Objective To explore the expression of membrane form of CD28 (mCD28) on T lymphoeytes and the serum level of soluble CD28 (sCD28) in elderly patients with primary non-small cell lung cancer (NSCLC) in order to investigate the relationship between age-related changes of CD28 and the development of NSCLC in elderly patients. Methods 63 elderly patients with NSCLC, 35 elderly patients with lung benign lesion, 30 elderly healthy donors, 30 young healthy donors, 20 young patients with lung benign lesion and 20 young patients with NSCLC were enrolled in this study. The mCD28 on T cells and the serum level of sCD28 were measured by four-color flow eytometric assay and enzyme linked immunosorbent respectively, and the relationship between CD28 and clinical characteristics of NSCLC was analysed Results The expression of mCD28 was decreased and the serum level of sCD28 was increased in elderly patients with NSCLC compared with the other groups (F= 184.25, P<0. 01 ; F= 365.40, P<0.01). The expression of mCD28 was significantly lower and the level of sCD28 was significantly higher in elderly healthy donors than those in young healthy donors and young patients with lung benign lesion (P<0. 05). There were no significantly statistical differences in expression of mCD28 and level of sCD28 between elderly healthy donors and elderly patients with lung benign lesion [(42.84±5.82)% vs. (46.09±-7.34)%, (39.38±6.02)μg/L vs. (35.84±5.02)μg/L, P>0. 05]. Logistic regression analysis showed that aging (OR=2. 432), down-regulation of mCD28 expression (OR=0. 876) and up-regulation of sCD28 level (OR= 1. 113) were the risk factors for lung cancer. In the elderly patients with NSCLC, there were significant differences in mCD28 expression and sCD28 level between stages Ⅲ-Ⅳ and stages Ⅰ-Ⅱ [(16. 51± 5.64)% vs. (24.41±8.24)%, (75.03±5.98) μg/L vs. (66.73±7.52)μg/L; t=4.497,4.794, both P <0. 01]. However, there were no significantly statistical differences among different pathological types (F=0. 609, 0. 302, both P > 0. 05). Conclusions The down-regulation of mCD28 expression and up-regulation of sCD28 level with advancing age play an important role in the oncogenesis and development of primary non-small cell lung cancer in the elderly patients.

Objective To investigate the seruln levels of solubl B7-H3(sB7-H3)in patients withbacteremia and in those with chronic hepatitis B(CHB),and its clinical significance.Methods Serumlevels of sB7-H3 from 50 bacteremia patients,78 CHB patients and 50 healthy individuals wero detected byEUSA,and its correlations with the quantities of leucocytes,neutrophils,lymphocytes and monocytes wereanalyzed.Results The average level of sB7-H3 in serum of bacteremia patients was(50.69±26.43)μg/ml which was higher than that of healthy group(P＜0.01);serum sB7-H3 level of patients with CHB was(18.07±7.28)μg/ml,and it had no significant difference compared with that in the healthy group(P＞0.05).Besides,positive correlations between the scrum sB7-H3 level in and the quantities of leucocytesand granulocytes were observed in bacteremia patients.Conclusions It suggests that sB7-H3 misht bepotentially used as a new parameter to improve the clinical diagnosis of bacteremia.

Objective To evaluate the effects of B7-H3 gene transfection on 18F-FDG uptake and 18F-FLT uptake in prostate cancer cells.Methods The absorption (A) values of untransfected prostate cancer(RM1) cells and B7-H3 gene-transfected RM1 (RM1-B7-H3) cells were detected at different culturing time points (0.5,1,2,3,4 and 5 d) with cell counting kit-8 (CCK-8) test.Cell cycle phase distribution of RM1 and RM1-B7-H3 cells was measured with flow cytometry.18F-FDG uptake of RM1 and RM1-B7-H3 cells was measured with γcounter and calculated under different conditions:5× 104-5× 106 cells; 0-11.0 mmol/L glucose; 20-120 min incubation in 37 ℃.18F-FLT uptake of RM1 and RM1-B7-H3 cells was measured in 1×106 cells under incubation for 100 min at 37 ℃.After administering anti-B7-H3 monoclonal antibody 4H7,18F-FDG uptake of RM1-B7-H3 cells was measured.The data were analyzed using one-way analysis of variance and two-sample t test.Results The A values of RM1-B7-H3 cells after being incubated for 1,2 and 3 d were higher than those of RM1 cells(1.59±0.23,2.26±0.15 and 2.01±0.60 vs 1.22±0.14,1.10± 0.09 and 1.04±0.15,t=3.923,19.228,4.467,all P＜0.01).There was no statistical significance between the 2 groups at other time points (t=-0.094,0.858,2.000,all P＞0.05).The ratios of RM1-B7-H3 cells in G1,S and G2/M phases were(32.96±2.56) ％,(39.11 ±2.57) ％ and (27.94±0.21) ％,respectively.The ratio of S phase in RM1-B7-H3 cells was higher than that in RM1 cells ((32.76±1.90)％,t=3.442,P＜ 0.05).18F-FDG uptake of the both cell lines decreased with the increase of glucose concentrations,while the uptake went up with the increase of cell number and incubation time.With the cell number of 1.0× 106,incubation time of 100 min and temperature of 37 ℃,the 18F-FDG uptake of RM1-B7-H3 and RM1 cells was (55.07±3.99)％ vs (44.16±3.60)％ (t=4.977,P＜0.01) ; and 18F-FLT uptake of RM1-B7-H3 and RM1 cells was (5.25±0.81)％ vs (3.33±0.64)％ (t=4.567,P＜0.01).After treated with antibody 4H7,18F-FDG uptake of RM1-B7-H3 cells ((45.36±2.92) ％) was lower than that of untreated group (F=10.001,P＜ 0.01).Conclusion B7-H3 gene transfection may promote the metabolism and proliferation of prostate cancer cells,and thereby increase the 18F-FDG uptake and 18F-FLT uptake.

