Effect of different cult ure conditions on differentiation of Treg and Th17 in PB-MC / 中国免疫学杂志

Objectiv e:To investigate the effect of different culture conditions on the differentiation of Treg and Th17 to lay a foundation for exploring the methods to reverse the immune tolerance induced by tumor microenvironment.Methods:The IL-6 gene was cloned and stablely transferred into the tumor cell line expressing TGF-β.The conditioned mediums ( CM) were prepared by collecting the culture supernatants of tumor cell lines with or without IL-6 expression and used in the in vitro culture of peripheral blood mononuclear cells ( PBMC ) .The changes of Treg and Th17 in PBMC treated with different CM were detected with flow cytometry ( FCM) .Results:The expression of TGF-βin BEL-7402 was higher than that in HepG2.Thus the BEL-7402 was selected for preparation of cell line stablely transfected with IL -6 gene.ELISA detection confirmed the effective expression of IL -6 by the identified cell lines.It was showed that the Treg increased in PBMC treated with culture supernatants of tumor cells .However,the presence of IL-6 reversed the increase of Treg and promoted the differentiation of Th 17.Conclusion: The culture supernatants of tumor cells increases the proportion of Treg.However,the presence of IL-6 in this CM can reverse the increase of Treg and raise the proportion of Th 17.

Progress in infection pathway and intracellular trafficking of adenovirus / 生物工程学报

The research on intracellular trafficking of adenovirus has been described mainly through observations of subgroup C adenoviruses in transformed cell lines. The basic elements of the trafficking pathway include binding to receptors at the cell surface, internalization by endocytosis, lysis of the endosomal membrane, escape to the cytosol, intracellular trafficking along microtubules, nuclear pore docking, and viral genome translocation into the nucleus. More than 80% of the adenovirus genome is delivered to the nucleus in a highly efficient manner in approximately 1 h. However, exceptions to this trafficking pattern have been noted, including: variations based on target cell type, cell physiology, and adenovirus serotype. This review summarizes mechanism of adenovirus infection pathway and intracellular trafficking, providinging a foundation for the development of clinical adenoviral vector.

Human umbilical cord-derived mesenchymal stem cells inhibit proliferation but maintain survival of Jurkat leukemia cells in vitro by activating Notch signaling / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on the proliferation and survival of Jurkat leukemia cells in vitro and explore the possible mechanism.</p><p><b>METHODS</b>Jurkat leukemia cells were co-cultured with hUC-MSCs isolated from human umbilical cord tissues by plastic adherence at a ratio of 10:1. The proliferation and survival of the co-cultured Jurkat cells, separated by immunomagnetic bead cell sorting on day 4, were evaluated by flow cytometry. Western blotting was performed to evaluate the activation of Notch signaling in the co-cultured Jurkat cells.</p><p><b>RESULTS</b>Jurkat leukemia cells co-cultured with hUC-MSCs for 4 days showed a lowered proliferation rate and cell cycle arrest at G0/G1 phase with a reduction in the cell apoptotic rate. Notch signaling pathway was activated in the co-cultured Jurkat cells as evidenced by an increased cellular expression of HES-1.</p><p><b>CONCLUSION</b>Co-culture with hUC-MSCs can inhibit the proliferation of Jurkat leukemia cells in vitro and protect the cells from apoptosis by activating Notch signaling, indicating a potential shielding effect of MSCs on leukemia cells.</p>

Influence of supernatant from human umbilical cord-derived mesenchymal stem cells on proportions of each human lymphoid subgroup / 中国免疫学杂志

Objective:To investigate the impact of human umbilical cord-derived mesenchymal stem cells on the activation ,the survival of human peripheral blood mononuclear cell ( hPBMC) and the proportions of each human lymphoid subgroup .Methods:PB-MC were isolated from healthy donors by density gradient centrifugation , then cultured in MSC-CM as treatment group after being acti-vated by OKT3.Each lymphoid subgroup proportion was analyzed by flow cytometry to observe the difference between treatment and control group .The effect of MSC-CM on activated PBMC for the production of IFN-γand IL-10 were tested by ELISA .The level of ap-optosis was assessed by flow cytometry with Annexin-V/PI as fluorescent marker .Results:Compared with the control group , MSC-CM down-regulated the ratio of CD4 +T cell to CD8 +T cell, and increased the proportion of CD4 +CD25 +CD127low Treg cell, thus other subgroup had no significant difference .MSC-CM inhibited the production of IFN-γby PBMC, but promoted the secretion of IL-10, and protected PBMCs from apoptosis when activated with OKT 3.Conclusion:hUC-MSC may play a role of immunosuppression by promo-ting the proliferation and activation of Treg cell .This kind of inhibitory activity is neither relied direct or indirect contact with the lym -phocytes , nor influenced by inducing immune cells apoptosis .

