Retroviral vector pLXSN was employed to introduce human r-Interferon (IFN-?) gene into four human hepa-tocellular carcinoma cell lines (HCC). The G418-resistant colonies were isolated and cloned. PCR and RT-PCR analysis indicated that the integration and expression of IFN-? gene was shown only in the transduced cells. Using a bioassay method, we found that all genetically modified HCC cells can secrete varied amount of IFN-?. The results of flow cytome-try showed that the cell surface expression of HLA class I molecules significandy increased following transduction. Moreover , we firsdy indicated that the increase in the expression of one specific HLA class I antigen, HLA-A2, was almost in the same magnitude as that of the total HLA class I molecules after transduction with IFN-r gene.

Objective To investigate the effects of leptin on Th17 cells and the possible mechanism. Methods The leptin-deficient ( ob/ob) mice and their homologous wild-type mice were used in the study.The percentages of Th17 cells in peripheral blood samples, spleen tissues and lymph nodes were measured by flow cytometry ( FCM) analysis.The splenic CD4+T cells, separated from the ob/ob mice and the wild-type mice by using magnetic beads,were respectively cultured with leptin at various concentrations and with anti-leptin neu-tralization antibody to evaluate the effects of leptin on Th17 cells.The quantitative real-time PCR was performed to analyze Th17 cell-related cytokines at transcriptional levels.The levels of IL-6 and IL-17A in the supernatants of CD4+T cell culture were measured with Luminex technology.Results Compared with the wild-type mice, the ob/ob mice showed lower percentages of Th17 cells in both peripheral blood samples and spleen tissues (0.49%±0.03%vs 1.29%±0.1%, 1.56%±0.22%vs 2.47%±0.11%).There was a decrease in the percentages of Th17 cells upon the in vitro treatment of CD4+T cells from wild-type mice with anti-leptin antibody.The per-centages of Th17 cells were increased in a dose-dependent manner upon the in vitro treatment of CD4+T cells from ob/ob mice with leptin.Moreover, the levels of IL-17A and IL-6 and the transcriptional levels of RORγt, IL-17A and IL-6 in leptin deficiency group were lower than those of wild-type group, but were increased upon the treatment with leptin.No significant difference with the transcriptional levels of TGF-βand IL-23 was ob-served between the two groups with and without intervention.Conclusion Leptin deficiency seriously hampered the generation of Th17 cells in mice and resulted in a decreased expression of RORγt, IL-17A and IL-6 at mRNA level.The treatment of CD4 T cells with leptin might promote the generation of Th17 cells through up-regulating the transcription of RORγt and IL-6.

Objective To explore the value of spectral imaging technique in dual-energy CT in differential diagnosis of the metastatic lymph nodes in thyroid carcinoma,squamous cell carcinoma metastatic lymph nodes and lymphoma in the neck.Methods In 30 patients with pathologically confirmed with a total of 79 cervical lymph nodes enlargement which were using dual energy scan .Then observed the change trend of the spectrum curve and comparison the three kinds of lymph node energy spectrum curve’s slope.Results In the 79 lymph nodes,the metastatic lymph nodes in thyroid carcinoma were twenty-three,squamous cell carcinoma metastatic lymph nodes were twenty-four and lymphoma were thirty-two.From 60 to 180 keV,with the increase of keV values,the three kinds of malignant lymph nodes of the corresponding CT value decreasing and the higher the keV value,the CT value decrease magnitude was small,and the spectrum curve was〞drop type〞.The slope spectrum curve of the metastatic lymph nodes of thyroid carcinoma in arterial phase and parenchymal phase were maximum,which were 1.23±0.41 and 0.85±0.33,respectively.The slope spectrum curve of lymphoma in arterial phase and parenchymal phase were least,whcih were 0.40±0.16 and 0.47 ±0.09.The slope spectrum curve of the squamous cell carcinoma metastatic lymph nodes in arterial phase and parenchymal phase were 0.88±0.10 and 0.62±0.28.The energy spectrum curve slope of the three kinds of malignant lymph nodes have statistical significance.Conclusion The energy spectrum curve slope of arterial phase and parenchymal phase has some significance lymph node metastasis of in thyroid carcinoma,the metastatic lymph nodes and lymphoma in the neck.

Objective To investigate the expression of exogenous γ-interferon gene in human hepatocellular carcinoma cells following retroviral transduction and the effect on the expression of surface HLA class Ⅰ molecules.Methods Retroviral vector pLXSN was used to introduce human γ-interferon (IFN-γ) gene into four different human hepatocellular carcinoma cell lines (HCC). The G418-resistant colonies were isolated and cloned. The integration and expression of IFN-γ gene were determined by PCR and RT-PCR analysis. A bioassay method was used to test the amount of IFN-γ secreted by gene modified HCC cells. The expression of HLA class Ⅰ molecules in HCC cells were analyzed by flow cytometry using indirect fluorescence staining.Results Four different HCC cell lines were successfully transduced with human IFN-γ gene using retroviral vector. The integration and expression of IFN-γ gene were shown only in the transduced cells. All four genetically modified HCC cells can secrete varied amount of IFN-γ and demonstrate a significant up-regulation of surface HLA class Ⅰ antigens. One specific HLA class Ⅰ antigen, HLA-A2, has almost the same degree of increase as that of the total HLA class Ⅰ molecules after transduction with IFN-γ gene.Conclusions Gene modification with IFN-γ gene can significantly enhance the expression of HLA class Ⅰ molecules in HCC cells and may increase its immunogenicity. These gene modified tumor vaccines can be helpful in tumor biotherapy.

