Study On The Reverse Effect And Molecular Mechanism Of Inhibiting B7/CD28Signal Pathway From Gene To Protein Level In Lupus-like Disease

B7-1molecule is an important co-stimulatory molecule expressed on the surface ofantigen-presenting cell (APC), and its combination to the receptor CD28can generatecostimulatory signals, which is essential to the primary immune response. Without thiscostimulatory signal, T cells will enter to a state of anergy, tolerance even apoptosis.Thehyperreaction of B7/CD28signal is closely related to the occurrence of autoimmunediseases.Systemic lupus erythematosus (SLE) is a common autoimmune disease whichetiology is uncertain characterized by T、B lymphocyte dysfunction, a variety ofantoantibodies, especially antinuclear antibody (ANA) and anti double-stranded DNA(dsDNA) antibody, immune complex formation and many organs damage.Lupusnephritis (LN) is one of the most common and serious complications of SLE, which isalso the primary cause leading to death of patients. Some studies have revealed thatB7/CD28signal participated in the pathogenesis of SLE.RNA interference (RNAi) is a kind of technology mediated by double strandedRNA with21-23nucleotides-small interfering RNA (siRNA), which can suppresshomologene expression by specific sequence, belonging to post-transcriptional genesilencing (PTGS). It can suppress definite gene expression quickly, specificly andefficiently.Specific antibody interaction with its ligand leads to inhibit or stimulate biologicsignal. Blocking antibody of B7molecule can prevent or down-regulate its interactionwith CD28, then inhibit the activation and response of T, B lymphocytes.In the present study, we established a C57BL/6J mouse model with lupus-likedisease induced by Pristane, which is similar to clinical pathogenesy and manifestationof patients with SLE. Based upon the model, we apply the lentiviral expression vector B7-1shRNA and mouse anti-human B7-1monoclonal antibody to the model mouse inorder to evaluate its reverse effect and molecular mechanism by blocking B7/CD28signal pathway, which could provide some experimental and theoretical data, even finda biologic intervention which is specific, efficient and harmfulless for SLE.Part Ⅰ Construction of lentiviral vector targeting mouse B7-1gene byRNA interference and evaluation its disturbing effect on theexpression of membrane B7-1moleculeObject: To construct lentiviral expression vector targeting mouse B7-1gene byshort hairpin RNA (shRNA), evaluate its disturbing effect on the expression ofmembrane B7-1molecule and select an optimal target sequence. Methods: Thecomplementary DNA containing both sense and antisense oligonucleotides of thetargeting sequence was designed, synthesized. After annealed, double-stranded DNAwas inserted into the LV3（pGLV-H1-GFP+Puro）vector.293T cells were cotransfectedwith pLV/helper-SL3、pLV/helper-SL4and pLV/helper-SL5. The titer of virus wastested according to the expression level of GFP. Mouse L929fibroblast cells wasinfected by the recombinant lentiviruses, the optimal target sequence was selectedaccording to the disturbing effect on the expression of membrane B7-1molecule byflow cytometry and immunofluorescence Results: DNA sequencing demonstrated thatlentivirus plasmid LV3-B7-1shRNA was constructed successfully. The titer of therecombinant lentivirus was1×10~8TU/ml, the best MOI for lentivirus infecting L929cell was60and infection efficiency was88.7%. As for the four shRNA templatesequence candidates, the optimal target sequence is B7-1shRNA-256, its silencingefficiency of the membrane B7-1molecule on L929cells reached the highest rate for73.2%. Conclusions: Lentiviral expression vectors targeting mouse B7-1gene by RNAinterference was constructed successfully. The recombinant lentivirus can effectivelysilence the membrane B7-1molecule on L929cells.Part Ⅱ Preparation and assay of mouse anti-human B7-1antibodyObject: To obtain pure B7-1antibody, analyze its titer and recognition of membrane B7-1molecule. Methods: Using of the4E5cell line our institutesuccessfully established, ascites were induced to produce the antibody from BALB/cmouse and purified with ProteinG affinity chromatography method. The Ig isotype of4E5was identified with the rapid test paper. Flow cytometry assay was used to test theability of4E5to recognize the membranes B7-1molecule on different cells. Theblocking effect on costimulatory signals of4E5was detected by MTT assay. Results:The positive rate of ascites formation in mice was about80%. Ascites harvest was5.9ml from each BALB/c mouse on average. The purified4E5from ascites was about2.3mg/ml. The4E5could specifically recognize membrane B7-1on cells of