Objective To evaluate the relationship of the hepatocyte growth factor (HGF) and the nuclear matrix protein 22 (NMP22) in urine and the stage and grade of bladder uroepithelium carcinoma.Methods A total of 48 post-operative patients (males 39, females 9) with bladder cancer enrolled in this study were perfused with THP. The voided urine of all the patients before and 6 months after perfusion were recovered selectively. HGF and NMP22 ELISA kits were used to detect bladder cancer. Results The recurrence rate was 12.5 %. The HGF level had positive correlation with the stage and grade of bladder uroepithelium carcinoma (P ＜0.05). The NMP22 level had positive correlation with the grade of bladder cancer. The sensitivity and specificity of HGF, NMP22 and cytology were 100 % (6/6), 83.3 % (5/6), 66.7 %(4/6) and 61.9 % (26/42), 57.1% (24/42), 97.6 % (41/42), respectively. Conclusion The HGF and NMP22 are both valuable tumor markers in the urine of bladder uroepithelium carcinoma. They have intimate relation with the stage and grade of bladder uroepithelium carcinoma. Hence combined with cytology, they could be selected as the significance level of early screening and diagnosing.

Objective To investigate the effect of B7-H3 on human hepatocellular carcinoma cell line HepG2 mediating regulation on human peripheral blood CD8+T cell activation,cell cycle and secretion of IL-17.Methods The expression of the B7-H3 on HepG2 cells was detected by RT-PCR and FCM respectively.B7-H3 was silenced by PGPU6/GFP/neo-B7-H3shRNA plasmid via cathodolyte liposome transfection method.CD8+T cells were sorted from healthy human peripheral blood with immunomagetic beads.The effect of HepG2 cells on activation,cell cycle and cytokine secretion of CD8+T cells which was stimulated by PHA or PMA respectively were analyzed by FCM.Results B7-H3 was highly expressed on HepG2 cells,and PGPU6/GFP/neo-B7-H3shRNA plasmid could effectively block down its expression.Otherwise,HepG2 cells could inhibit the expression of CD69,the early activation phenotype of T cell,blockade B7-H3 on HepG2 cells could significantly attenuate the inhibitory effects.Likewise,blockade B7-H3 on HepG2 cells apparently reversed the inhibitory effects of HepG2 cells on CD8+T cell cycle through down-regulating the cell number in G0/G1 phase and up-regulating the cell number in S phase;Moreover,HepG2 cells caused a sharp increase of IL-17 which was secreted by CD8+T cells and the level of IL-17 was further up-regulated after blocking down B7-H3.Conclusion HepG2 cells highly expressed B7-H3 that could promote the inhibitory the effect of HepG2 on expression of CD69 and cell cycle of CD8+T cells.HepG2 cells were able to up-regulate the level of IL-17 secreted by CD8+T cells,in which B7-H3 played an inhibitory role.

AIM: To establish a quantitative measurement method of ECV-304 cell migration in a scratch wound model in vitro. METHODS: The method of ECV-304 cell scratch wound model was improved. The ECV-304 cell migration was measured quantitatively using a computer-assisted video microscopic method with image-analysis system. The effects of heparin and thrombospondin-1 on ECV-304 cell migration were observed in the scratch wound model in vitro. RESULTS: The average width and length of cell scratch wound was (0.95?0.08) mm and (51.20?3.40) mm respectively in 120 experiments. The peak of cell migration reached about 24 h after culture. The stimulated effect of heparin and inhibited effect of thrombospondin-1(TSP-1) on cell migration were observed. CONCLUSION: A quantitative measurement method of ECV-304 cell migration in vitro is established. The stimulated and inhibited effects on ECV-304 cell migration were proven respectively using this method.

