| In normal condition, the important costimulatory molecule, B7, which is expressed on antigen presenting cells(APCs) combining with its receptor CD28 expressed on T cells can provide an essential costimulatory signal for the immune response of T cells and activation/function of T cell-dependent B cells. But under a pathological state hyper-activation of the B7/CD28 signaling pathway can lead to the hyper-function and abnormal immune response of T cells and B cells, which results in the occurrence of autoimmune diseases.The autoimmune disease systemic lupus erythematosus(SLE) characterized by immune system disorder, such as the emergence of a variety of auto-antibodies and immune complex formation/deposition as well as inflammatory lesions in many tissues and organs. Among the above-mentioned, lupus nephritis(LN) deemed as the most common and serious complication in SLE and a main cause of death has received extensive attention. Up to now, several studies have demonstrated an abnormal B7/CD28 costimulatory signal and the hyper-function of T cells and B cells caused by the former play an significant role in the pathological process of LN.Using specific antibody to bind/block B7 molecules and to intervene in B7/CD28 signaling pathway at protein level can interrupt or impair the abnormal T-cell/B-cell immune response and help to restore normal immune tolerance, which has the positive significance to alleviate the pathological damage in LN. Accordingly, RNA interferencemediated by 21~23bp small interfering RNA(si RNA) which can sequence specifically at gene level inhibit B7 expression on APCs and cause the absence or down-regulation expression of B7 molecules is also capable of blocking or inhibiting B7/CD28 costimulatory signal and the abnormal activation and response of T cells and B cells.In the present study, using a hybridoma cell line(clone 1D1) successfully established in our institute which is capable of secreting mouse anti-human B7-2monoclonal antibody, we produced and prepared purified antibody and then identified its biological characteristics in vitro. In addition, murine B7-2 gene RNAi recombinant lentivirus were constructed and the antagonism effect on B7/CD28 costimulatory signal was verified as well. Moreover, we employed a chemical method using Pristane to develop C57BL/6 induced murine lupus-like nephritis model which is similar to the natural pathogenesis and pathological symptoms of human SLE. Simultaneously, we analysed splenic immune cell activation, auto-antibodies and immune complex deposition and renal inflammatory injury to assessed stability and reliability of this model. Followed the above work, we used anti-B7-2 monoclonal antibody and murine B7-2 gene RNAi recombinant lentivirus to exercise immune intervention on the C57BL/6 murine lupus-like nephritis model, whereafter immunological serological and histological dynamic detection and analysis, we evaluated the immune intervention effect and molecular mechanism by inhibiting B7/CD28 costimulatory signal and affecting the chain reactions of APC-T-B as well as to analysis and compare the alleviated/reversal effect on renal pathological injury in mice of the two different interventions, in order to provide some experimental and theoretical data to assist the understanding of immune cell activation and pathological damage mechanism in the development of SLE, even help to find a new biologic intervention for systemic lupus erythematosus.Part ⅠPreparation and characterization of mouse anti-human B7-2 moleculefunctional monoclonal antibodyObject: To prepare functional mouse anti-human B7-2 monoclonal antibody, and study its biological characteristics. Methods: Using a hybridoma cell line named 1D1 secreting mouse anti-human B7-2 monoclonal antibody that was established in our institute, we used an improved method to induce ascites fluid in BALB/c mice and to produce antibody. After the B7-2 monoclonal antibody we got was purified via Protein G immunity chromatography, its capability to recognize B7-2 molecules expressed on different cells was analyzed by flow cytometry assay. Further more the antagonism effect on B7/CD28 costimulatory signal of B7-2 monoclonal antibody was measured via MTT assay. Results: We got approximately 80% positive rate of ascites fluid formation in BALB/c mice, and obtained an yield of ascites fluid averagely 5.5m L per mouse There are averagely 2.5mg B7-2 antibody in per milliliter ascites fluid, and this antibody can recognize and bind B7-2 molecules expressed on the membrane of L929-B7-2 fibroblast cells, Raji and mouse spleen cells with the respective positive binding rates of 94.5%, 98.2% and 52.7%; in addition, the antibody is capable to suppress costimulatory signal mediated by B7-2 molecules and inhibiting the growth and proliferation of L929-B7-2 cells and peripheral blood T lymphocytes(p < 0.05)Conclusion: we successfully obtained mouse anti human B7-2 molecule functional monoclonal antibody which has the ability of antagonizing B7/CD28 costimulatory signal.Part ⅡConstruction of lentiviral expression vector and preparation ofrecombinant lentivirus for murine B7-2 gene RNA interferenceObject: To construct