AIM: To investigate the produce of intracellular cytokine following short-term in vitro stimulation with vMIP and LPS, and discuss the effect of vMIP to cellular immunity. METHODS: The methods of Cross-linking of radioactivity, ELISA and four-colors flow cytometer were used to test the level of the secretion of chemokine IL-12 and intracellular cytokine IFN-? and IL-4. RESULTS: After treated the PBMCs with vMIP-II, the levels of secretion of IL-12, IFN-? and IL-4 were reduced in the present of LPS by competitively combining chemokine receptor; vMIP promoted CD4+T cell to secrete IL-12, IFN-? and IL-4. CONCLUSION: vMIP-II can protect systemic response of immunity and reduce extremely inflammation by down-regulating proinflammation.

Objective:To establish an efficient method for expression and purification of vMIP-Ⅱ which encoded by a viral gene and ho-mogued human chemokine in active single strand in E.coli cells. Methods: Cloning with bi-enzyme restriction and lysising bacteria with cold osmoic shock were used for expression,MBP affinity chromatography of fusion protein and self-selective of recombinative peptide for final purification. The expressing and purifying products were detected with SDS-PAGE and Western blotting and identified with inhibitory adhesion experiment for its activity.Results: Fusion protein MBP-vMIP was effeciendy expressed in E. coli at secretive type, and self-cleaved to sparate the vMIP-Ⅱ. The final product single strand vMIP-Ⅱ was active for blocking chemokine receptor CCR5.Conclusion:This is an effective method for obtaining viral chemokine vMIP-Ⅱ of recombinant single strand.The recombinant vMIP-Ⅱ may be useful for the study of diseases involving in chemokiine receptors such as HIV infection,rejection of transplantation and chronic inflammation,etc.

Objective: IFN-y is produced by both activated T and NK cells in response to mitogen or antigen and has a broad range of immunoregulatory activity. IL-12 has been described as a strong inducer of IFN-?production and promotes the differentiation of naive CD4+T cells toward the Th1 phenotype, priming them for IFN-?production, and consequent induction of cell-mediated immunity. Aim is to know endogenous production of IL,12 from PBMC inducing production of IFN-?in vitro, which is involving in mechanism of T cells to be activated. Methods: Induced IFT-? secretion from human PBMC by stimulated with anti-CD3 , PHA, anti-CD3 plus anti-CD28 and antigen(MLC) Also inhibited IFN-?production by neutralizing antibodies to IL-12 and IL-12R?1 significantly. Results: IFN-?secretion from human activated PBMC is endogenous IL-12dependent, and activated T cells induce the production of IL-12 from APC by a mechanism involving the interaction between CD40L on T cells and CD40 on APC. Conclusion: These results suggest that endogenous IL-12 plays an important role in the normal host defense against infection by a variety of intracellular pathogens and also plays a central role in the genesis of some forms of immunopathology including autoimmune diseases and transplantation rejections.

AIM: To demonstrate the susceptibility of cell apoptosis varies during the progress of cell malig- nant transformation from human being in vitro. METHODS: A SV40T - transfected human bronchial epithelial im- mortalized cell line (called M) was selected in this work, which has acquired some characteristics of malignant trans- formation at the later passage. The alterations of apoptosis and bcl- 2, P53 genes between early and later passage of M cells were investigated by means of TDT labeling in situ, chromosome FISH, RNA and protein testing, etc. RE- SULTS: Incidence of apoptosis induced by cis - platin was significantly lower in later than in early passages of M. Levels of bcl - 2 mRNA and protein in later passages were higher than early passages of M, and overxpression of bcl -2 was accumulated following the development of cellular malignancy. P53 protein level was as high in early as in later passages. CONCLUSION: Overexpression of bcl - 2 decreases the cellular sensitivity to apoptotic inductors plays an important role during progress of carcinogenesis in human bronchial epithelial cancers. The inactivation of P53 protein in the SV40 - T transfected M cell line may be one of reasons of bcl - 2 overexpression, but not associated with the accumulation of bcl - 2 expressed level during cell transformation.

Death with dignity is now not legislation in our country .This paper mainly discussed about some barriers to the legalization of death with dignity in China , from the viewpoint of Chinese traditional ideas , the lack of death education , risk of abusing , the subject change of the informed consent right , doctor-patient communica-tion and trust lsot and so on .It is proposed that our country should perfect the medical security system , strengthen the education of death at the same time and help the citizen set up the view of science .Outside, still need to fur-ther deepen the reform of medical system in our country , the maintaining patient ’ s autonomy and right of choosing , protect the informed consent right of patients .Create the doctor-patient relationship of mutual trust .