Objective To study the impacts of B7-H3 molecule on the proliferation of T lymphocytes in different activation conditions and on the secretion of relevant cytokines.Methods Peripheral blood samples were collected from healthy subjects to separate peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation.T lymphocytes were isolated from some of the PBMCs and purified with T Cell Enrichment Kit.PBMC and purified T lymphocytes were activated by anti-CD3/CD28 monoclonal antibodies (McAb) in vitro.Flow cytometry analysis was used to detect the expression of CD28 and CTLA-4 on T lymphocytes at different time points for further analyzing the activation states of T lymphocytes.On this basis, human B7-H3-Fc fusion protein was added into the mixed co-cultivation system on days 0, 1, 2, 3 and 4, and Hu-Fc fusion protein was used as isotype control.CCK-8 method was performed to detect the proliferation of T lymphocytes in each group.ELISA method was used to detect the secretion of cytokines (IL-2, IL-10 and IFN-γ) and to analyze the immune responses induced by stimulating T lymphocytes at different states of activation with B7-H3.Results B7-H3 molecule significantly inhibited the quiescent T lymphocytes from secreting IL-2 and IL-10, but had no significant impact on IFN-γ secretion.Moreover, it significantly promoted the activated T lymphocytes to secret IL-2 and IFN-γ, but had no obvious impact on IL-10 secretion.Results of the cell proliferation assay showed that B7-H3 molecule inhibited the in vitro proliferation of T lymphocytes in the PBMC, but had no obvious impact on purified T lymphocytes.ConclusionThe regulatory effects of B7-H3 molecule on the immune functions of T lymphocytes vary with the activation states of T lymphocytes.

Background:The abnormal expression of costimulatory molecules is closely related to immune escape of tumor cells. Programmed death-1(PD-1)is an important negative costimulatory molecule,and can induce and maintain the immune tolerance of tumor cells by binding with related ligands,thus promotes the development and progress of tumor. Aims:To investigate the expression of peripheral blood PD-1 and its clinical significance in patients with colorectal cancer. Methods:Eighty-eight patients with colorectal cancer from March 2015 to September 2015 at the First Affiliated Hospital of Soochow University were enrolled,and 16 healthy volunteers were served as controls. Level of soluble PD-1(sPD-1)was determined by ELISA. The expression of PD-l on CD3 + T cells in peripheral blood was measured by flow cytometry. Results:Level of sPD-1 and expression of PD-l on CD3 + T cells in peripheral blood in colorectal cancer patients were significantly higher than those in controls(P ﹤ 0. 05). Level of sPD-1 in colorectal cancer patients was closely related to tumor differentiation,lymph node metastasis and Dukes’stage( P ﹤ 0. 05),but not related to gender,age and tumor location(P ﹥ 0. 05). Conclusions:Peripheral blood PD-1 is highly expressed in patients with colorectal cancer,and is positively correlated with tumor stage and metastasis. The detection of PD-1 is helpful to estimate the progress of colorectal cancer,and it may become a new tumor marker or a target for anti-tumor targeted therapy.

Objective:To prepare functional monoclonal antibodies against human 4-1BBL molecule and analysis of their biological characteristics.Methods:Female BALB/c mice of 6-8 weeks old were immunized with 4-1BBL transfectant (L929/4-1BBL) as immunogen.The spleen B cells of the mice were fused with sp2/0 and hybridoma cells were screened with 4-1BBL transfectant (L929/4-1BBL) by FCM.The biological characteristics of antibody were investigated by rapid isotyping analysis,karyotype analysis,competitive inhibition test etc.Furthermore,the growth of monocytes in vitro was determined by cell number counting and the cytokine concentration in the supernatants was assayed by ELISA.Results:One hybridoma cell line named 3E7 was obtained,which had the property of secreting anti-human 4-1BBL monoclonal antibody continuously and steadily.This mAb specifically recognized human 4-1BBL molecules.The experimental results manifested that mAb 3E7 could effectively enhance the growth of monocytes and the high level IL-6 and TNF-? secretion.Conclusion:One hybridoma cell line which can secret a functional mouse anti-human 4-1BBL mAb has been developed successfully.This mAb can specifically recognize human 4-1BBL and regulate growth and functions of monocytes in vitro.

Objective To explore the change of CD4+CD25+Foxp3+ regulatory T (Treg) cells during aging and the relation with lung tumor. Methods The Lewis lung cancer model was set up in C57BL/6 female mice, and the 36 mice were divided into young health group, middle-aged health group, elderly health group, young tumor group, middle-aged tumor group and elderly tumor group. The percentages of CD4+CD25+Foxp3+ Treg in CD4+ T cells in mice spleen cells were measured by flow cytometry, for reflecting the quantity of CD4+CD25+Foxp3+ Treg cells. And the level of Foxp3 mRNA in splenocyte was tested by real-time PCR method. Results The level of CD4+CD25+Foxp3+/CD4+ T cells and the quantity of Foxp3 mRNA were higher in tumor groups than in healthy groups(both P＜0.05 ). Besides, in the healthy groups, there were statistical differences in the level of CD4+CD25+Foxp3+/CD4+ T cells (F=47.70, P=0.000) and the quantity of Foxp3 mRNA among the different months groups. Accumulation of the CD4+CD25+Foxp3+ Treg cells was accompanied with aging, the elderly mice contained a significantly larger population of CD4+CD25+Foxp3+ Treg cells in their spleen when compared with the younger counterparts, and the highest was the elderly tumor group. So it was with the functional gene Foxp3 mRNA (F=6.56, P=0.009). Conclusions The results suggest a close relationship of the change of CD4+CD25+Foxp3+Treg cells with aging and the genesis and development of lung tumor.