| ObjectiveHuman dendritic cells(hDCs) are the professional antigen-presenting cells,they combined with peptide antigens through its surface major histocompatibility complex and presented antigens to T cells promoting T cell activation and proliferation, in addition, they promote B cell activation and regulation of humoral immunity. Mature DC surface express MHC molecules, adhesion molecules,cytokines(such as IL-12), which largely promote the body’s immune response, this characteristic of DC was applied in anti-tumor immunotherapy and anti-infective therapy.Mature DC immunization can effectively break self tolerance at the cellular level,i.e.,activate self antigen-specific CTLs,but rarely causes antoimmune pathologies against normal tissues and tumors,suggesting that the use of higher-affinity self peptides,enhancement of DC maturation and TLR agonists activate autoreactive T cells fail to break tolerance and induce pathological antitumor immunity at the host level.It indicated that the condition of breaking self tolerance in which cellular level(autoreactive CTL activation) and host level(autoimmune pathology) is different,even though in persistent inflammatory stimulus,signaling suppressor can effectively prevent autoimmune pathology which induced by excessive activation of DC and autoreactive CTL.Under the conditions of suppressing immune response in a natured state, autoimmune tolerance is still exist and may promoted by variety of mechanisms,such as regulatory T cells and various inducing factors secreted by tumor cells,thus obstructing the application and efficiency of DC-based vaccine.Gilboa E has demonstrated that DCs are effective in inducing antitumor responses in mice.However,the result of DC vaccine trials have been largely disappointing with very low rates of objective clinical responses.The major challenge now is to find a noval way to elicit-effective T-cell responses to self-antigens preferentially expressed by tumor cells.SOCS1family regulates the secretion of cytokines through the Janus kinase-signal transducers and activators of transcription(JAK-STAT) pathway.This family consists of eight members,SOCSl to SOCS7and CIS (cytokine-inducible SH2-containing protein),which share a number of features including the presence of a src homology(SH2) domain and a highly conserved carboxyl terminal SOCS box.SOCS1,a prototype molecule of the SOCS family,was initially defined as a suppressor of cytokine signaling.The molecular mechanisms of SOCS1-mediated function have been subsequently identified by studies using gene knockout mice and gene silencing technology.As part of a negative feedback regulation,SOCS1downregulates cytokine signaling through direct inhibition of JAK tyrosine kinase and the signaling cascade of activated cytokine receptors,thereby attenuating cytokine-initiated signal transduction.Moreover,other studies have demonstrated that SOCS1also downregulated TLR signaling through direct and indirect mechanisms.Both cytokine receptor and TLR signaling pathways mediate important functions in survival,maturation and differentiation of various types of cells,and in the regulation of immune function.Abnormal expression of SOCS1in tumor cells has been detected in various human cancers,where it is associated with dysregulation of cytokine receptor and TLR signaling to promote cell transformation.Recent studies on the function of SOCS1in tumor cells have revealed its novel role in carcinogensis.In antigen presenting cells(APCs),SOCS1is considered as a classic presentation attenuator.APC-secreted cytokines,whose signal transduction is negatively regulated by SOCSl,influent not only APCs but also the differentiation and function of T cells in microenvironment.SOCSl controls signaling of many cytokines,including IFN-γ,IFN-a,IL-2,IL-4,IL-6,IL-7,IL-10,IL-12,IL-15and IL-21utilizing a feedback loop mechanism which are important for immune response.For example,IL-2promotes the generation of CD4+T cell memory.IL-7is important for CD8+T-cell survival and CD4+memory cell homeostasis.IL-12conditions CD8+T-cell for long-term immunity by increasing sensitivity to IL-7and IL-15.Meanwhile,SOCS1also directly influences the differentiation,maturation and function of T cells through other mechanisms. Because of its critical role in immunomodulation,SOCS1has been studied in several immune-related disease,including HIV infection,systemic lupus erythematosus,rheumatoid arthritis,multiple sclerosis etc.We hope to unfetter IL-12and the downstream cytokine signaling through suppressing SOCS1in DCs which can break of the host level of self-tolerance and become the new direction of tumor vaccine.Hong B etc. have been confirmed silencing of dendritic cells SOCS1gene can enhance specific anti-tumor immunity in vitro and in vivo.Therefore, we hope to utilize construction of SOCS1gene siRNA lentiviral vectors to inhibit the expression of SOCS1gene in dendritic cells and provide a method for further study in DC-based antitumor vaccine.There are chemically synthesized siRNA to supress SOCS1gene in human dendritic cells in which way it can further study the the hDC immune function changes,but it is unstable,transient and the efficiency of transfection is low,so we constructed a lentiviral recombinant plasmid inhibition of SOCS1gene,compared to synthesized siRNA,it can suppress SOCS1gene for a longer time and had a higher efficiency. We hope our studied can lay a foundation for further study in the effect of DC enhanced anti-tumor immune response.Methods1.Human SOCS1oligonucleotide design and synthesis Screened out of a nucleotide sequence according to the literature as RNAi targets and designed synthesizing oligonucleotide strand, Bbs Ⅰ and Xho Ⅰ restriction Enzyme cutting site were inducted to the terminal of