Objective:To evaluate the effect of nicastrin (nct) gene on the biological functions of melanocytes in zebrafish.Methods:By using a morpholino oligonucleotide (MO) technology, a nct-MO sequence targeting the zebrafish nct mRNA was designed, so was a MO control (ctrl-MO) sequence. Then, the enhanced green fluorescent protein (EGFP) mRNA with MO target sequence at its 5′ end was synthesized, and co-microinjected with the nct-MO or ctrl-MO sequence into the zebrafish embryos to verify the silencing efficiency of nct-MO and observe changes in developmental phenotypes in zebrafish. With wild-type zebrafish as a blank control group, real-time fluorescence-based quantitative PCR (RT-PCR) was conducted to determine the mRNA expression of melanin synthesis-and notch signaling pathway-related genes, including mitfa, tyr, tyrp1a, tyrp1b, dct, pmela, notch1a, notch1b and hey1 genes. One-way analysis of variance was used for the comparison of means among multiple groups, and least significant difference (LSD) - t test for multiple comparisons. Results:Eight hours after zebrafish fertilization, green fluorescence was observed in the zebrafish embryos in the ctrl-MO+EGFP mRNA group, but not in the nct-MO+EGFP mRNA group or blank control group. Forty-eight hours after fertilization, the proportion of pigmented area among the whole area of the tail of zebrafish larvae was significantly lower in the nct-MO group (0.169 ± 0.083) than in the ctrl-MO group (0.258 ± 0.042, t=3.202, P=0.005) , and disorderly pigment distribution in the tails was observed in the nct-MO group. RT-PCR revealed significant differences in the mRNA expression of pmela, tyrp1a and hey1 genes among the nct-MO group, ctrl-MO group and blank control group (all P < 0.05) , but no significant difference was observed in the mRNA expression of mitfa, tyr, tyrp1b, dct, notch1a or notch1b genes among the 3 groups (all P>0.05) ; the relative expression levels of pmela and tyrp1a mRNAs were significantly lower in the nct-MO group (0.708 ± 0.028, 0.558 ± 0.136, respectively) than in the ctrl-MO group (1.023 ± 0.142, 1.016 ± 0.134, respectively, both P < 0.05) . Conclusion:The nct gene may affect biological functions of melanocytes by regulating melanin synthesis in zebrafish.

BACKGROUND: Hematopoietic stem cells have an ability of multi-directional differentiation and can be differentiated into megakayocytes in the presence of suitable hematopoietic growth factors and lineage special growth factors.Inducing of CD34+ stem cells to megakaryocytes in vitro involves many changes in gene expression, especially the signal transduction genes.OBJECTIVE: To study the expression of signal transduction genes after differentiation from stem cells into megakaryocytes.DESIGN: Open experiment.SETTING: Department of Tumor Immunology, Fujian Provincial Tumor Hospital.MATERIALS: 60-145 mL of cord blood was collected from normal labor fetuses under sterile conditions by using the blood-bag collective method. Patients and their relatives provided the confirmed consent. Reagents and equipments were detailed as follows: VarioMACS segregation apparatus, CD34+ Isolation Kit, SCGM serum-free medium, CD monoclonal antibodies, cytokines, human genome Oligo Set Version 2.1 from the CapitalBio Corporation, and LuxScan 10K/A bi-passage laser scanning apparatus. The researchers obtained consent from the local ethic committee.METHODS: This experiment was carried out in the Department of Tumor Immuonlogy, Fujian Provincial Tumor Hospital and the CaptialBio Corporation. from July 2005 to June 2006. RNA was extracted separately from CD34+ cells purified from cord blood and CD41+ cells induced from the same cord blood. The cDNA probes were synthesized by reverse transcription and purified. CD34+ cells before culturing and CD41+ cells after culturing were labeled with Cy3-dCTP and Cy5-dCTP respectively. Labeled DNA was dissolved in 30 μL hybridization fluid and hybridized at 42 ℃ over night MAIN OUTCOME MEASURES: The differential gene expression between haematopoietic stem cells and megakaryocytes involving cell signal transduction were detected by using DNA microarray analysis. According to the gene chip results,17 differentially expressed genes were selected for further analysis by RT-PCR.RESULTS: In this experiment, 3 522 differentially expressed genes were screened, including 1705 up-regulated genes and 1 817 down-regulated genes. In the 3 522 differentially expressed genes, there were 343 genes involved in cell signaling, 150 genes involved in transcription regulation, 21 genes involved in cell differentiation. The expression of the CD61 gene increased 369.83 fold, the expression of the CD41 gene increased 27.38 fold, and the expression of PF4 gene increased 24.06 folds; The expression of mitogen-activated protein kinases (MAPKs), G protein-coupled receptors (GPCRs) and members of RAS oncogene family increased mostly. The expression of the genes involved in STAT pathways, both SOCS1 and JAK2 were up-regulated, but STAT5A was down-regulated. As determined by the gene chip results, 17 differentially expressed genes were selected for further analysis by RT-PCR. GAPDH was used as the internal control, and RT-PCR results were in agreement and confirmed the finding from the gene chip expression results.CONCLUSION: Thrombopoeitin (TPO) and other hematopoietic growth factors may enhance the proliferation and differentiation of megakaryocytes derived from cord blood stem cells by GPCRs-Ras-MAPK pathway.