Cone beam computer tomography (CBCT) has advantages of high precision, low radiation and high image quality. It has been developing quickly since it was applied clinically. In order to control X-ray TUBE HEAD effectively in Dental CBCT, X-ray TUBE HEAD Control System was designed and realized in this study. This control system is the core of CBTC system, which includes the communication between CBCT system and computer, the control of X-ray tube head by CBCT system main control board and the synchronization between main control board and the flat panel detector. Control circuit of the control system and corresponding operating software were designed with PIC16F877A as the core. This control system has been put into use in current CBCT system successfully.
Algorithms ; Cone-Beam Computed Tomography ; instrumentation ; Equipment Design ; Software

Objective To assess the curative effect of percutem transilluminated with negative pressured on the potaried technique on the treatment of venous ulcer in lower extremity.Methods The clinical date of 300 cases involving 300 legs with venous ulcer in lower extremity,who underwent the percutum transilluminated negative pressured potaried technique using TRIVEXTM Ⅱ potaried system or the percutum transfixion surgical treatment from October 2005 to June 2009,were analyzed.Three hundred cases were randomly divided into potaried group and transfixion group.In potaried group,there were 190 cases involving 190 legs treated with TRIVEXTM Ⅱ potaried system.In transfixion group,110 cases involving 110 legs treated with percutum transfixion.The clinical indexes of skin infection rate and skin necrosis rate,shrinkage rate of wound area and skin depigmentation rate,ulcer healing rate and ulcer recurrence rate were calculated to assess the clinical curative effect on day 5,day 20,day 120 and day 360 after operation respectively.Results The rates of skin infection and skin necrosis were significantly decreased in potaried group compared with transfixion group on day 5 after operation (P0.05).Ulcer recurrence rate was remarkably lower in potaried group than that in transfixion group on day 360 (P

OBJECTIVETo detect the expression levels of co-inhibitory molecules, including CTLA-4, LAG-3, PD-1 and CD39, on CD4⁺ T cells in peripheral blood or tumor tissues from NSCLC patients and to investigate their potential internal relationships with the progression of NSCLC.

METHODSEighty-eight patients including 53 NSCLC, 17 disease control cases and 18 healthy controls were studied. All the peripheral blood and 13 cases of tumor and tumor-adjacent tissues from surgically treated NSCLC patients were obtained. The expression levels of co-inhibitory molecules CTLA-4, LAG-3, PD-1 and CD39 were assayed by flow cytometry (FCM).

RESULTSThe ratios of CD4⁺ CTLA-4⁺ T cells, CD4⁺ LAG-3⁺ T cells, CD4⁺ PD-1⁺ T cells and CD4⁺ CD39⁺ T cells in the peripheral blood of NSCLC patients were (2.49 ± 2.43)%, (2.47 ± 3.50)%, (12.94 ± 5.96)% and (6.78 ± 5.21)%, respectively, the ratio of CD4⁺ CTLA-4⁺ T cells was significantly higher in the peripheral blood of NSCLC patients than that in the disease controls and healthy controls (P < 0.05) . The ratio of CD4(+)PD-1⁺ T cells was also highly raised in the peripheral blood of NSCLC patients than that in the healthy controls (P < 0.05). Further stratified analysis indicated that the ratio of CD4⁺ PD-1⁺ T cells was (13.21 ± 5.96)% in NSCLC patients entering stages III and IV, also significantly increased as compared with that of (11.06 ± 3.42)% in the patients undergoing stages I and II (P < 0.05). More CD4⁺ CTLA-4⁺ T cells, CD4⁺ LAG-3⁺ T cells and CD4⁺ PD-1⁺ T cells were verified in the cancer tissues (5.07 ± 2.11)%, (7.86 ± 3.24)% and (40.20 ± 18.84)%, respectively, than those in their matched peripheral blood (3.13 ± 1.01)%, (2.65 ± 1.48)% and (15.79 ± 5.69)%, (P < 0.05 for all), and especially, CD4⁺ CTLA-4⁺ T cells and CD4⁺ PD-1⁺ T cells were also highly increased than those in matched cancer-adjacent tissues (P < 0.05 for all).

CONCLUSIONSThe increased expression levels of co-inhibitory molecules CTLA-4, LAG-3 and PD-1 on CD4⁺ T cells in peripheral blood and tumor tissues may be one of the mechanisms related to immune escape of tumor cells, acceleration of disease progression and poor prognosis in NSCLC patients.

