Study Of Containing With Fc Gamma RⅡb Gene Lentivirus Vectors In MRL/Lpr SLE Mice

Objective Packing containing mouse FcγRⅡB lentivirus vectors, detecting the expression in B cell surface and study its expression in MRL/Lpr SLE mice.Methods The lentiviral expression plasmid TRE-FcγRⅡB, lentivirus vector Tet regulating lentiviral packaging plasmids were co-transfected HEK293 T cells were expressing viral packaging and regulate virus were measured and regulate the expression of viral titer viral infection. The MRL / lpr SLE mice prevent animal(weeks around eight weeks) were divided into 40 groups virus solution 100 ul, 200 ul virus solution set, preventive empty virus group(excluding FcγRIIb gene), the prevention of the control group(saline group) Four groups of 10, then the model animal(weeks 16 weeks) group of 40 were randomly divided into groups virus 100 ul, 200 ul group, empty viral treatment group(excluding FcγRIIb gene) and therapy control group(saline group) n = 10. By intravenous injection of the virus were transduced into MRL / lpr SLE mice, continuous injection four weeks, once a week. After the injection, was observed after the end of the reflection mention collected mouse urine protein urine test mice, the orbital angular vein blood was taken about two weeks later, the mice 500μL antinuclear antibodies and anti-double stranded DNA content, complete blood after death mice, each group of mice grinding fresh spleen lymphocytes after separation MACS FcγRIIb detect the expression of the gene m RNA and protein on B cells and for kidney, lymph nodes, liver biopsy. At the same time in each group by Western blot and protein phosphorylation Western blot, to study the possible mechanisms Btk B cell pathways on Ship1 Pyn total protein, and phosphorylated proteins was observed. SPSS16.0 software for statistical data treatment groups were compared using t test, p <0.05 was considered statistically significant. Chart utilizing Graph Pad Prism 5.0 software to draw.Results 1. Expression and regulation viruses drops of up to 106 TU/m L.2.MRL / lpr SLEFcγRⅡB m RNA and protein level test results: the prevention and treatment of viral empty meaningless compared with the control group, virus group(including 100μL and 200μL group) and empty virus group increased FcγRⅡB m RNA and protein compared to.3.MRL / lpr SLE mouse urinary protein content of anti-double stranded DNA antibody test results: the prevention and treatment of viral empty meaningless compared with the control group, compared to the urine virus group(including 100μL and 200μL group) and empty virus group protein, anti-double stranded DNA content decreased.3.MRL / lpr SLE mouse urinary protein content of anti-double stranded DNA antibody test results: the prevention and treatment of viral empty meaningless compared with the control group, compared to the urine virus group(including 100μL and 200μL group) and empty virus group protein, anti-double stranded DNA content decreased.4. 4.1Btk group B cells, Lyn meaningless compared with empty virus, preventing viral group Btk(including 100μL and 200μL group) significantly reduced protein, p <0.01 compared with empty virus group, with statistical significance. Prevention 200μL total Btk protein group was significantly lower compared with 100μL; Btk treatment group B cells of each histone unchanged. Prevent the virus group Lyn 100μL protein content increased significantly compared with the empty virus group, p <0.01, statistically significant; 200μL group decreased significantly compared with 100μL; compared to the treatment of viral groups Lyn 100μL group and 200μL group decreased significantly. Ship1 total protein prevention and treatment of various groups change.4.2Prevention of empty virus group Btk phosphorylation, Lyn Ship1 phosphorylation and phosphorylation levels were relatively unchanged. Prevention of viral 200μL group phosphorylated Btk, phosphorylation and phosphorylation of Lyn Ship1 empty virus group compared with the expression level was significantly reduced, p <0.01, prevent virus 100μL group compared with empty virus meaningless. Prevention of viral 200μL group significantly decreased compared with 100μL. Treatment of empty virus group Btk phosphorylation, Lyn Ship1 phosphorylation and phosphorylation levels were relatively unchanged. Treating viral 200μL group Btk phosphorylation and phosphorylation Ship1 and empty virus group increased significantly compared, p <0.01, statistically significant. Treating viral 100μL group phosphorylated Lyn and empty viral protein was significantly increased compared to group, p <0.01, statistically significant. 200μL and 100μL viral treatment group was significantly higher compared.5.The pathology results5.1: Kidney: HE staining showed that the prevention of kidney cortex of mice, extensive inner medullary vascular congestion expansion around the lymphocyte infiltration; visible increase in the number of intracellular glomerular lesions, volume slightly increased or normal glomerular surrounded by lymphocytes infiltration. Treated kidney cortex and medulla generalized vascular congestion expansion, especially in medulla and glomerular obvious, between arteries and arcuate arteries, not the whole bureaucratic, surrounded by a large number of lymphocytes; diffuse renal medulla small ball mild proliferative changes, glomerular lesions seen in the number of cells increased, the volume slightly; the ball around a large number of lymphocytes, the glomerular capillary group atrophy, sclerosis. AIDS prevention and treatment groups(100μL and 200μL) compared with the respective control group lymphocytic infiltration, diffuse glomerular proliferative changes light.5.2: Liver: Prevention mice loose cytoplasm of hepatocytes breadth, a large number of lymphocyte infiltration around the central vein, part of the regional central vein and hepatic sinusoid expansion; part of the regional portal area fibrosis, dilation of small blood vessels and a large number of lymphocytes. Treatment of mice loose cytoplasm of hepatocytes widespread around the central veins infiltration of lymphocytes and split lobule, areas central vein and hepatic sinusoid dilatation, dilation of small blood vessels and a large number of lymphocytes, some regions serious disease, liver large number of lymphocytes infiltrating lobular and lobular division, showing liver punctate necrosis, lobular almost substituted lymphocytes, lobular structural damage. AIDS prevention and treatment groups(100μL and 200μL) compared with the respective control group less lymphocytic infiltration.5.3: lymph nodes: Prevention mice lymph node mild proliferative changes, clear lymph inherent structure, a large number of lymphocytes in the lymphoid follicles, occasionally germinal centers; Mice lymph nodes in the proliferative changes, mainly manifested as lymphoid nodules increase in size, lymph follicular hyperplasia, proliferation of lymphocytes, a large number of regions of the visible part of histiocytosis, almost no germinal centers, so that the cortex and medulla boundaries unclear, severe lesions seen lymph inherent structure disappeared. AIDS prevention group(100μL and 200μL) than the control group no significant proliferative changes. Viral treatment group(100μL and 200μL) than the control group histiocytosis less, in which even the 200μL virus injection group.Conclusion:Successfully packaged lentiviral vector of FcγRⅡB. By transfection to MRL / lpr SLE mice overexpressing reduced antinuclear antibody MRr / lpr mice, anti-double stranded DNA and urine protein content, delaying the development of its pathology, up on B cell suppression receptor expression may be a new approach for the treatment of systemic lupus erythematosus.