AIM: To elucidate the effects of losartan on the expression ofmatrix metalloproteinases-2, JNK1/2 and proliferation in cardiac fibroblast. METHODS: Neonatal rat cardiac fibroblasts were cultured. The cells proliferation was determined by MTT. To determine effects of AngⅡ on JNK1/2 activity, cells were incubated (for 0, 2, 5, 10, 30, 60, 120 min) in serum-freemedia with AngⅡ, and the other group fibroblasts were exposed to serum-free media with or without AngⅡ and losartan (AngⅡ 100 nmol/L, AngⅡ 100 nmol/L+losartan 100 nmol/L, losartan100 nmol/L, losartan for 45 min before). Cells protein was collected with MBST buffer. The relative abundance of MMP-2, JNK1/2 and p-JNK1/2 in cells was determined by immunoblotting. The secretion of MMP-2 in media of cell culture was determined by ELISA. RESULTS: AngⅡ increased the proliferation of CFB in a dose-dependent manner, whereas losartan decreased the proliferation of CFB stimulated by AngⅡ in a dose-dependant manner, too (P＜0.05). The relative abundance of JNK1/2 was highest in AngⅡ of the 2-min-stimulated group. AngⅡincreased expression of JNK1/2 and MMP-2 protein (P＜0.05), on the contrary, losartan inhibited JNK1/2 and MMP-2 protein expression.CONCLUSION: AngⅡ induce the increase of proliferation of CFB, expression of JNK1/2 and MMP-2 in CFB, and losartan inhibits these effects of AngⅡ.

Objective:To study the alteration of K-ras mutations in different stages of colorectal cancer(CRC) and its influence on the progression of CRC. Methods:The 20 paraffin-embedded tissues, including primary foci, metastatic lymph nodes, remoter metastatic foci, colorectal adenoma, and normal colorectal tissues, were collected from 20 patients with colorectal cancer. The sequence of PCR-amplified products were analyzed. Results:The wild K-ras gene was expressed in normal colorectal tissues. The mutation frequency of K-ras gene was 20.0% (4/20) in colorectal adenoma, 30.0% (6/20) in primary foci, 25.0% (5/20) in metastatic lymph nodes, and 30% (6/20) in remote metastatic lesions. In the samples with K-ras mutations, the consistency of the types of K-ras mutations between primary foci and colorectal carcinoma, lymph node metastatic lesions, remote metastatic lesions was 0.0%(0/4), 40.0%(2/5), and 50.0%(3/6), respectively.Conclusion:The colorectal adenoma, metastatic lymph nodes and remote metastatic lesions were not suited for K-ras analysis as routine samples in clinical practice. If the samples of primary lesions were not available, the detection results of metastatic lymph nodes and remote metastatic remote lesions will provide some reference values. K-ras gene had several different mutations in the progression of CRC.

Objective: To explore the significance of Th17 in hepatocellular carcinoma, expecially with HBV infection.Methods:Cytometric bead array(CBA) was employed to detect 5 cytokines(IL-2,IL-4,IL-6,IFN-γ,IL-17A)from 39 tumor and non-tumor tissues of HCC and combined clinical data for comparative statistic analysis.Results:The expression of IL-2,IL-4,IFN-γin liver cancer tissue[(4.61±0.28),(3.37±0.58),(3.08±1.08)pg/ml,respectively] was significant lower than non-cancer tissue [(5.57±0.59),(3.77±0.70),(3.69±1.20)pg/ml,respectively].Otherwise,the expression of IL-6,IL-17A in cancer tissue [(280.09±254.68), (2.66±1.66) pg/ml, respectively] was higher than non-cancer [(6.58 ±1.92), (1.49 ±0.98) pg/ml, respectively].And,whatever cancer or non-cancer tissue,the expression of IL-17A in tissue[(3.45±1.86)pg/ml] with high HBV load (>1 000 U/ml) was significant higher than tissue with low HBV load[(1.97±1.16)pg/ml].Conclusion: IL-17A was highly expressed in HCC,and IL-2,IL-4,IFN-γmay inhibit its expression,and IL-6 may promote it.Hepatitis B virus infection may promote Th17 expression,thereby reducing patient′s prognosis.

