Objective To study the effects of Genistein-magnetic-nanoparticles at different concentrations on the growth and expression of focal adhesion kinase(FAK) in human hepatocellular carcinoma cell line HepG2.Methods Activities of HepG2 cells treated by Genistein-magnetic-nanoparticles were examined by MTT assay.The expression of FAK mRNA and protein was respectively detected by immunohistochemistry staining and reversed transcriptase polymerase chain reaction(RT-PCR).Results Genistein-magnetic-nanoparticles at a concentration of 10-80mg/L inhibited the proliferation of HepG2 cells,with obvious concentration-dapendent and time-dependent effect relationships(P

Objective:To investigate the expression of indoleamine 2,3-dioxygenase and the distribution of Treg cells in breast cancer and tumor draining lymph nodes (TDLNs) and to explore the relationship between them.Methods:26 cases of breast cancer and 10 cases of breast benign diseases were collected from Tianjin medical university cancer hospital.RT-PCR was used to detect the mRNA of IDO in breast cancer,TDLNs,benign diseases and normal breast tissues.Immunohistochemistry was used to detect the expression of IDO and Foxp3 proteins in the same tissues. Results:The mRNA and the percentage of IDO~+ cells [(19.59±7.65)%] in TDLNs were higher than in breast cancer [(13.16±7.82)%] (P<0.05),while in the breast cancer were higher than in benign diseases [(3.24±1.30)%] and normal breast tissue [(2.70±1.53)%] (P<0.05).Expression of IDO protein in breast cancer was associated with tumor clinical stage and lymph nodes metastasis while had no relationship with tumor diameter,ER,PR and Her2 status.The percentage of Foxp3~+ cells in breast cancer [(3.50±1.04)%] was higher than in benign diseases [(0.71±0.42)%] (P<0.05) and normal breast tissue [(0.55±0.34)%],and that of the TDLNs [(6.13±2.31)%] was higher than in breast cancer [(3.50±1.04)%] (P<0.05).The percentage of IDO~+ cells was positive correlated with the distribution of Treg cells in breast cancer(r~2=0.449,P<0.05)and TDLNs (r~2=0.454,P<0.05).Conclusion:Expression of IDO in breast cancer is upregulated.The high level expression of IDO is accompanied by increasing Treg cells in breast cancer and TDLNs,which suggests that breast cancer can recruit Treg cells by expressing IDO to participate the immune tolerance.

Objective To study the effects of Genistein - magnetic - nanoparticles at different concentrations on the growth and ex-pression of focal adhesion kinase (FAK) in human hepatocellular carcinoma cell line HcpG2. Methods Activities of HepG2 cells treated by Genistein - magnetic - nanoparticles were examined by MTT assay. The expression of FAK mRNA and protein was respectively detected by immunohistochemistry staining and reversed transcriptase polymerase chain reaction (RT - PCR) . Results Genistein - magnetic -nanoparticles at a concentration of 10 -80mg/L inhibited the proliferation of HepG2 cells, with obvious concentration - dapendent and time - dependent effect relationships (P < 0.05). After treatment with various concentrations of Genistein - magnetic - nanoparticles for 48h, the relative level of FAKmRNA of Genistein - magnetic - nanoparticles and control groups was 1.242 ± 0.031,1.213 ± 0.443,0.834 ± 0.044,0.652 ± 0.039,0.446 ± 0.041, and 1.443 ± 0.053 (F = 21.97 ,P < 0.05), respectively. The expression of FAK protein in the cells was decreased, which showed an obvious a concentration - effect relationship. Conclusion Genistein - magnetic - nanoparticles can reduce the mRNA and protein expressions of FAK and inhibit the proliferation of HepG2 cells.

