| Background&Objective:The induction of antigen-specific T-cell tolerance in the thymus and itsmaintenance in the periphery is crucial for the prevention of autoimmunity. As wellas their stimulatory function, there is growing evidence that dendritic cells, acting asprofessional antigen-presenting cells, also maintain and regulate T cell tolerance inperiphery. This control function is exerted by certain maturation stage and subsets ofdifferent ontogeny, and can be influnenced by immunomodulatory agents. Dendriticcells (DCs) are essential for initiating primary immune responses, they also play acentral role in the initiation and perpetuation of immune-mediated disease. DCs notonly initiate T cell responses, but are also involved in the induction of tolerance. Thefunctional properties of DC are strictly dependent on their state of maturation. In thesteady state in the absence of pathogenic invasion or inflammation, DCs exist in animmature form. Immature DC is inclined to induce immunological tolerance, butmature DC can be inclined to induce immunolgical response. DC derived from bonemarrow can induce immunolgical response and them derived from lymph. Recentlymany scholars have taken advantage of the potentia of immunological tolerance in immature DC, then use genetic modification, antibody technology, antisenseoligonucleotides and culture in vitro to keep DC in an immature stage. TolerogenicDC can be obtained and were abroadly applied to solid organ transplantation and theprevention and treatment of autoimmune diseases. For example; murine bonemarrow DC modified by IL-4 gene can effectively reduce onset morbility ofCollagen Induced Arthritis mice; DC modified by IL-10 gene can significantlyprolong survival of cardiac transplantation; IL-12 gene was silenced by RNAiapproach in DC, Tolerogenic DC will be obtained, etc.Although DC plays a vital role in immune-mediated disease, there is a littlenumber of DC in the body, in detail, there exist less than one percent DC in wholeperipheral blood mononuclear cell, DC was called trace cell population. In 1992, DCcan be amplificated in large number under the condition of certain cytokine, that is,low dose GM-CSF and IL-4 can induce large amount DC from DC bone marrowprecursor. Based on the large number DC culture, a key gene, NF-κB/RelB, whichcan contribute the development and maturation of DC was silenced by RNAi, themost effectively RelB shRNA was screened and was packaged into lentivirusparticle. Bone marrow derived DC was infected by lentivirus, RNAi RelB DC can begained. This kind of DC can be further identified in morphology, phenotype andimmune function and then to get a novel type tolerogenic DC, that is, RNAi RelBDC. This DC can be discussed further in the mechanism of induction of T cellimmune tolerance and hopefully provided some theories data to the successfulapplication to treatment and prevention of graft rejective reaction and autoallergicdisease.Methods:1. Culture in vitro and identification of mice bone marrow derived Dendritic cellsBone marrow was flushed from the femur and tibia of C57BL/6 mice, red blood cells were lysed, and remaining cells were plated in RPMI 1640 (plus 10% FCS,2mM L-glutamine) at 2×106 cells/mL/well. The medium was supplementd withculture supernatant with recombinant mouse granulocyte-macrophagecolony-stimulating factor and interlukin-4 (rmGM-CSF, rmlL-4). Using handfulscreening combined with CD11c positive immunomagnetic beads selection, DCderived from bone marrow was amplified. DC was divided into two groups here.One is called control DC group, that is, immature DC, DC culture for day 6 washarvested and selected by CD11c immunogenic microbeads; The other is calledLPS-DC group, namely mature DC, LPS was added in DC culture for day 5 at thefinal concentration of 1μg/mL for 24h, in day 6, DC was harvested. Both groups'DCwere detected and identification from cell morphology, cell surface marker, allogenicmixed lymphic reaction and RelB gene expression level, etc.2. Construction and identification of RelB-Lentivirus vector by RNAiRelB gene expression level depended on different mature stage of DC, twosites' RelB specific sequences were designed by using on line software through theinternet, that is, http://rnaidesigner.invitrogen.com/maiexpress/. In our experiment,U6 lentivirus expression system was used to construct lentiviral expression vector ofour target gene RelB shRNA, after sequencing, this vector was packaged intolentivirus partical in 293FT cell. Three lentivirus were obtained, they are specific toRelB, R2 and R5, nonspecific to RelB, T6, respectively. The titer was detected. DCculture for day 4 was used to infect, RelB gene expression level was detected byRT-PCR and immunofluorescence method. Control group was immature DC.3. Induction of bone marrow tolerogenicDC and study of its immunological functionRelB was silenced in murine bone marrow DC. The following experiment wasset up to discuss the bioimmuofunctions of RelB-silenced bone marrow DC. Theexperiment was divided into four groups. That is,①Control of immature DC; ②Control of mature DC