Objective To observe the expression of cytotoxicity and receptors on NK cells in breast cancer,and investigate the impact of soluble MICA(sohble MHC class Ⅰ-related molecules A,sMICA) on NK cells receptors expression and cytotoxicity.Methods ELISA was used to examine the sMICA in peripheral blood.The expressions of activated receptor(NKG2D),killer inhibitory receptor (KIR)(CD158b) and NK cells were identified by flow cytometry(FCM).Cytotoxicity of NK cells to breast cancer were tested by MTT.Results sMICA was (205.36±71.27)ng/L in breast cancer patients and 81.6 % samples were detected.There were positive correlations between sMICA levels with breast cancer stages.There were no difference of NK cells percentage between breast cancer and healthy person.The cytotoxicity of NK cells and expressions of NKG2D were obviously lower in breast cancer with sMICA(+) than in healthy person,but CD158b was higher in healthy person.After cultured with sMICA,NK cells cytotoxicity decreased from(76.2±6.7)% to(48.4±4.1)% and the expression of NKG2D reduced from(92.5±7.1)% to (62.5±6.4)%,but the the expression of CD158b increased from(10.6±3.2)% to (43.6±3.4)%.IL-15 up regulated the expression of NKG2D and NK cells cytotoxicity,but decreased the expression of CD158b by co-culturation with IL-15 and sMICA in sMICA+ patients with breast cancer.Conclusion sMICA reduced the expression of NKG2D and increased the expression of Kill,which lead to the down regulation of NK cells cytotoxicity.IL-15 can reverse this effect.

Objective To assess the adhesive behavior of dual-targeted microbubbles carrying both Sialyl Lewisx and anti-ICAM-1 monoclonal antibodies in vitro. Methods Selectin-targeted (with Sialyl Lewisx) microbubbles (MB-S),ICAM-1-targeted (with anti-ICAM-1 monoclonal antibodies) microbubbles (MB-Ⅰ),and dual-targeted (with both ligands) microbubbles(MB-D) were prepared by attaching the ligands to the biotinylated lipid-microbubbles via multi-step avidin biotin bridging chemistry. A parallel plate flow chamber combined with a novel automated tracking algorithm,were used to analyze the transient velocities,rolling and firmly adherent numbers of microbubbles at various shear stress (0. 6,2.0 and 4.0 dyn/cm2)over 6 min. Microbubbles detachments were tested by ramping up the shear stress at 30 s intervals. Results At 0.6 dyn/cm2 shear stress, the rolling numbers of MB-S and MB-D were remarkably more than that of MB-I( P＜0.05), while at 2.0 and 4.0 dyn/cm2 MB-S performed higher rolling efficiency as compared with either MB-I and MB-D ( P＜ 0.05). In all flow conditions, the adhesive numbers of MB-D to the targets were obviously greater than those of MB-S and MB-I ( P＜ 0.05). Half-maximal detachment decreased gradually in MB-I, MB-D and MB-S by turns ( P＜ 0.05). Conclusions MB-I, MB-S and MB-D have different adhesive behaviors. MB-I exhibites primarily firm adhesion with low rolling efficiency, while MB-S reveales unstable or transient adhesion with high rolling efficiency,and MB-D exhibites firm adhesion with high rolling efficiency. MB-D may be suitable for molecular imaging in high-flow vessels.

Objective:To explore the role of NKG2D ligand MHC-I related molecule A (MICA) in chemotherapy combined with NK cell immunotherapy in patients with advanced esophageal cancer after surgery. Methods:A total of 90 patients with esophageal cancer from Fujian Provincial Tumor Hospital were divided into three groups after surgery:40 patients of chemotherapy alone, 25 patients of chemotherapy combined with NK cell therapy with negative expression of MICA (MICA-group), and 25 patients of chemotherapy combined with NK cells therapy with positive expression of MICA (MICA+group). The efficacy was then compared. Results:Compared with the chemotherapy alone and MICA-groups, the positive rates of CD3+, CD4+T cells, NK cells, and the CD4+/CD8+ratio in peripheral blood from MICA+group were higher than those before treatment (64.2%± 6.4%vs. 51.3%± 5.6%, 39.8%± 8.2%vs. 29.5%± 3.2%, 25.3%± 2.1%vs. 16.4%±4.3%, 1.4%± 0.5%vs. 1.1%± 0.7%;P<0.05). Meanwhile, the levels of T-reg cells were lower than those before treatment (6.3%± 4.5%vs. 17.3%± 2.4%, P<0.05). No significant difference was observed between the disease control rate and response rate. Chemotherapy-induced neutropenia and peripheral neurotoxicity symptoms were significantly improved, and time to progression (TTP) and overall survival (OS) were significantly prolonged (P<0.05). No statistically significant difference was observed between the chemotherapy alone group and MICA-group (P>0.05). Conclusion:Treatment with chemotherapy and autologous NK cells on patients with advanced esophageal carcinoma and MICA positive expression can be safely transfused with only minor side effects and can effectively improve a patient's immune system, quality of life, and survival.