Daudi、Raji、H1299、spleen cells of mouse with the positive rate of88.2%、92.7%、20.6%,48.5%,respectively. In addition,4E5could inhibit the growth and proliferation of L929-B7-1ce11s and PBTCs through blocking the B7-1costimulatory signals with MTT assay.Conclusions: Mouse anti-human B7-1monoclonal antibody which can efficiently blockthe B7-1costimulatory signals in vitro was successfully obtained.Part Ⅲ Establishment and evaluation of mouse lupus nephritis modelObject: To establish and evaluate of lupus nephritis model in C57BL/6J miceinduced by Pristane. Methods: Thirty female mice aged6-8weeks were divided intotwo groups randomly. The model-making group was injected with0.5ml Pristane byintraperitoneal while the control group with0.5ml saline.The activation ofmacrophages、dendritic cells、granulocytes and B cells in spleens and the expression ofcostimulatory molecule CD86and MHC-II on B cells were measured byimmunofluorescence and FCM at day10after injection. ANA、anti-dsDNA antibodiesin serum and proteinuria were detected monthly after injection.8months after injection,all mice were killed and kidneys were slided and stained with H&E or FITC-labeledIgG to observe the evidence of glomerulonephritis histopathologically. Results:1.10days after injection, macrophages、dendritic cells、granulocytes and B cells in spleenswere activated, positive expression were7.27±0.85%、4.72±0.68%、9.82±1.26%and17.79±0.65%respectively, significantly higher than that of control group (p<0.05).Meanwhile, the expression of costimulatory molecule CD86and MHC-II in CD21+Bcells up-regulates to38.69±3.14%and55.33±2.58%, which was also significantly higher compared to the control group (p<0.05).2. The concentration of ANA andanti-dsDNA antibody in mice sera showed the same trend. The concentration of ANAwas up-regulated from30%to100%, after3months injection to8months by dynamicmonitoring. Anti-dsDNA antibody was up-regulated from20%to80%.3. Four monthsafter injection, the urine protein of model group was detected with+～++（300～1000mg/L）in20%model-making group mice. Finally++～++++（1000～20000mg/L）in100%model mice were tested as time went by.4. Immunofluorescence intensity ofimmune complex in model-making group mice was strong positive.5.Kidneyhistopathology analysis showed that glomerular volume enlarged severely, the numberof cell significantly increased, and fibrous tissue appeared, interstitial tissue wasinfiltrated by lymphocytes. Conclusions: The manifestation of mouse lupus nephritismodel induced by Pristane was similar to human SLE with characteristic immune cellsactivation and kidney damage.Part Ⅳ Studyon the reverse effect and molecular mechanism ofinhibiting B7/CD28signal pathway from gene to protein level inlupus-like diseaseObject: To explore the formation of the lupus like disease model and the reverseeffect in pathological injury and its molecular mechanism by different interventionmethods with the adoption of B7-1shRNA lentiviral vector and B7-1antibody to inhibitB7/CD28signal pathway on gene and protein level. The curing effects of the specificantibody on the related diseases were also studied. Methods:1.The treatment methodsof experimental animal groups: C57BL/6J female mice aged6-8weeks were randomlydivided into8groups. A: Negative control group, in which each mouse was injectedwith0.5ml saline by intraperitoneal. B: Model-making group, in which each wasinjected with0.5ml Pristane by intraperitoneal. C：B7-1antibody early interventiongroup, in which each was injected with0.5ml Pristane by intraperitoneal at first, theninjected B7-1monoclonal antibody200μg through tail intravenous on day1,3,5,8,15,and once a month in next three months. D: Antibody isotype control group, in whicheach was injected with same doses of IgG1. E: Lentivirus interference group, in whicheach was injected with0.5ml Pristane by intraperitoneal at first, then injected 0.4x10~8TU of LV-B7-1shRNA on day1,60through tail intravenous. F. Lentivirus blankgroup, in which each was injected with0.5ml Pristane by intraperitoneal at first, theninjected LV-NC shRNA through tail intravenous on day1,60. G: positive agent controlgroup（CTX）, in which each was injected with0.5ml Pristane by intraperitoneal at first,then injected with0.5ml CTX（counted by60mg/kg each time）on day1、8、22、29、43、50、64、71、85、92. H：B7-1antibody delayed intervention group, in which eachwas injected with0.5ml Pristane and injected same dosage and frequency of B7-1antibody after4month when proteinuria and autoantibody emerged.2. Disturbing effectof recombinant lentivirus on the expression of membrane B7-1molecule in spleenantigen present cells: on day10after first infection, spleen cells of mice were collected.The percentage of