lentiviral expression vector for murine B7-2 gene RNA interference, and prepare recombinant lentivirus. Methods: We chose three target sequence segments for murine B7-2 gene RNA interference, then designed and synthesised the template oligonucleotides of short hairpin RNA. Followed the construction of recombinant lentiviral expression vector, with the help of liposome lentiviral expression vector together with packaging plasmids and envelope plasmids were co-transfected into 293 T cells to produce recombinant lentivirus. After that we used ultracentrifugation to get concentrated lentivirus and employ biological assay to check the titer of recombinant lentivirus and the generation of replication-competent lentivirus. Results: Sequencing confirmed that the lentiviral expression vector for murine B7-2 gene RNAi was constructed successfully. After co-transfecting 293 T cells by those lentiviral plasmids, we acquired recombinant lentivirus, which was then concentrated by ultracentrifugation and improved titer to 1~ 3×108TU/m L. Further more, the results of biological assay demonstrated no replication-competent lentivirus generate during the infection of recombinant lentivirus, which confirmed its good biosafety. Conclusion: We constructed three kinds of lentiviral expression vectors and prepared the recombinant lentivirus with good biosafety for mouse B7-2 gene RNAi(LV-139、LV-425、LV-848) successfully.Part ⅢInterference effect of recombinant lentivirus for murine B7-2 gene RNAi on the expression of membrane B7-2 molecules in dendritic cellsObject: To select the murine B7-2 gene RNAi recombinant lentivirus with best interference effect and to detect its antagonism effect on B7/CD28 costimulatory signal Methods: Mouse bone marrow cells were isolated and then induced to differentiate into DCs with the help of GM-CSF and IL-4; DCs were stimulated sequently or 48 hours by lipopolysaccharide to maturate, followed by the identification of its morphology and phenotype(CD11c, B7-1, B7-2 and MHCⅡ). We analyzed the infection efficiency of LV-NC on DCs at different multiplicity of infection(5,10,20,40,60) via flow cytometry to ascertain the optimal conditions for infection. LV-139, LV-425 and LV-848 infected DCs at the determined optimal conditions, then the interference efficiency on B7-2molecules expression was analysed by flow cytometry aiming to select a recombinant mouse B7-2 gene RNAi lentivirus with the best interference effect. MTT assay was applied to measure the ability of mouse B7-2 gene RNAi recombinant lentivirus to antagonize B7/CD28 costimulatory signal and to inhibit the growth and proliferation of DCs and mouse spleen T cells. Results: We obtained murine bone marrow-derived DCs in vitro successfully, which had typical dendritic protrusions and expressed CD11c(68.5%), B7-1(71.6%), B7-2(74%) and MHC Ⅱ(84%). Flow cytometry analysis demonstrates that LV-NC can effectively infect DCs with a infection efficiency of86.4%; the interference efficiency of LV-139, LV-425, LV-848 on B7-2 molecules expression in DCs at MOI 80, were respectively 66.7%, 67.2% and 63.8%. MTT assay also showed that recombinant lentivirus LV-425 can antagonize B7/CD28 costimulatory signal and inhibit the growth and proliferation of DCs and mouse spleen T cells significantly(p< 0.05). Conclusion: Mouse B7-2 gene RNAi recombinant lentivirus LV-425 can antagonize B7/CD28 costimulatory signal.Part ⅣDevelopment and characterization of a murinelupus-like nephritis modelObject: To develop and characterize of a C57BL/6 murine lupus-like nephritis model induced by Pristane. Methods: Female C57BL/6 mice aged 6~8 weeks were intraperitoneally injected with 0.5m L Pristane to develop a murine lupus-like nephritis model. Ten days after injection of Pristane, the activation of macrophages, dendritic cells, granulocytes and B cells in mouse spleen were detected by flow cytometry, as well as the expression of B7-2 and MHC Ⅱon CD21+ B cells. Blood were sampled from orbital plexus of living mice every month, to detect ANA and anti-ds DNA antibody in serum via immunofluorescence assay. Morning urine were sampled every month to measure the level of proteinuria using Albustix test strips. Eight months later after Pristane injection and model making, mice were humanely killed. Kidneys were slided to stain by H&E or PE-Ig G and then we observed the evidence of immune complex deposition and glomerulonephritis histopathologically. Results: 1.Ten days later, in model making group splenic Mφ, DCs, granulocytes and B cells all activated remarkablely compared to that in the control group(p<0.05), which the positivity rates of CD11 b, CD11 c, Gr1 CD21 were 7.35 ± 0.80%, 3.78 ±0.56%, 9.85 ± 1.13% and20.94±1.24% respectively Meanwhile, the expression rates of CD86 and MHCⅡon CD21+B cells were up-regulated to 56.31±2.24% and 67.15±3.62%, also significantly higher than control group(p<0.05). 2. At the third month, the detection results of ANA and anti ds DNA antibody began to appear positive with 30% and 10% positive rates separately, as well the increasing of positive rate and titer can be seen over time. When time came to the Eighth month, ANA detection results of all mice in model-making group were positive and the positive rate of ds DNA antibody rised to 89%. 