Objective To investigate the effect of Angelica polysaccharides on the balance of Th17/T-regulatory(Treg)cells and the expression of related inflammatory factor proteins in the bone marrow of mice with aplastic anemia.Methods 50 male BALB/c mice were randomly divided into a normal group,model group,cyclosporine A(CsA)group,and angelica polysaccharide low-(APS-L)and high-dose(APS-H)groups.A mouse model of aplastic anemia was prepared by combining irradiation with allogeneic lymphocyte infusion.After modeling,the mice were orally administered with corresponding drugs for 28 days.The general condition,weight changes,and spleen index of the mice were observed.Blood samples were taken to test for changes in basic hematological parameters of peripheral blood,and hematoxylin and eosin staining was used to evaluate the pathological changes in mouse bone marrow tissue.An ELISA method was applied to detect bone marrow TGF-β1.The expression levels of proteins such as IL-10,IL-17A,and IL-6 were analyzed,and flow cytometry was used in detecting the bone marrow Th17/Treg cell ratio.Results Compared with the normal group,the model group mice showed clumsiness during activity;lethargy;pale eyelids,lips,and ears;and reduced food and water intake.Both body weight and spleen index were significantly decreased.Peripheral red blood cell,white blood cell,and platelet counts and hemoglobin concentration were significantly reduced.A disordered bone marrow tissue structure and significantly reduced proliferation of hematopoietic tissue were seen.The expression of IL-17A and IL-6 proteins was significantly upregulated,and the expression of TGF-β1 and IL-10 protein was significantly downregulated.The proportion of Th17 cells in bone marrow significantly increased,the proportion of Treg cells significantly decreased,and the ratio of Th17/Treg cells significantly increased(P＜0.01).Compared with the model group,the general condition of the APS-L and APS-H groups improved,showing an increase in food and water intake;a significant increase in body weight and spleen index;a significant increase in peripheral red blood cells,white blood cells,platelets,and hemoglobin concentration(P＜0.05);an improvement in bone marrow tissue structure;and an increase in hematopoietic tissue proliferation(P＜0.05).The expression of IL-17A and IL-6 proteins was significantly downregulated.The expression of TGF-β1 and IL-10 protein was significantly upregulated(P＜0.05 or P＜0.01).The proportion of Th17 cells in bone marrow significantly decreased,the proportion of Treg cells significantly increased,and the ratio of Th17/Treg cells significantly decreased(P＜0.01).Conclusions Angelica polysaccharides can improve bone marrow hematopoietic function in aplastic anemia mice,and its mechanism of action may include the regulation of Th17/Treg cell balance.

Objective: To investigate the effect of myeloid-derived suppressor cells (MDSCs) from mice bearing breast cancer on the function of normal B cells. Methods:ABABL/c mouse 4T1 breast cancer model was established. The spleen MDSCs of tumor-bearing mouse and normal mouse spleen B cells were sorted by magnetic beads, and the sorted MDSCs and B cells were co-incubated. Flow cytometry was used to test the effect of MDSCs on the expressions of B cell surface molecules, including PD-1, PD-L1, CTLA-4, CCR6, CD62L and MHCⅡ; ELISA assay was used to detect the secretion of IgA, IgM and IgG by B cells; BrdU kit was used to detect B cell proliferation; andAnnexin V/PI staining was used to detect B cell apoptosis. B cells in the co-culture system were again sorted by magnetic beads and were then co-cultured with T cells; BrdU kit was used to detect T cell proliferation, and Annexin V/PI was used to detect T cell apoptosis. Results: Compared with B cell control group, the expression of PD-L1 on B cells in B+MDSC group was increased (P<0.01), while the expressions of PD-1, CTLA-4, CCR6, CD62L and MHC Ⅱ were all decreased (all P<0.01); The IgA, IgM and IgG secreted by B cells were significantly increased (all P<0.01); the proliferation of B cells was increased (P<0.01) and the apoptosis was decreased (P<0.01). Compared with the T cell control group, the proliferation of T cells in the B+MDSC (1:5) +T group was significantly reduced (P<0.01); however, there was no significant difference in T cell apoptosis. Conclusion: MDSCs from breast cancer bearing mice promotes B cell proliferation and inhibits B cell apoptosis, and the MDSC-induced B cells can inhibit T cell proliferation.