CD11b+B7-1+、CD11c+B7-1+and CD21+B7-1+double positivecells were analyzed by immumofluorescence method and FCM to explore thesilencing effect of B7-1shRNA lentivirus infection on target molecules.3. Activationanalysis of immune cells in spleen: spleen cells were collected from mice10days afterfirst treatment. The positive expression percentages of Mφ、DC、Gr1+and B cells aswell as the expressions of membrane costimulatory molecules CD86and MHC-II onCD21+B cells were analyzed by immumofluorescence method and FCM.4. Expressionof ANA、anti-dsDNA antibodies in serum were detected monthly after injection.5. Urineprotein analysis: urine protein was detected by Albustix test paper monthly.6.Concentration analysis of cytokines in serum: the concentration of IL-4and IFN-γ inserum was determined by ELISA.7. The immune complex deposit in kidney wasanalyzed by direct immumofluorescence method.8. Renal tissues from mice wereanalyzed by light microscope and transmission electron microscope. Results:1. AfterB7-1shRNA lentivirus interference,positive expression of CD11b+B7-1+,CD11c+B7-1+and CD21+B7-1+cells in spleens were58.43±2.87%,60.18±3.08%,52.85±2.75%,respectively, significantly lower than the control group (p<0.05), suggestion thatB7-1shRNA lentivirus could efficiently inhibit the target molecule expression on APC.2.After intervented by lentivirus and early monoclonal antibody, the Mφ, DC, granulocyteand B cell in spleens down-regulated, significantly lower than the model-making group(p<0.05). Meanwhile, antibody treatment group showed lower than B7-1shRNAlentivirus interference group.3. ANA was detected in part of mice both in B7-1shRNAlentivirus interference group and antibody early intervention group with titer lower than 1:00in all after3months, significantly lower than the model-making group(1:00-1:300). ANA down-regulated in all of B7-1shRNA lentivirus interference group,antibody early intervention group and antibody delayed intervention group, and ANA ofthe three groups was significantly lower than the model-making group (1:000-1:3000)(p<0.05), suggestion that ANA could decrease by B7-1shRNA lentivirus interference orantibody intervention, further more, antibody early intervention group showed lowerANA comparing with B7-1shRNA lentivirus interference group and antibody delayedintervention group (P<0.05).4. Anti-dsDNA antibody was detected in model-makinggroup in4months with low titer around1:10, another4months later, it changed into1:100in90%mouse, higher than the other3groups (B7-1shRNA lentivirus interferencegroup, antibody early intervention group and antibody delayed intervention group),butwithout significant statistical differences (P>0.05).5. IL-4showed0.84±0.52pg/ml and0.88±0.64pg/ml while IFN-γ was3.06±0.38pg/ml and3.02±0.55pg/inB7-1shRNA lentivirus interference group and antibody early intervention grouprespectively, all significant lower than model-making group(P<0.05),but withoutstatistical differences comparing to antibody delayed intervention group.6. In4th month,the urine protein was detected with+～++(300～1000mg/L) in20%mice ofmodel-making group. In8th month, it changed to++～++++（1000～20000mg/L）in allthe mice of model-making group.Proteinuria was±～+++（≤3000mg/L）inB7-1shRNA lentivirus interference group and antibody delayed intervention group,significant lower than model-making group (P<0.05). The urine protein was lowest inB7-1early intervention group, it’s significant lower than B7-1shRNA lentivirusinterference group and antibody delayed intervention group（P<0.05）with the urineprotein content±～++(≤1000mg/L).7. Immunofluorescence intensity of IC in theintervention group mice was weaker than the model-making ones. The kidneys ofcontrol group mice had no significant pathological changes and IC could not be detected.Comparing all the groups except control group, B7-1early intervention group showedleast IC.8. Kidney histopathologically detection showed less damage in B7-1earlyintervention group, B7-1shRNA lentivirus interference group was much severer, and themodel-making group was the severest.9. Ultrastructure of transmission electronmicroscopy analysis showed less change in B7-1early intervention group, B7-1shRNAlentivirus infection group was much severer, and the model-making group was the severest.Conclusions: Both specific RNA and antibody could inhibit the B7-1moleculemediated signal pathway, down-regulate immune cell activation and reversepathological injury. B7-1antibody had more intervention effect than B7-1shRNAlentiviral vector. Further, specific antibody had preventive effect as well as therapeuticeffect against lupus nephritis. Earlier antibody intervention had better therapeutic effectsthan delayed one.