Specific TCRαβgene modification promotes anti-tumor reactivity of T cells / 中国免疫学杂志

Objective:To investigate effects of tumor specific TCR gene Vα12.2-Vβ7.1 modification on recognition of tumor antigen and activation of anti-tumor reactivity of T cells.Methods: T cells were transduced using recombinant Ad 5F35-TRAV-TRBV adenovirus ,and multiplicity of infection was optimized.Specific lysis of T cells was evaluated by calcein release assay.The frequency of apoptotic cells in target cells was detected by Annexin V /PI double-labeled FACS.The expression of FasL on T cells was analyzed by FACS.The secretion of cytokine IFN-γand IL-2 of T cells was determined by ELISA assays.Results: The highest tranduce efficiency was obtained at MOI 100 by recombinant Ad5F35-TRAV-TRBV adenovirus.The frequency of TCRVα12+Vβ7+cells reached above 25%3 days after transduction.TCR gene modification enhanced the ability of T cells to lyse HLA-A2+AFP+target cells(P<0.001), the ability of T cells to induce HepG-2 apoptosis(P<0.001),and expression of FasL on T cells(P<0.001).TCR gene modification also enhanced T cells to secret IFN-γafter coculture with antigen positive tumor cells ( P<0.001 ).Conclusion: Specific TCR gene modification by recombinant adeno virus effectively promotes T cells to recognize antigen positive tumor cell and exert anti -tumor reac-tivity.

Survivin-2B induces apoptosis of human breast cancer cells / 中国病理生理杂志

AIM:To explore the role of survivin-2B in the process of tumor cell apoptosis .METHODS:The survivin-2B gene was cloned into pcDNA3.1 vector and the recombinant plasmid pcDNA3.1-survivin-2B was obtained.Hu-man breast cancer MCF7 cells were transfected with pcDNA3.1 and pcDNA3.1-survivin-2B using Lipofectamine 2000.The cell cycle was determined by propidium iodide staining , and the apoptosis was detected by annexin V/7-AAD staining 48 h after transfection.Meanwhile, tatal RNA was extrated and multiplex polymerase chain reaction based on GenomeLab GeXP Genetic Analysis System was performed to detect the expression of 21 tumor-related genes .RESULTS: Flow cytometry analysis indicated that over-expression of survivin-2B promoted the apoptosis and cell cycle arrest of MCF 7 cells.Compared with control group , totally 10 differential expressed genes were related to the over-expressed survivin-2B, among which 2 were up-regulated and 8 were down-regulated. The expression of aldehyde dehydrogenase 4 family member A1 (ALDH4A1) was 48%down-regulated, and the expression of protein regulator of cytokinesis 1 (PRC1) was 1.08 folds up-regulated.CONCLUSION:Survivin-2B induces the expression changes of some tumor-related genes, which results in the apoptosis and G 2/M arrest of MCF7 cells.

Development of a novel assay for cytotoxicity test by Calcein-AM release / 中国免疫学杂志

Objective:To develop and optimize a novel assay for determination of cytotoxicity based Calcein -AM release.Methods:The target cells stained by Calcein-AM dye,then effectors and targets were incubated at E/T ratios from 30∶1-1∶1 for 4 h at 37℃,and the supernatant of reactions were detected by Fluorescence-Measurement to analyze specific cytotoxity.Results:The optimal excitation and emission wave lengths of Calcein were 485 nm and 515 nm.Dilutions of target cells stained by Calcein-AM had a linear relationship with measured fluorescence values.The Calcein-AM dye used to stain the living cells was shown to have a low spontaneous leakage rate-less than 15% in 4 hours at 37℃.Cytotoxicity activity of CIK showed a significant and positive correlation with E/T ratio when incubated at 4 h.Conclusion:The developed cytotoxicity test by Calcein-AM release is accurate and can avoid the application of radioactive reagents.

HLA-A2-restricted point-mutated Survivin peptide elicited specific cytotoxic T lymphocyte reaction against hepatocarcinoma / 中华微生物学和免疫学杂志

