Objective RNA interference (RNAi) expression vector was constructed to inhibit the focal adhesion kinase(FAK)expression in colon carcinoma HT29 cells, and then the sensitivity of the cells to 5-fluorouracil (5-FU) was determined. Methods One specific pair of oligonucleotides with short hairpin and its negative control sequence were designed and synthesized based on FAK cDNA sequences, then they were inserted into pGenesil-l vector to generate the recombinant plasmids. After identification by the restriction endonuclease and DNA sequencing, the recombinant plasmids were transfected into HT29 cells by lipofectamine TM 2000. The stable transfected cells were selected in a medium containing geneticin G418. The change in FAK expression in HT29 cells before and after RNA interference was detected by reverse transcription and polymerase chain reaction (RT-PCR) analysis and SP immunocytochemistry technique. Sensitivity of HT29 cells to 5-FU was determined by MTT assay. Results The recombinant plasmids coincided completely with the designs in the restriction map and the sequence analysis. After FAK being targeted by RNA interference, immunocytochemistry showed the protein expression of FAK was reduced dramatically, and RT-PCR revealed FAK mRNA expression was down-regulated by 76.94% compared to that of untransfected cells. MTT assay also showed that the sensitivity of HT29 cells to 5-FU in transfected pGenesil-1-FAK vector cells was increased, while IC50 declined remarkably (P

Objective To investigate the antitumor activity of Gefitinib, a selected epidermal growth factor receptor-tyrosine kinase inhibitor, on human colorectal cancer cell lines in vitro, and to explore the relationship between the inhibitory effect of Gefitinib on cancer cells and the expression of epidermal growth factor receptor (EGFR). Methods The growth inhibitory effects of Gefitinib, which expressed as the half growth inhibition dose IC50, on colorectal cancer cells were assessed by MTT assay. EGFR mRNA expression was detected by reverse transcriptional PCR (RT-PCR). Western blot was used to determine the expression of EGFR protein as well as its phosphorylated forms (p-EGFR). Results Gefitinib inhibited growth of all the six colorectal cancer cell lines in vitro with an IC50 range from 6.5 to 172.7?mol/L. Lovo cell line, with an IC50 value less than 10?mol/L, was the most sensitive one to Gefitinib, HT29 and SW480 were moderate sensitive to 10?mol/L

Objective To study the NF-?B activity in colon carcinoma HT-29 and Lovo cells treated with different crude extracts of Abrotani herba obtained by column chromatography with macroporous resin.Methods The decoction of Abrotani herba absorbed by macroporous resin AB-8 was mounted into the column and then eluted with distilled water and 30%,50%,75% and 95% alcohol.To select appropriate concentrations for treatment,HT-29 cells were pretreated for 24 hours with each elution phase of the rude extracts in different concentrations(0,50,100,200,400 and 600?g/ml),and then their activity was detected by MTT.The HT-29 and Lovo cells were thereafter cultured in DMEM complete media respectively containing distilled water extract and 30%,50%,75% and 95% alcohol extracts in the concentrations as obtained by MTT assay,and the cells in control group were cultured in DMEM only.The cells were then harvested and the nucleic proteins were extracted for measurement with electrophoretic mobility shift assay(EMSA).Changes in NF-?B activity in HT-29 and Lovo cells treated with different concentrations of crude extracts were observed by EMSA.Results Viability of HT-29 cells treated with 400 and 600?g/ml crude extracts were significantly lower than those treated with 0-200?g/ml crude extracts(P0.05).Conclusion Crude extracts of Abrotani herba,extracted by column chromatography with macroporous resin and eluted with 30% alcohol,can inhibit NF-?B activity in colon carcinoma HT-29 and Lovo cells.

Objectives To explore the effect of NF-?B signaling pathway on multicellular resistance(MCR) to 5-Fu in colon carcinoma cells.Methods The multicellular spheroids(MCS) of colon carcinoma HT-29 cell line were cultured by using liquid overlay technique,and the monolayer cells by using routine procedure.The morphology of MCS was observed with light microscope and scanning electronmicroscope(SEM).The expression of proliferating cell nuclear antigen(PCNA) was determined by immunohistochemistry.24 hours after adding 50?g/ml SN50 to spheroids cells,the 50% inhibiting concentration(IC50) for 5-Fu was determined by using radial outgrowth assay.The activity of NF-?B was measured by using electrophoretic mobility shift assay(EMSA) and the specific bands were observed.Apoptosis of spheroids cells,which were pretreated with 5?g/ml 5-Fu for 24 hours,was identified by TUNEL,the apoptosis related proteins caspase-3,Bcl-2 and Bax were detected by Western blotting and analyzed by semi-quantitative method.Results A necrotic core formed in the centre of MCS,and the peripheral cells were actively proliferating.Compared with the monolayer cultured cells,the activity of NF-?B was increased in MCS.When the activity of NF-?B in spheroid cells was inhibited by SN50,5-Fu readily induced cells to apoptosis(15.75%?1.02% vs 8.71%?0.73%,P