Objective To investigate the phenotype and the immunoregulatory function of CD8αα+TCRαβ+regulatory T cells in peripheral blood samples from mice.Methods The distribution profile and the phenotype of CD8αα+TCRαβ+regulatory T cells in C57BL/6 mice were detected by flow cytometry.The cytokines released by CD8αα+TCRαβ+regulatory T cells upon the stimulation with anti-CD3 antibody were analyzed by cytometric bead array.The in vitro immunosuppressive activity of CD8αα+TCRαβ+regulatory T cells on activated CD4+T cells was analyzed by using flow cytometry and carboxyfluorescein succinimidyl ester ( CFSE ) .An adoptive cell transfer assay was set up to evaluate the immunoprotective effects of CD8αα+TCRαβ+ regulatory T cells in a mouse model of experimental autoimmune encephalomyelitis ( EAE) .Results CD8αα+TCRαβ+regulatory T cells were detected in liver, spleen and peripheral blood samples collected from na?ve C57BL/6 mice.Compared with CD8αβ+TCRαβ+regulatory T cells, CD8αα+TCRαβ+regulatory T cells showed a memory-activated phenotype of CD25+CD122high CD44high CD62Llow CD69high NK1.1+DX5+.CD8αα+TCRαβ+regulatory T cells could produce IL-2 after 24 hours stimulation with anti-CD3 antibody, followed by producing IFN-γ, TNF-α, IL-4, IL-17A and traces of IL-6 and IL-10. In vitro, CD8αα+TCRαβ+regulatory T cells specifically suppressed the proliferation of activated CD4+T cells ( P<0.01 ).Moreover, they could delay the onset of EAE in mice and reduce clinical score (P<0.01).Conclusion CD8αα+TCRαβ+regulatory T cells were a unique population with immunoregula-tory function, which could be used as a potential therapeutic target in the treatment of autoimmune disease.

Objective To investigate the effects of the genetic polymorphisms in osteoporosis-related genes and the gene-gene interaction on bone mineral density (BMD) and osteoporotic fractures.Methods Thirty-nine single nucleotide polymorphism (SNP) sites in 23 genes that related to bone mineral density ( BMD ) and osteoporotic fractures were scanned in 683 Shanghai Han postmenopausal women.TaqMan SNP Genotyping Assay or Sequenom Mass ARRAY System were applied for genotyping analysis.The relation of these SNP sites with BMD and osteoporotic fractures were analyzed.Results Altogether,12 SNPs in 9 candidate genes ( rs7524102 and rs6696981 in ZBTB40 gene,rs9479055 in ESR1 gene,rs6993813,rs6469804,and rs11995824 in OPG gene,rs3736228 in LRP5 gene,rs1107748 in SOST gene,rs87938 in CTNNB1 gene,rs1366594 in MEF2C gene,rs7117858 in SOX6 gene,and rs10048146 in FOXL1 gene) were associated with BMD at lumbar spine(L1-L4) or total hip.In addition,rs11898505 in SPTBN1 gene was related to osteoporotic fractures ( OR 0.522,95％ CI 0.326-0.838,P =0.007 ).Gene-gene interaction involving rs1038304 in ESR1 gene,rs1366594 in MEF2C gene,and rs10048146 in FOXL1 gene was associated with osteoporotic fractures ( P =0.010 7 ).Conclusions ( 1 ) SNPs in gene ZBTB40,ESR1,OPG,LRP5,SOST,CTNNB1,MEF2C,SOX6,FOXL1,and SPTBN1 are associated with BMD of lumbar spine or total hip,as well as osteoporotic fractures.(2) Gene-gene interaction involving rs1038304,rs1366594,and rs10048146may contribute to the risk of osteoporotic fractures.

Flow Cytometry acting as a popular technology based on single cell with high throughput and high content in all-round life science fields besides clinical diagnosis , has been launched for more than 52 years, With the advancement of fluidic path , photic path ( including novel labeling dye ) together with signal collection , transformation and algorithm et al .Particularly , traditional flow cytometry combined with mass spectrometry, microfluidic technology, image principle,Raman spectrometry and so on, resulted in flow cytometry being applied in hematology , immunology, oncology with an enhanced performance .Meanwhile, the improvement of standardization and quality control will boost the flow cytometry in clinical application .

Objective: To investigate the expression and molecular mechanism of dickkopf 1 ( DKK1 ) in tongue squamous cell carcinoma (TSCC) by bioinformatics method and molecular biology experiments． Methods: The patients information wasdownload from TCGA-TSCC database，the differentially expressed genes between the cancer and normal tissues were screened by NetworkAnalysed site，the key genes and clinical prognosis were identified through Kaplan-Meier analysis and Lasson regression，the functions and pathways of differentially expressed genes were gained by GO and KEGG database，the expression of DKK1 mRNA and protein in TSCC as well as its relationship with clinicopathological features were analyzed by UALCAN database and immunohistochemistry．Western blot assay was conducted to detect the protein expression of DKK1 in TSCC cells，and siRNA was used to konck down the expression of DKK1 protein in Cal27 cells. Results : The three key genes DKK1，CYP19A1 and IRX4， which were highly expressed in tongue squamous cell carcinoma and the survival rate of TSCC patients with high expression group was poor，were screened through NetworkAnalysed ，Kaplan-Meier analysis and Lasson regression method．UALCAN database showed that the mRNA level of DKK1 in TSCC tissues was higher than that in normal tissues，and its high expression was significantly correlated with clinical stage，histological grade and lymph node metastasis of TSCC patients．The immunohistochemistry assay suggested that the positive rate of DKK1 protein in clinical stage Ⅲ + Ⅳ TSCC tissues was significantly higher than that in stage Ⅰ + Ⅱ TSCC tissues．In addition， the expression level of DKK1 protein in TSCC tissues was significantly higher than that in adjacent tissues．Western blot assay also showed that the protein expression of DKK1 in TSCC cell Cal27 was much higher than normal oral epithelial cell HOEC．When knock down the protein expression of DKK1 in Cal27，the expression of β-catenin、p- p65 和 p65 werealso reduced． Conclusion DKK1 is highly expressed in tongue squamous cell carcinoma tissues and cells and plays an important role．It may be a new target for early diagnosis and drug treatment of TSCC.