Objective To examine the association between serum lipids,including total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), and the risk and progression of breast cancer in postmenopausal and premenopausal women. Methods A review analysis of female patients who underwent breast cancer surgery and blood lipid metabolism testing in Tianjin One Hospital, from January 2013 to October 2013, was performed. A total of 1 081 patients were included in the final analysis. The control group consisted of 2 981 women without breast cancer. We collected all of the cases' demographic,pathology, lymph nodes metastasis information, was used to testify the difference of serum lipid level between patient and control group, also the postmenopausal and pre-menopausal groups. Results The average age of the patients and control subjects were (51.6±0.3) and (51.0±0.2) years, respectively. Serum TC and LDL-C levels in the patient group, (5.16±0.03) and (3.28±0.26) mmol/L, respectively, were significantly higher than in the control group, (5.02±0.01) and (2.51±0.01) mmol/L, respectively (t values 3.89 and 4.81 and P<0.001). HDL-C levels in the patient group, (1.60±0.01) mmol/L, were significantly lower than in the control group, (1.65±0.01) mmol/L (t=3.90, P<0.001). Similar observations were made in postmenopausal patients. Serum TC and LDL-C levels in the postmenopausal patient group, (5.48±0.04) and (3.27±0.03) mmol/L, respectively, were significantly higher than in control subjects, (5.24±0.02) and (2.71±0.02) mmol/L, respectively (t values 4.75 and 15.30, all P values <0.001). HDL-C levels in postmenopausal patients, (1.60±0.02) mmol/L, were significantly lower than in the control group, (1.69±0.01) mmol/L (t=4.85,P<0.001). In the breast cancer patient group, those at pathological stages 0-Ⅱ had lower TG levels than those at Ⅲ-Ⅳ. These values were (1.19±0.05) and (1.43± 0.09) mmol/L, respectively (t=2.69,P<0.001). Meanwhile, patients with no lymph node metastases had lower TG levels than the lymph node-positive group, with values of (1.15 ± 0.05) and (1.37 ± 0.07) mmol/L, respectively (t=2.53,P=0.012). Conclusion We found that dyslipidemia may affect the incidence of breast cancer, particularly among postmenopausal women. Serum lipids may promote cancer progression through higher TG and low HDL-C levels.

Objective To examine the association between serum lipids,including total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), and the risk and progression of breast cancer in postmenopausal and premenopausal women. Methods A review analysis of female patients who underwent breast cancer surgery and blood lipid metabolism testing in Tianjin One Hospital, from January 2013 to October 2013, was performed. A total of 1 081 patients were included in the final analysis. The control group consisted of 2 981 women without breast cancer. We collected all of the cases' demographic,pathology, lymph nodes metastasis information, was used to testify the difference of serum lipid level between patient and control group, also the postmenopausal and pre-menopausal groups. Results The average age of the patients and control subjects were (51.6±0.3) and (51.0±0.2) years, respectively. Serum TC and LDL-C levels in the patient group, (5.16±0.03) and (3.28±0.26) mmol/L, respectively, were significantly higher than in the control group, (5.02±0.01) and (2.51±0.01) mmol/L, respectively (t values 3.89 and 4.81 and P<0.001). HDL-C levels in the patient group, (1.60±0.01) mmol/L, were significantly lower than in the control group, (1.65±0.01) mmol/L (t=3.90, P<0.001). Similar observations were made in postmenopausal patients. Serum TC and LDL-C levels in the postmenopausal patient group, (5.48±0.04) and (3.27±0.03) mmol/L, respectively, were significantly higher than in control subjects, (5.24±0.02) and (2.71±0.02) mmol/L, respectively (t values 4.75 and 15.30, all P values <0.001). HDL-C levels in postmenopausal patients, (1.60±0.02) mmol/L, were significantly lower than in the control group, (1.69±0.01) mmol/L (t=4.85,P<0.001). In the breast cancer patient group, those at pathological stages 0-Ⅱ had lower TG levels than those at Ⅲ-Ⅳ. These values were (1.19±0.05) and (1.43± 0.09) mmol/L, respectively (t=2.69,P<0.001). Meanwhile, patients with no lymph node metastases had lower TG levels than the lymph node-positive group, with values of (1.15 ± 0.05) and (1.37 ± 0.07) mmol/L, respectively (t=2.53,P=0.012). Conclusion We found that dyslipidemia may affect the incidence of breast cancer, particularly among postmenopausal women. Serum lipids may promote cancer progression through higher TG and low HDL-C levels.

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.

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 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 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: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.