Objective To investigate the anti-hepatocarcinoma(anti-HCC) function of HLA-A2-restricted point-mutated Survivin peptide induced CTLs.Methods The HLA-A2-restricted Survivin nonapeptides were evaluated using bioinformatics software.The binding affinity of Survivin peptide to HLA-A2 molecular was determined with flow cytometry analysis.After peptide-induced CTLs were generated in vitro,flow cytometry and ELISA were performed to detect the levels of IFN-γ,which were secreted by reactive CTLs.Peptide-induced CTLs were co-cultured with hepatoma cell lines HepG2 and BEL-7402.The rates of tumor cells lysis were assayed using CytoTox 96(R) and the morphological changes of tumor cells were observed with inverted phase contrast microscope.Results Point-mutated Survivin nonapeptide Sur79M2 (KMSSGCAFL) was filtered out,which was shown higher scores compared with the wild-type peptide Sur79.Consistent with the results of software analysis,Sur79M2 showed higher binding ability in T2 binding assays.At the same time,Sur79M2-induced CTLs could release a large number of IFN-γ after incubated with target cells rather than Sur79.When co-cultured with HCC cell lines HepG2 and BEL-7402,Sur79M2-induced CTLs effectively lysis HepG2 on HLA-A2-restricted manner without killing effect on BEL-7402 that do not express HLA-A2 molecules.Conclusion Sur79M2 could elicited specific cytotoxic T lymphocytes in vitro,which were able to specifically kill HCC cell lines on HLA-A2-restricted manner.

Modeling of p53 signaling pathway based on S-system equations / 生物医学工程学杂志

p53, as a transcription factor, is an important tumor suppressor gene and plays the key role in the p53-dependent gene regulatory network. Therefore, it is important to understand its biological function at the level of the whole system. In this paper, based on KEGG database and related literatures in English and Chinese, the interaction mode and quantitative relationship of the related molecules involved in p53 signaling pathway were extracted. By using S-system equations and 'Simulink' toolbox of Matlab7.0, a dynamic model of p53 signaling pathway was developed, and the dynamic regulatory characteristics of p53 signaling pathway were analyzed on model simulation. The results were in accord with the literatures and could reflect quantitatively the complex regulatory relationship between the interacting molecules involved in p53 signaling pathway. In addition, model simulation helped us find and identify the key molecules in this signaling pathway. Thus, this model can be used as a basis for the follow-up study of the relationship by precise and quantitative assessment.

The experimental study of the cytotoxicity to breast cancer cell line using modified T lymphocyte by TCRVpT.1 gene / 中国免疫学杂志

Objective: To investigate the influence of transfection of TCRVJ37.1 gene in cytotoxicity of PBMC of healthy donor to breast cancer cell line MCF-7/S. Methods :pcDN*31vp'1 packaged by lipofectamine were transfected to healthy donor's PBMC,Flow Cytometer Analysis and MTT Colorimetric Assay were used respectively to test the expression of pcDNA3.1VB7.1 gene and the cytotoxicity of PBMC before and after gene transfection of healthy donor to breast cancer cell line MCF-7/S. Results: The expression of TCRVJ37.1 gene after transfection was obviously higher.There was distinguish differences of cytotoxicity before and after gene transfection. Conclusion:The modified healthy donor's PBMC by TCR gene could have stronger cytotoxicity to breast cancer cell line.

The usage of TCRV? gene and activation of PTK signal pathway in McAb costimulated PBLs and induction of in vitro apoptosis of hepatoma cells / 中国免疫学杂志

Objective:To study the expression of TCRV? subfamily which specially recognize the hepatoma cell antigen and the apoptosis of hepatoma cell induced by McAb costimulated PBLs.Methods:The change of the phenotype of PBLs was studied by flow cell cytometry and the level of the expression of TCRV? was studied by RT-PCR and Southern blot,the PTK by western blot.The hypermicroscopic ultrastructure was observed through transmission electron microscope.Results:The level of CD3 and CD8 of PBLs was significantly increased after acted with hepatoma cells,while there was no change in CD4.The expression of TCRV?7 of PBLs was dramaticly increased and peaked at 4 days.PTK increased correspondently,to 58% compared with 11% in control.Besides anti-CD3 McAb induced lymphocyte apoptosis,the mediated apoptosis of hepatoma cells was found in the other three groups.Conclusion:TCRV?7 was the tumor antigen specific T cell receptor,and it activate the PTK signal pathway.The McAb activated lymphocytes initiated apoptosis in hepatoma cells.

Biological effect of recognizing hepatoma antigen TCRV?7 subfamily after cloning and transgene / 中国免疫学杂志

Objective:To study the biological effects of TCR to hepatoma cell by transfection V?7 to lymphocytes. Methods:TCRV?7 gene was amplified by RT PCR and cloned to expression vector pLXSN. The recombinant was transferred into lymphocytes by Lipfectin Reagent transfection,then the lymphocytes were co cultured with hepatoma cells.The phenotype of lymphocytes was detected on the Flow Cytometry and the ultrastructure of the hepatoma cells was showed by electronic microscope.Results:The lymphocyte amount with TCRV?7 expressing in those being transfected was much more than those no transfection.Apoptosis appeared in the hepatoma cells.Conclusion:TCRV?7 subfamily can recognize hepatoma antigen and stimulate T cell.