stimulated by LPS;③RelB-silenced bone marrow DC;④RelB-silenced bone marrow DC stimulated by LPS. Bioimmunofunction can bediscussed from cell morphology, cell surface molecular expression and cellularimmune function including IL-12 secretion capacity, Th1/Th2 cytokine secretioncapacity and MLR.Results:1. A large number DCs can be successfully induced from mouse bone marrowprecusor cells under the condition of rmGM-CSF and rmlL-4. There are 1×107～2×107 BM-DC from two mice in culture for day 6. The positive ratio of CD11c inBM-DC is up to 70％through handful screening, while it can be improved to beyond90％combined with immunomagnetic microbead positive selection. Afteridentification by flow cytometry, MHC classⅡ, costimulating molecule CD86 andCD40 expressed at low level on the surface of control DC.It was similar to immatureDC, that is immature DC. DC stimulated by LPS expressed three molecules aboveon the surface at a high level, it has mature DC characteristic, that is mature DC.There was round, multifold wrinkles, less protuberance, less organelles, but manyphagocytic matter in intra-cellular. While in LPS-DC group, there are lessphagocytic matter, lots of cytochondriome and rough endoplasmic reticulum in intracellular, retraction of cytoplasmic processes. ELISA assay showed IL-12 secretioncapacity in control DC was lower than that in LPS-DC (P＜0.01). The similar resultswere showed in MLR and RelB gene expression.2. Two target sites of RelB (Genbank no. NM009046) were designed and synthesed,annealed at temp 95℃, detected annealed product on 4％agar gel electrophoresis.Ligation reaction was set up between annealed fragments and pENTRTM/U6,transformed into competent cell TOP10, positive clone was screened throughkanamycin (final concentration: 50μg/mL), sequencing using U6 forward primer (5'-GGACTATCATATGCTTACCG-3'), positive result was obtained. Recombinationreaction was set up between target positive plasmid and lentivirus, then transformedcompetent cell Stb13 through selecting on ampicillin plate, another medicinenegative selection was carried out on the Chloramphenicol LB plate, sequencing,finally, sequencing was carried out to verify the positive recombinator using primerforward U6. Positive recombinator and ViraPowerTM packaging mixtures wereintroduced to 293FT cell line mediated by lipid LipofectamineTM 2000, supernatantfluid was collected and stored at -80℃. Packaged lentivirus titer was determined byserial dilution method and titers of 6×105TU/mL (Transducing Unit (TU)/mL) weredetermined. Packaged lentiviruses of two sites targeting RelB and one negativecontrol T6 were used in RNAi assay in bone marrow immature DC. DC surfacemolecular expression level in above three groups was identified by flow cytometry,DC with infected by lentivirus R5 has the lowest surface molecule CD40. RT-PCRresult showed the lowest RelB gene expression level among three experimentalgroups. Immunofluorescence staining told us RelB protein was low expression leve.That is, Site R5 targeting RelB has the best interfering result.3. There was round or ellipticalness cytomorph, lots of vesicle structure, phagocyticforeign substance, cellular nucleus deviation, less lysosome and cytochondriome,less surface stick and relative regular morphous in DC infected by lentivirus of siteR5 targeting RelB. Low expression levels of MHC classⅡ, CD86 and CD40 wereappeared on the surface of DC infected by Lentivirus R5. IL-12 secreting level wasdetected at a low level by ELISA in group of RNAi RelB DC.and MLR assays toldus there was a weak stimulating T cell proliferation ability in group of RNAi RelBDC. There are a bias from Th0 to Th2 in the MLR, cytokines of IL-2 and IFN-γwere suppressed, while cytokines of IL-4 and IL-10 was dominant.Conclusions: 1. Under the low dose rmGM-CSF and rmlL-4, DCs were proliferated from mousebone marrow precursors at a large number. High purification BM-DC can beobtained from immunomagnetic beads column of mouse CD11c, purify of DC wasabove 90％. Studies of immunolgic function and RelB gene expression level inimmature and mature DCs both told us immature DC was inclined to induceimmunotolerance and had a low RelB expression level, while mature DC wasinclined to inducing immune response and had a high RelB expression level, in aword, RelB expression level has some relationship with mature state of DCs.2. Lentivirus vectors of targeting mouse RelB gene for RNAi were successfullyconstructed. Two targeting sites of R2 and R5 were successfully cloned. Lentivirusvectors were packaged and detected their titers successfully.3. A novel DC was constructed sucessfully, that is, RNAi RelB DC. Site R5 oftargeting RelB had the highest effect. From the cytomorph, cell surface molecularexpression and cytoimmunofunction point, RNAi RelB DC is a kind of immature DC,and This DC can't be stimulated by LPS to develop mature state. This researchresults told us this DC is a kind of tolerogenic DCs.Maybe a broad application ingraft rejective reaction and prevention and treatment of autoimmune diseases in nearfuture.
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