the value of contrast-enhanced ultrasound (CEUS) quantitative technique in evaluating the perfusion of hepatic microcirculation in acute hemorrhagic shock (HS),and to investigate the value of CEUS quantitative analysis in HS diagnosis and treatment.Materials and Methods Sixty healthy adult New Zealand white rabbits of either gender were randomly divided into three groups for establishing mild,moderate and severe HS models,respectively.Before modeling and 30 min after stable modeling,liver CEUS examination was performed,and the original images were stored.Blood test of lactic acid,liver function,and liver biopsy for pathological examination were conducted after CEUS.Finally,the arrival time (AT),time to peak (TTP),rising time (RT),peak intensity (PI) and area under the curve (AUC) were analyzed offiine.Results Compared with pre-modeling,AUC decreased in mild HS group (P＜0.05);TTP and RT were delayed,but PI and AUC decreased in moderate and severe HS groups (all P＜0.05);AT was delayed in severe HS group (P＜0.05).The differences of TTP,RT,PI and AUC between the groups of mild,moderate and severe HS were significant (P＜0.05).Compared with pre-modeling,lactic acid in three HS groups increased significantly,the liver function indexes were changed to different degrees,and the degree of liver cell pathological changes was closely related to the degree of HS.Conclusion CEUS can quantitatively evaluate the changes of hepatic microcirculation induced by HS at different degrees.

Purpose To investigate the value of contrast-enhanced ultrasound (CEUS) in monitoring hepatic parenchymal perfusion quantitatively during resuscitation after hemorrhagic shock (HS).Materials and Methods Forty-five labeled rabbits were randomly divided into five groups.One group was normal control group,and the other four groups were established reversible HS modes by using modified Wiggers method.In the four model groups,one group was chosen as the shock group,and the other three groups received colloid (hydroxyethyl starch) for resuscitation.The three resuscitation groups were named as 1 h group,4 h group,24 h group according to resuscitation time.The arterial blood was collected for testing blood lactic acid (LAC) in each group.The CEUS was performed to form time-intensity curve (TIC) for quantitative analysis.At the end of the experiment,the rabbits were sacrificed and the liver tissues were examined for pathology.Results Compared with the normal control group,the arrival time (AT) and time to peak (TTP) of the shock group were significantly prolonged,and the peak intensity (PI) and area under curve (AUC) of the shock group decreased,all with significant difference (all P＜0.05);in the 1 h resuscitation group,the PI and AUC increased,and the AT decreased,all with significant difference (all P＜0.05);in the 4 h and 24 h resuscitation group,the PI and AUC increased,and the AT and TTP decreased,but all without significant difference (all P＞0.05).Compared with the shock group,the PI and AUC of the 1 h resuscitation group increased,and the AT of 1 h resuscitation group decreased,all with significant difference (all P＜0.05);all the tested parameters of the 4 h and 24 h resuscitation group were significant different (P＜0.05).Conclusion CEUS can quantitatively evaluate the changes of hepatic parenchymal perfusion during HS resuscitation,which has a certain reference for clinical treatment.

Objective:To evaluate the efficacy and safety of dienogest (DNG) in the treatment of refractory endometriosis-associated pain (REAP).Methods:In this study, REAP was defined according to the following criteria: (1) the pain duration was ≥12 months and visual analogue scale (VAS)≥60 mm; (2) the previous treatments with over two medicines like oral contraceptives and levonorgestrel-releasing intrauterine system failed to achieve satisfactory relief of pain, with VAS reduction less than 50%; with gonadotropin-releasing hormone agonist or mifepristone, the pain could be controlled temporarily, but it recurred after discontinuation of medicines; (3) the pain could not be relieved by surgery or even repeated surgeries. In the present study, 48 patients with REAP were treated with DNG 2 mg/day orally and the clinical outcomes were retrospectively analyzed. The VAS scores, levels of CA 125, estradiol, FSH, LH and changes in the size of endometriotic lesions before and after treatment were compared respectively. The side effects were also analyzed. Results:The average duration of DNG treatment was (20.1±12.8) months. After 3 months of medication, the VAS score was significantly reduced from (77.9±15.8) mm to (20.8±10.7) mm ( P<0.01), and CA 125 level was significantly reduced from (95±139) kU/L to (38±45) kU/L ( P<0.05). The effects were maintained with continuation of DNG treatment. Endometriotic lesions tended to shrink, after 12 months of DNG treatment, the size of ovarian endometriomas was reduced significantly from (3.1±1.0) cm to (1.9±1.2) cm ( P<0.05). The mean level of estradiol was maintained at 124.82-221.04 pmol/L and levels of FSH and LH did not change significantly during the treatment. The major side effect was irregular bleeding (75%, 36/48). Conclusions:DNG could effectively relieve REAP and is a well-tolerated therapy. It may supply an alternative option for patients with REAP.