Positive regulative factors of PI3K/Akt signaling pathways is often activated in ovarian cancer, whereas the function of negative regulator PTEN is often defective. These two kinds of regulators cooperatively regulate tumor cell proliferation and apoptosis in closely association with tumor angiogenesis and invasion and metastasis. It has been suggested that PI3K-Akt pathway is not only related to prognosis of tumor patients but can also be used as novel targets for tumor therapy.

Objective:To study the changes of iNOS expression in periodontal tissues of rats with periodontitis in orthodontic tooth movement. Methods:48 SD rats at 10 weeks were divided randomly into two groups: periodontitis teeth group and normal teeth group. The maxillary first molar was pulled mesially both in the two groups. 4 rats were executed each time after activation of pull-spring 1, 2, 3, 7,14 and 21d both in two groups. iNOS immunohistochemistry staining were done to compare the expression variations. Results: There were significant differences between the two groups in 2,3, 7, 14 and 21 d. The periodontitis teeth group had stronger positive signals. Conclusion: Periodontitis will increase the expression of iNOS in the rats periodontal tissue during tooth movement, and influence the reconstruction of periodontium.

To find a new source of seed cells for constructing tissue-engineered intervertebral disc, nucleus pulposus (NP) cells and mesenchymal stem cells (MSCs) were isolated from New Zealand white rabbits. The nucleus pulposus cells population was fluorescence-laelled and co-cultured with MSCs with or without direct contact. Morphological changes were observed every 12 h. Semi-quantitative reverse transcriptase-polymerase chain reaction was performed to assess the expression levels of Sox-9, aggreacan and type II collagen every 24 h after the co-culture. MSCs treated with direct contact rounded up and presented a ring-like appearance. The expression of marker genes was significantly increased when cells were co-cultured with direct contact for 24 h. No significant change was found after coculture without direct contact. Co-culture of NP cells and MSCs with direct contact is a reliable method for generating large amount of NP cells used for cell-based tissue engineering therapy.
Aggrecans/metabolism ; Cell Differentiation ; Cells, Cultured ; Coculture Techniques ; Collagen/metabolism ; Gene Expression ; Gene Expression Regulation ; High Mobility Group Proteins/metabolism ; Intervertebral Disk/*cytology ; Mesenchymal Stem Cells/*cytology ; Mesenchymal Stem Cells/metabolism ; Models, Biological ; Reverse Transcriptase Polymerase Chain Reaction ; SOX9 Transcription Factor ; Tissue Engineering/instrumentation ; Tissue Engineering/*methods ; Transcription Factors/metabolism

0.05).IGFR-1? expressed in all the 6 cell lines,with the highest expression in SW480(P0.05),but a gefitinib-induced and dose-dependent increase in p-IGFR-1?,not IGFR-1?,expression was observed in HT29 cells(P0.05),but the expression of IGFR-1? was higher in HCT116 than in Lovo and HT29(P

Granuloma, Pyogenic ; Humans ; Infant, Newborn ; Male ; Mouth Diseases

Objective To monitor the changes of resting energy expenditure in ventilated critically ill children,to compare the results of standard equations and indirect calorimetry (IC) in predicting energy expenditure,and to investigate the possible influence factors of the metabolic status of the critically ill children.Methods From September 2012 to September 2013,56 critically ill children on assisted ventilation and fitting the requirements of IC in pediatric intensive care unit of Shanghai Children's Medical Center were enrolled in this prospective study.IC measurements were performed using metabolic cart on day 1,4,7,10 after trachea intubation.General clinical data of these children were recorded.Results 130 IC measurements were performed in the 56 children.The measured resting energy expenditure (MREE) did not exhibit significant differences among day 1,4,7,and 10 (P =0.379).Although there were no significant differences between MREE and energy expenditure predicted with Schofield and WHO equations (P =0.917,P =0.995),the agreement was poor between the measured and predicted values (R2 =0.185,R2 =0.322).The metabolic status of the children on day 1 of ventilation was only correlated with age (P =0.000) and height (P =0.027),not with severity of underlying diseases or clinical outcomes.Conclusions MREE of IC method in ventilated critically ill children did not significantly change over time in this study.A poor agreement was observed between equationpredicted energy expenditure and MREE.IC measurement of resting energy expenditure is recommended for guiding individual nutritional support among critically ill children so as to improve clinical outcome.