Objective: To analyze the circulating endothelial cells (CECs) and circulating endothelial precursors (CEPs) in peripheral blood of tumor patients. Methods: CECs and CEPs were enumerated in 57 tumor patients and 15 healthy controls by 3-color flow cytometry. The serum level of angiogenesis related cytokines(VEGF, bFGF)was determined by ELISA method. Results: In tumor patients, mean value of CECs and CEPs was (0.378?0.047)% and (0.059?0.013)% respectively. The serum level of VEGF and bFGF were (295.58?59.56) ng/L and (28.73?7.40) ng/L. The amount of CECs/CEPs positively correlated with the serum level of VEGF and bFGF. Conclusions: The CECs/CEPs and serum level of VEGF and bFGF of tumor patients was higher than those of normal controls. VEGF and bFGF may participate into the course of endothelial progenitors mobilization.

Objective:For developing the study of MUC1/Y vaccine,constructed th eukaryotic expression vectors expressing MUC1/Y.Used it to modify the DC inducint CTL in order to treat the gastrointestinal cancers.Methods:Obtained the MCU1/Y cDNA full length cloned it into pIRES2 EGFP that expressing green fluorescence protein and pcDAN3 1+ respectively.Selected 8 cases of gastrointestinal cancer whose HLA phenotype was A2,inducing DC using rhIL 4 combined with GM CSF in vitro.Transfected DC with pcDAN3.1 MUC1/Y then co cultured DC with T cell to induce CTL (named as T pcDAN3 1 MUC1/Y).At the same time,used pIRES2 EGFP MUC1/Y to detect transfection efficiency.SW620 (HLA A2+,MUC1/Y+),the gastric cancer cell line was used as specific target cell;Lovo(HLA A2 ,MUC1/Y+)and Raji(HLA A2 ,MUC1/Y )were used as unspecific target cells.The cytolytic of specific CTL was measured by LDH releasing assay.The apoptosis of target cells were detected by ANNEXIN V FITC kit.The ability of IFN ? releasing of T cells was measured by ELISA.Results:The transfection efficiency of plasmid was about 8%.There was significant difference between the cytolytic activity of T pcDNA3 1 MUC1/Y to SW620,Lovo and Raji.The cytolytic activity was about (42 8?6 15),(27 26?1 96)% and (22 73?2 15)% respectively.Compared with T IL 2)CTL induced by PBMC stimulated of IL 2(100 U/ml)) and T pcDNA3 1(CTL induced by DC transfected by pcDNA3 1+),the cytolytic activity of T pcDNA3 1 MUC1/Y against SW620 cell line showed a significant increase.The results of ANNEXIN V-FITC experimences showed that T pcDNA3 1 MUC1/Y could induce apoptosis of specific target cell.Conclusion:The expressing of MUC1/Y mRNA has important sense in gastrointestinal cancer.Constructed eukaryotic vector pIRES2 EGFP MUC1/Y that contains full length MUC1/Y cDNA could be used to study the transfection efficiency and select the positive clone;pcDAN3 1 MYC1/Y could induce powerful CTL immunoresponse.

Objective:Analyze the influence of hematopoietic stem cell mobilization on circulating endothelial cells (CECs) and circulating endothelial precursors (CEPs).Methods:CECs and CEPs were enumerated in 68 tumor patients and 15 healthy controls by 3-color flow cytometry. 11 cases underwent PBSC (peripheral blood stem cell) mobilization by combination chemotherapy and G-CSF (granulocyte colony-stimulating factor).Results:CECs and CEPs in tumor patients were 0.378%?0.103% and 0.059%?0.013% respectively,which were higher than that of normal controls.CECs/CEPs in peripheral blood (PB) were increased after G-CSF mobilization.Conclusion:Hematopoietic and endothelial progenitors can be mobilized into the PB by treatment with growth factor G-CSF.

Objective:To observe ability of DC vaccine transfected with tumor cell total RNA to induce specific antitumor immunity in lung cancer patients in vitro.Methods:DCs were generated from lung cancer patients' peripheral blood mononuclear cells(PBMC).Total RNA was isolated from lung cancer tumor cell line Calu-6 by Trizol.Autologous DCs transfected with Calu-6 total RNA by liposome were used to induce specific CTL proliferation.Specific cytotoxicity and IFN-? secretion were measured by LDH assay and ELISA method.Results:Transfected DCs exhibited dramatically increased expression of specific membrane markers and function-associated molecules,and were more potent in stimulating allogenous or autologous T cell proliferation than that of control DCs.Specific CTLs induced by transfected DCs showed higher cytotoxicity than LAK against Calu-6 antigens positive target cells.When sensitized lymphocytes were restimulated by transfected DCs again,IFN-? secretion enhanced significantly.Conclusion:Lung cancer patient's autologous DCs transfected with tumor total RNA are effective vaccines in stimulating specific antitumor T-cell immunity in vitro.