3. At the fourth months, 30% mice of model-making group had developed 300mg/L~3000mg/L(+~++) proteinuria Following the positive rate and severity increased over time, at the8 th month, 100% mice had developed 1000mg/L~20000 mg/L(++~++++) proteinuria4. The results of direct immunofluorescence assay demonstrated that glomerular capillary, mesangial region appears strong positive immune complex deposition. Kidney histopathology analysis using H&E stain showed segmental or diffuse proliferative glomerulonephritis were developed in C57BL/6 murine lupus-like nephritis model induced by Pristane. Conclusion: C57BL/6 murine lupus-like nephritis model induced by Pristane shows an manifestation similar to human SLE, and it is a stable and dependable lupus-like nephritis model.Part ⅤStudy on the immune intervention effect and molecularmechanism of B7-2 molecule specific antibody and RNAirecombinant lentivirus in a murine lupus-like nephritis modelObject: To compare the immune intervention effect on lupus nephritis formation and pathological injury by inhibiting B7/CD28 costimulatory signal via anti-B7-2monoclonal antibody and recombinant lentivirus for murine B7-2 gene RNAi in a murine lupus-like nephritis model and to explore the corresponding molecular mechanism. Methods: Early B7-2 antibody intervention group: first of all each mouse was injected once time with 0.5m L Pristane by intraperitoneal, then 200μg B7-2antibody was given through tail intravenous injection per mouse every time on day 1, 35, 8, 15 and once a month in next three months; Delayed B7-2 antibody intervention group: mouse was injected with same dosage and frequency of B7-2 antibody at the 4th month when proteinuria and autoantibody emerged. Recombinant lentivirus for murine B7-2 gene RNAi intervention group: each mouse was injected with 0.5m L Pristane by intraperitoneal followed by the intravenously injection of 0.5 × 108 TU LV-425 everymouse on day 1 and 60; Meanwhile, a normal control group, isotype antibody control group, chemical drugs(CTX) control group and the recombinant lentiviral control(LV-NC) group were set up. 10 days later after model making, the silencing effect of recombinant lentivirus for murine B7-2 gene RNAi on B7-2 molecules expression in mouse splenic APCs and the activation of splenic cells were analyzed by flow cytometry; Expression of ANA and anti-ds DNA antibodies in serum and content of proteinuria in morning urine were detected monthly via indirect immunofluorescence assay and Albustix test strips respectively. At the 8th, IFN-γ and IL-4 in mouse serum was measured via ELISA. Further more, auto-immune-complex deposition in mice kidneys was checked via direct immumofluorescence assay and the pathological examination of renal tissues was also carried out using light microscope and transmission electron microscope. Results: Ten days after Pristane and recombinant lentivirus for murine B7-2 gene RNAi injection, the positivity rates of B7-2 molecules on mouse splenic APCs and activation degree of splenic cells were significantly lower than that in model making group(p<0.05) Meanwhile, both of B7-2 antibody early intervention and recombinant lentivirus LV-425 intervention decreased the level of ANA and ani-ds DNA antibody as the detection results were all negative in those two groups at the third month. Moreover, intervention using B7-2 antibody and LV-425down-regulated the positive rate and titer of ANA/anti-ds DNA antibody significantly when compared to model making group(p<0.05), also we can see the mice in antibody early intervention group showed much lower ANA/anti-ds DNA antibody level compared to recombinant lentivirus LV-425 intervention group and B7-2 antibody delayed intervention group at the 8th month(p<0.05). The content of IFN-γ and IL-4 in mouse serum was also reduced significantly compared with model making group after the intervention using B7-2 antibody and LV-425 at the 8th month. After intervention using antibody and recombinant lentivirus for murine B7-2 gene RNAi, mice all developed less severe proteinuria compared to that in model making group(p<0.05), in addition significant reduction of proteinuria compared to B7-2 antibody delayed intervention group and LV-425 intervention group can be seen in B7-2 antibody earlyintervention group(p < 0.05). Intervention using B7-2 antibody and recombinant lentivirus LV-425 both reduced immune complex deposition and alleviated pathological damage in mice kidneys, concomitantly, B7-2 antibody early intervention demonstrated much better immune intervention/alleviating effect. Conclusion: B7-2 monoclonal antibody and recombinant lentivirus for murine B7-2 gene RNAi LV-425 could both supress B7/CD28 costimulatory signal, down-regulate immune cells activation and alleviate pathological injury in C57BL/6 murine lupus-like nephritis model. B7-2antibody shows better intervention effect than recombinant lentivirus for murine B7-2gene RNAi, it can play not only a preventive effect but also a alleviating effect against lupus nephritis. |
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