Antigens, CD ; metabolism ; Apyrase ; metabolism ; CD4-Positive T-Lymphocytes ; metabolism ; CTLA-4 Antigen ; metabolism ; Carcinoma, Non-Small-Cell Lung ; diagnosis ; metabolism ; Disease Progression ; Flow Cytometry ; Humans ; Lung Neoplasms ; diagnosis ; metabolism ; Programmed Cell Death 1 Receptor ; metabolism

Objective To investigate the effects of leptin on Treg cells and the possible mecha-nism. Methods Leptin-deficient ( ob/ob) mice and homologous wild-type mice were used in this study. The percentages of Treg cells in spleen tissues and peripheral blood samples were measured by flow cytometry ( FCM) . Differences in Treg cell functionality were compared between the two groups. Splenic CD4+T cells, separated from the ob/ob mice and the wild-type mice by magnetic beads, were respectively cultured with leptin and anti-leptin neutralization antibody to evaluate the effects of leptin on Treg cells. Quantitative real-time PCR was performed to analyze the expression of Treg cell-related cytokines at transcriptional level. The levels of IL-10 and TGF-β in the supernatants of CD4+T cell culture were measured with Luminex technolo-gy. Results Compared with the wild-type mice, the ob/ob mice showed higher percentages of Treg cells in both peripheral blood samples and spleen tissues [(11. 56 ± 0. 72)％ vs (5. 47 ± 0. 81)％, (10. 16 ± 0.93)％ vs (6.29±0. 69)％]. Treg cells isolated from the ob/ob mice had stronger immunosuppressive effects on the proliferation of effector T ( Teff) cells and the secretion of TNF-α and IFN-γ than those from the wild-type mice [TNF-α:(1. 6±0. 2)％ vs (2. 4±0. 5)％, IFN-γ:(4. 3±0. 3)％ vs (7. 2±1. 2)％]. The percentages of Treg cells were decreased from (12. 2±1. 8)％ to (7. 6±0. 9)％ upon the in vitro treat-ment of CD4+ T cells from the ob/ob mice with leptin and the immunosuppressive effects of Treg cells were also weakened. However, the percentages of Treg cells were increased from (7. 8±0. 85)％ to (13. 1± 1. 5)％ upon the in vitro treatment of CD4+T cells from the wild-type mice with anti-leptin antibody and the immunosuppressive effects of Treg cells were improved as well. Moreover, the expression of Foxp3, IL-10 and TGF-β at transcriptional level and the levels of IL-10 and TGF-β in the ob/ob group were higher than those in the wild-type group. Conclusions Leptin deficiency significantly promoted the generation of Treg cells in mice and resulted in an increased expression of Foxp3, IL-10 and TGF-βat mRNA level and elevat-ed levels of IL-10 and TGF-β. The treatment of CD4+T cells with leptin might inhibit the generation of Treg cells through down-regulating the transcription of Foxp3, IL-10 and TGF-β.

Epidemiological and animal studies indicate that pre-existing diabetes increases the risk of Parkinson's disease(PD).However,the mechanisms underlying this association remain unclear.In the present study,we found that high glucose(HG)levels in the cerebrospinal fluid(CSF)of diabetic rats might enhance the effect of a subthreshold dose of the neurotoxin 6-hydroxydopamine(6-OHDA)on the development of motor disorders,and the damage to the nigrostriatal dopaminergic neuronal pathway.In vitro,HG promoted the 6-OHDA-induced apoptosis in PC12 cells differentiated to neurons with nerve growth factor(NGF)(NGF-PC12).Metabolomics showed that HG promoted hyperglycolysis in neurons and impaired tricarboxylic acid cycle(TCA cycle)activity,which was closely related to abnormal mito-chondrial fusion,thus resulting in mitochondrial loss.Interestingly,HG-induced upregulation of pyruvate kinase M2(PKM2)combined with 6-OHDA exposure not only mediated glycolysis but also promoted abnormal mitochondrial fusion by upregulating the expression of MFN2 in NGF-PC12 cells.In addition,we found that PKM2 knockdown rescued the abnormal mitochondrial fusion and cell apoptosis induced by HG+6-OHDA.Furthermore,we found that shikonin(SK),an inhibitor of PKM2,restored the mito-chondrial number,promoted TCA cycle activity,reversed hyperglycolysis,enhanced the tolerance of cultured neurons to 6-OHDA,and reduced the risk of PD in diabetic rats.Overall,our results indicate that diabetes promotes hyperglycolysis and abnormal mitochondrial fusion in neurons through the upre-gulation of PKM2,leading to an increase in the vulnerability of dopaminergic neurons to 6-OHDA.Thus,the inhibition of PKM2 and restoration of mitochondrial metabolic homeostasis/pathways may prevent the occurrence and development of diabetic PD.

Leptin system plays an important role in lung inlfammation and tumorigenesis, but the precise function in the tumormicrovironment and the prognosis value of the leptin system in lung cancer have not been fully elucidated. hTis re-view will focus on the relationship between leptin system and non-small cell lung cancer (NSCLC), in which special attentions will be paid on the expression of leption and its receptor (LepR) in peripheral blood and tumor tissues, leptin-related signal transduction pathways, the interaction between leption and regulatory T cells (Treg) and the gene polymorphisms of leptin and leptin receptor, and possibly provide new strategies for diagnosis and treatment of NSCLC.