Objective:This work aims determine the expression of the neurotensin (NTS) gene in hepatocellular carcinoma (HCC) subgrouping using immunohistochemical staining (IHC) as well as to evaluate the correlation between the activation of NTS/IL-8 pathway in HCC and inflammatory response in microenvironment and epithelial mesenchymal transition (EMT) in cancer and in the prognosis of patients. Methods:Tumor tissues and corresponding adjacent normal tissue were collected from 64 cases of HCC patients. The expression levels of NTS protein and multiple inflammation and EMT-related proteins, including IL-8, VEGF, MMP9, CD68, E-Cadherin,β-Catenin, and Vimentin, were examined in 64 cases of paraffin-embedded HCC tissues using the immunohistochemistry (IHC) staining method. The clinical outcome and overall survival (OS) among 64 cases of HCC patients were compared. Results:We found that the frequency of NTS-expressing tissues among all HCC samples was 17.19%(11/64). Significantly increased IL-8 protein was confirmed in 90.91%of NTS+HCC samples and was positively correlated with the levels of NTS protein in cancer tissues (P=0.036), which implied the dysfunctional activation of NTS/IL-8 pathway in HCC. The levels of VEGF and MMP9 were significantly correlated with the co-expression of NTS and IL-8 in HCC. Evident features of EMT, including decreased membrane expression of E-Cadherin and increased accumulation of cytoplasmicβ-Catemin and Vimentin, were found in NTS+IL-8+samples. The co-expression of NTS and IL-8 in cancer was significantly correlated with the clinical outcomes of patients, as the mortality rate of NTS+IL-8+HCC patients is 2.5-fold higher than that of others after surgery (P=0.022).Accordingly, the OS of NTS+IL-8+HCC patients significantly decreased (24.65±4.45 m vs. 75.79±16.32 m, P=0.013), and these patients are at a higher risk of death at an expected hazard ratio (HR) of 3.457. Conclusion:The NTS/IL-8 pathway is dysfunctionally activated in a subgroup of HCC samples. Highly expressed NTS is associated with increased inflammatory response in microenvironment, enhanced EMT in cancer, and worse prognosis in HCC patients.

Objective:To highlight the developmental process of 3D cell culture technology system, which is more suitable for isolating and identifying lung cancer stem-like cells than 2D cell culture technology system, and to explore the application of 3D cell cultures in the evaluation of proliferation, apoptosis, invasion, and drug resistance of lung cancer. Methods:Cells (104/well) from the human lung adenocarcinoma cell lines A549 and RPMI 1640 were cultured in complete medium containing 10%fetal bovine serum. Cell suspension was cultured in a BME basal medium. A growth curve was drawn after 7 d of culture. The stem-like cell was identified through a mammosphere culture, drug resistance and invasion assay, and flow cytometry. Data of A549 cells cultured in 3D and 2D tra-ditional cell culture technologies were compared. Results: Cells from the 3D cell culture had higher tumor formation rates [(20.75 ± 0.85) d vs. (60.25 ± 1.49) d, P<0.01)] and tumor sphere formation (28.50%± 1.17%vs. 8.67%± 0.80%, P<0.01) than those from the 2D cell culture. Moreover, cells from 3D cell culture were more invasive and resistant to therapy (58.17%± 2.19%vs. 41.70%±5.81%in 48 h, P<0.01;33.27%±5.76%vs. 27.30%±4.25%in 72 h, P<0.01). Phenotype experimental results demonstrated that the CD44 and CD326 cells were double-positive, whereas the CD24 cell was negative. Conclusion:The proportion of stem-like cells in A549 cell line after 3D cell culture significantly increased compared with 2D cell culture. The 3D cell culture can promote the proliferation of lung cancer stem cells.