Objective To induce the immune tolerance of heart grafts with infusion of isogeneic bone marrow mesenchymal stem cells (BMSCs) in heart transplant rats.Method Donor Wistar rats and recipient F344 rats were randomly divided into 4 groups:acute rejection group (group A),Wistar rats as the donors and F344 rats as the recipients for heart transplantation; low dose cyclosporin A(CsA) group (group B),recipient F344 rats given low dose CsA; BMSCs group (group C),recipient F344 rats given isogeneic BMSCs; BMSC and low dose CsA group (group D),the recipient F344 rats given isogeneic BMSCs and low dose CsA.The serum cytokine levels were determined,and the donor heart pathological changes and survival were observed postoperatively.The relative level of Foxp3 mRNA expression in the spleen of the recipient F344 rats was also observed.Result The blood levels of interleukin-2 (IL-2) and interferon-γ(INF-γ) were significantly reduced,but IL-4 and IL-10 levels were increased (P＜0.05),and the survival time of donor heart was significantly prolonged in group D as compared with groups A,B and C (P＜0.05 for all).Heart pathological examination revealed a mild acute rejection in group D,moderate acute rejection in groups B and C group,and severe acute rejection in group A respectively.The expression of Foxp3 mRNA was significantly lower in group A than in groups B,C and D (P＜0.05 for all),and that in group D was significantly higher than in groups B and C (P＜0.05 for both),but there was no significant difference between between groups B and C (P＞0.05).Conclusion Intravenous administration of BMSCs can alleviate immunorejection in heterotopic rat heart transplantation.Low-dose CsA acts synergistically with BMSCs to significantly inhibit acute rejection after heart transplantation.The partial mechanisms involve the suppressive effect of BMSCs on the expression of Foxp3 mRNA and modulation on cytokine.

Objective To explore the relevance of expression of MHC class Ⅰ-related molecules A (MICA) molecule and NK cells immunotherapy in esophageal cancer patients after operation.To analyze the significance of MICA expression in NK cell immunotherapy.Methods 100 patients of esophageal cancer were divided into 3 group,surgical alone group,MICA negative with NK therapy group (MICA-group) and MICA positive with NK therapy group (MICA+ group).The immunity indicators and tumor markers including the levels of CD3+,CD4+ T cells,ratio of CD4+/CD8+, NK cells,Treg cells,the levels of Th1/Th2/Th17 cytokine,the antibody IgA,IgM,IgG and the tumor markers of CEA,SCC,CA199,CYFRA21-1 were detected before treatment and after treatment 60 days.Results The positives rates of CD3+,CD4+ T cells,NK cells and the ratio of CD4+/CD8+ in peripheral blood from MICA+ patients group were higher than those of before treatment [(68.3±7.6) ％ vs (56.2±4.1) ％,(39.8±8.2) ％ vs (30.8±4.7) ％,(22.2±4.7) ％ vs (18.7±5.5) ％,(1.49±0.30) vs (1.15±0.61),P ＜ 0.05],meanwhile the levels of Treg cells was lower than those of before treatment [(8.1± 4.0) ％ vs (13.4±4.5) ％,P ＜ 0.05].There was no statistical significant difference of positive rate of CD8+ T cells [(26.9±6.2) ％ vs (27.8±7.1) ％,P ＞ 0.05].The levels of Th1 cytokin (IL-2,IFN-γand TNF-α) increased and Th2 cytokin (IL-4,IL-6 and IL-10) decreased after treatment.The level of Th17 cytokine was not different significantly (P ＞ 0.05).The content of IgA,IgM,IgG in MICA+ group were effectively improved after treatment.The tumor markers CEA,SCC,CA199,CYFRA21-1 had no statistically change before and after treatment.Conclusion The results indicate that NK cells immunotherapy can enhance the cellular immunity and humoral immunity of MICA positive esophageal cancer patients after operation.