Objective:To evaluate the role of DNA methyltransferase in acute lung injury in septic mice.Methods:Forty-eight healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (group Sham), sham operation+ DNA methyltransferase inhibitor group (group Sham+ 5-Aza), sepsis group (group Sepsis) and sepsis+ DNA methyltransferase inhibitor group (group Sepsis+ 5-Aza). Sepsis model was developed by cecal ligation and puncture (CLP) in anesthetized mice.Mice were sacrificed at 24 h after CLP, and lung tissues were obtained, DNA was extracted to determine the global DNA methylation by colorimetry, and RNA was extracted to detect the expression of DNA methyltransferase (DNMTl, DNMT3a, DNMT3b) mRNA by real-time fluorescent quantitative polymerase chain reaction, the wet/dry lung weight ratio (W/D ratio) was measured, the histopathological changes of lung tissues were determined by HE staining, the contents of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), high-mobility group box 1 protein (HMGB1) and malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase were measured by enzyme-linked immunosorbent assay. Results:Compared with group Sham, the global DNA methylation was significantly increased, the expression of DNMT1 and DNMT3a mRNA was up-regulated, the lung injury score, W/D ratio, and contents of IL-6, TNF-α, HMGB1 and MDA were increased, and activities of SOD and CAT were decreased at 24 h after CLP in group Sepsis and group Sepsis+ 5-Aza ( P<0.05), and no significant change was found in the indexes mentioned above in group Sham+ 5-Aza ( P>0.05). Compared with group Sepsis, the global DNA methylation was significantly decreased, the expression of DNMT1 and DNMT3a mRNA was down-regulated, the lung injury score, W/D ratio, contents of IL-6, TNF-α, HMGB1 and MDA were decreased, and the activities of SOD and CAT were increased in group Sepsis+ 5-Aza ( P<0.05). Conclusions:DNA hypermethylation mediated by DNMT1 and DNMT3a is involved in the process of acute lung injury in septic mice.

Objective To construct a lentiviral vector delivering the Nicastrin (NCT) gene-targeted short hairpin RNA (shRNA) and determine gene-silencing efficiency of the vector in the human immortalized keratinocyte cell line HaCaT,and to construct a NCT gene-silenced HaCaT cell model to lay an experimental foundation for subsequently studying effects of NCT gene silencing on biological behavior of keratinocytes.Methods Three NCT gene-targeted shRNAs were designed and inserted into the pGLV3/ H1/GFP + Puro vector to construct three recombinant plasmids,which were then confirmed by sequencing.Recombinant plasmids combined with lentivirus packaging plasmids were co-transfected into 293T cells to obtain lentivirus particles,and the virus titer was determined.Cultured HaCaT cells were divided into 3 groups:blank group receiving no treatment,negative control group infected with the empty vector LV3-shNC,interference groups infected with lentivirus NCT-shRNA1,-shRNA2,-shRNA3,respectively.Flow cytometry was performed to determine transfection efficiency,and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine efficiency of target gene silencing in HaCaT cells,so as to select the most efficient interference sequence.Results Sequencing analysis indicated that recombinant lentiviral vector NCT-shRNA was constructed successfully.After co-transfection of recombinant plasmids and lentivirus packaging plasmids into 293T cells,the titer of recombinant lentivirus particles was about 109 TU/ml.Flow cytometry showed that the transfection efficiency was greater than 95％.qRT-PCR revealed that the NCT mRNA expression was obviously down-regulated in the interference group compared with the negative control group,and NCT-shRNA1 was the most efficient sequence with interference efficiency being 75％.Western blot analysis showed that the inhibition rate of NCT protein expression was 71.7％ in the shRNA1 group compared with the negative control group.Conclusion The most efficient NCT-shRNA interference sequence is screened out,and the recombinant lentiviral vector NCT-shRNA and an NCT gene-silenced HaCaT cell model are both constructed successfully.