the strand. The oligonucleotide sequences are as follows:SOCS15’-CACGCACTTCCGCACATTC,-3’; negative control:5’-TTCTCCGAACGTGTC ACGT-3’.2. Construction of SOCS1shRNA expression recombinated plasmid phosphorylated synthetic oligonucleotides5’end and annealed to form a Bbs Ⅰ Xho Ⅰ restriction enzyme sites of double-stranded DNA, and connected into LV1lentiviral vector to construct the SOCS1siRNA recombinant.Recombinant plasmid after connection then transformed to competent E.coli DH5a,PCR amplification was used to initial identification, and then plasmid kit was used to extract plasmid, and further identified by DNA sequencing.3.Packaging and production of the expression construction Cultured293T cells through conventional methods, transfered293T cells into a culture dish before the day of transfection,cotransfected293T cells with SOCS1-siRNA lentiviral packaging compound, refreshed cells after24h transfection with DMEM containing of10%fetal bovineserum.Collected supernatant after48h transfection and centrifuged5min with the speed of5000r/min,then filtered the supernatant with the0.45μm PVDF membrane,stored at-80℃refrigerator.4.Determination the titer of the virus Inoculated well-growing293T cells to96well plates,after24hours infected cells with viral solution.When cells were almost stable after72h infection,counted fluorescent cells through a fluorescence microscopy to calculate the viral titer.5.DC culture Withdrawaled healthy adult volunteer peripheral blood,diluted with the same amount of physiological saline and then separated by Ficoll,collection of lymphocytes, use the AIM-V medium, adjusted the cell concentration to5×105/ml,cultured in6-well plates, each well3ml. After cultured adherent5h in5%CO2incubator at37℃,gently washed away the suspended lymphocytes, added to each well3ml AIM-V medium containing of GM-CSF (final concentration800U/ml).Refreshed medium every other day,each well added3ml AIM-V medium containing of GM-CSF (final concentration800U/ml and IL-4(final concentration800U/ml).On day5,in addition to GM-CSF and IL-4,also added TNF-a (final concentration20ng/ml).On day7collected DC and observed them morphology under the microscope.6.Gene transfection Viral transfection when DC cultured for3days. Transfection experiment consistsd of three groups:blank group,siRNA negative group,siRNA group. The blank group considered as the control group which was used for detecting the background level of SOCS1gene expression in dendritic cells,the siRNA-negative group was used to detect whether plasmid affect SOCS1gene expression when it was transfected into cells. Each group had two wells,each well of siRNA group added40μl virus solution which was diluted to106and a final concentration of5μg/ml Polybrene, each well of siRNA negative control group was added to40μl control virus solution, and a final concentration of5μg/ml of Polybrene, blank group added the same amount of the AIM-V medium,each well of all groups added to960μl AIM-V medium containing of GM-CSF(the final concentration800U/ml) and IL-4(final concentration of800U/ml).After12h incubation refreshed medium,each well added to3ml AIM-V medium containing of GM-CSF (final concentration800U/ml) and IL-4(final concentration800U/ml).Refreshed medium every other day.7.RT-PCR detect the expression of SOCS1mRNA Observed the expression of EGFP under a fluorescence microscope after DC was transfected with96h.8.Western blot detect the expression of SOCS1protein After DC was transfected with96h,respectively collected three groups hDCs,the total proteins of DCs were extracted by RIPA whole-cell lysate,determined the concentration of proteins by BCA method.Each of30μg samples of these proteins proceed8%SDS-PAGE gel electrophoresis,trarsmembrane,lutation,and incubation at4℃with anti-SOCSl antibody,TBST washed the membrane three times, interacted with HRP labeled secondary antibody,chemiluminescence imaging method was used to development after TBST repeated washed the membrane three times,X-ray exposed the film,used gel imaging analysis system to analyse the film.9.Statistically analysis We used SPSS13.0softwar to analysis the results.The measurement date was defined as mean±standard deviation (X±S). We used analysis of variance (one-way ANOVA) to analyse the experimental data of RT-PCR and Western Blot, and set the inspection level α=0.05.Results1.Morphological Observation of DC We observed a large number of mature dendritic cells isolated from peripheral blood,DCs are in different forms and various lengths of dendritic protrusions,gradually constituted of colonies in7days culturing.2.Observed transfection efficiency by fluorescence microscopy We respectively observed the transfection efficiency by a fluorescence microscopy after48h and96h transfection.The fluorescent cells increased significantly after96h transfection compared with48h.We observed that the fluorescent cells were more than70%by naked eye which initially indicated the lentiviral vector was constructed successfully.3.The expression of SOCS1mRNA was detected by RT-PCR RT-PCR results showed that compared with blank group and siRNA negative group,the expression of SOCS1mRNA was decreased by72%.The melt curves indicated that the products by PCR were specific amplified products by which quantitated were accurate and credible.4.The expression of SOCS1protein was detected by Western blot Compared with blank group and siRNA negative group,the expression of SOCS1protein in siRNA group was significantly decreased by Western blot.(P<0.05)ConclusionWe successfully transfected dendritic cells with a construction of SOCS1gene siRNA lentiviral vector and compared with normal dendritic cells,the expression of SOCS1gene was significantly suppressed. |
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