BACKGROUND: Cancer stem cells have been investigated as new targets for colorectal cancer (CRC) treatment. We recently reported that CD133+ colon cancer cells showed chemoresistance to 5-fluorouracil through increased survivin expression and proposed the survivin inhibitor YM155 as an effective therapy for colon cancer in an in vitro study. Here, we investigate the relationship between survivin and CD133 expression in surgically resected CRC to identify whether the results obtained in our in vitro study are applicable to clinical samples. METHODS: We performed immunohistochemical staining for survivin and CD133 in surgically resected tissue from 187 stage II or III CRC patients. We also comparatively analyzed apoptosis according to survivin and CD133 expression using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. RESULTS: The results of the Mantel-Haenszel test established a linear association between nuclear survivin and CD133 expression (p = .018), although neither had prognostic significance, according to immunohistochemical expression level. No correlation was found between survivin expression and the following pathological parameters: invasion depth, lymph node metastasis, or histologic differentiation (p > .05). The mean apoptotic index in survivin+ and CD133+ tumors was higher than that in negative tumors: 5.116 ± 4.894 in survivin+ versus 4.103 ± 3.691 in survivin– (p = .044); 5.165 ± 4.961 in CD133+ versus 4.231 ± 3.812 in CD133– (p = .034). CONCLUSIONS: As observed in our in vitro study, survivin expression is significantly related to CD133 expression. Survivin may be considered as a new therapeutic target for chemoresistant CRC.
Apoptosis ; Colonic Neoplasms ; Colorectal Neoplasms* ; Deoxyuridine ; Fluorouracil ; Humans ; In Vitro Techniques ; Lymph Nodes ; Neoplasm Metastasis ; Neoplastic Stem Cells

Objectives: To explore the differences in signal pathway and gene expression related to the pathogenesis of congenital microtia by the in-depth analysis of DNA methylation profiling of auricular chondrocytes from congenital microtia patients. Methods: Genome wide methylation profile of congenital microtia was obtained by MeDIP chip technology, and analyzed by Gene ontology (GO) and Pathway analysis. The gene expression levels of Wnt1 and Wnt11 were evaluated by Real-time PCR in the auricular cartilage from the healthy side and affected side of the congenital microtia patients , and healthy controls. Results: The GO and Pathway assay showed that Wnt signal pathway was enriched in differential methylated levels. The Wnt1 and Wnt11 genes were with higher methylation in the promoter region and CpG islands in healthy control group than that in microtia group, in addition the methylation level in the affected side auricular cartilage was lower than that in the healthy side. There was no difference in Wnt1 and Wnt11 gene expression in microtia patients and healthy controls. The higher Wnt11 gene expression was detected in the affected side residual cartilage tissues than in the healthy side cartilage tissues of the same congenital microtia patient. Conclusions The over expression of Wnt11 during embryonic development might be associated with the pathogenesis of congenital microtia. The mechanism of the difference in methylation levles of Wnt11 affecting pathogenesis of congenital microtia needs further research.

Objective:To construct a chronic obstructive pulmonary disease (COPD) assessment test (CAT) score prediction model based on a deep network fused with air data, and to explore its significance.Methods:From February 2015 to December 2017, the outdoor environmental monitoring air data near the residential area of the patients with COPD from the Respiratory Outpatient Clinics of Peking University Third Hospital, Peking University People′s Hospital and Beijing Jishuitan Hospital were collected and the daily air pollution exposure of patients was calculated. The daily CAT scores were recorded continuously. The CAT score of the patients in the next week was predicted by fusing the time series algorithm and neural network to establish a model, and the prediction accuracy of the model was compared with that of the long short-term memory model (LSTM), the LSTM-attention model and the autoregressive integrated moving average model (ARIMA).Results:A total of 47 patients with COPD were enrolled and followed up for an average of 381.60 days. The LSTM-convolutional neural networks (CNN)-autoregression (AR) model was constructed by using the collected air data and CAT score, and the root mean square error of the model was 0.85, and the mean absolute error was 0.71. Compared with LSTM, LSTM-attention and ARIMA, the average prediction accuracy was improved by 21.69%.Conclusion:Based on the air data in the environment of COPD patients, the fusion deep network model can predict the CAT score of COPD patients more accurately.