Dissertation
Dissertation > Medicine, health > Surgery > Orthopaedic Surgery ( movement system diseases,orthopedic surgery ) > Joint disease and injury

The Effects of mPDL1-hIg on Collagen-Induced Arthritis

Author WangGuoHua
Tutor WuXiongWen
School Huazhong University of Science and Technology
Course Immunology
Keywords mPDL1-hIg CIA Cell proliferation IL-17 IL-23
CLC R684
Type PhD thesis
Year 2009
Downloads 51
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Background programmed death receptor of -1 (programmed death 1, PD-1) is a member of the CD28 family. Activation of T cells and B cells expressing PD-1. PD-1 body equipped with the programmed death receptor 1 (programmed death of ligand PDL1) or programmed death receptor ligand 2 (programmed death ligand 2, PDL2), passing the inhibitory costimulatory signal. T cells and B cells play an important role in the pathogenesis of rheumatoid arthritis. T cells as an important negative regulator of B cell activation molecules, PD-1 may be the target of a treatment of rheumatoid arthritis (rheumatoid arthritis, RA). mPDL1-hIg is composed by mouse PDL1 ectodomain and human IgGFc fusion proteins, having the biological activity of the mice PDL1. MPDL1-hIg containing human IgGFc was thus mPDL1-hIg can be relatively easily detected and purified. And, mPDL1-hIg human IgGFc theoretically prolong the half-life of mPDL1-hIg mice PDL1 extracellular segment favor mPDL1-hIg mice PDL1 ectodomain exert its effect in the body. Ⅱ collagen-induced arthritis (Collagen Ⅱ-induced arthritis, CIA) is widely used to study the pathogenesis of human rheumatoid arthritis experimental arthritis model. The purpose of new thinking successful to build mPDL1-hIg gene eukaryotic expression vector, then get stable to express mPDLl-hIg CHO cell lines, which on the basis of mPDL1-hlg explore nPDL1-hIg effect on the CIA, to explore the treatment of RA , new methods, and experimental evidence. After the interaction of the the method ① activation normal mice to erythrocytes in the spleen cells with mPDL1-hIg and then with fluorescein-labeled anti-human IgG secondary antibody, flow cytometry activated normal mouse spleen cells to erythrocytes mPDL1 -hIg binding rate. ② calf collagen type II 2 mice immunized spleen cells to erythrocytes after CFSE staining, and then denatured calf collagen type II stimulation. Cell proliferation the of flow cytometry mPDL1-hIg on denatured calf type II collagen-stimulated cells the ELISA assay mPDL1-hIg denatured calf collagen type II stimulated expression of IL-17 and IL-23. The ③ calf type II collagen-immunized mice induced CIA, and calf collagen type II immunized mice were randomly divided into 2 groups: the experimental group and the control group, the experimental group of mice treated with mPDL1-hIg control mice employing IgG therapy. Daily observation starting from day 0, the mice (second immunization time determined as 0 day). CIA mice, and human IgG treatment (4) on the ninth day, taking mPDL1-hIg treatment of CIA mice serum ELISA assay of serum IL-17 and IL-23 expression. ⑤ On the ninth day, the serum, were sacrificed by cervical dislocation mPDL1-hIg treatment of CIA mice treatment of CIA mice, and human IgG, and then quickly clip paw tissue, quickly placed in 10% formalin solution, fixed claws organization; then fixed good paws organization moved to 10? the TA solution decalcification; the rinse after decalcification completed, dehydration, embedding into a wax block, the last slice and HE staining. ⑥ mPDLl-hIg treatment and therapy of human IgG to erythrocytes of CIA mice spleen cells after CFSE staining, then denatured calf type II collagen stimulation, flow cytometry mPDL1-hIg treatment of denatured calf type Ⅱ collagen to red blood cells stimulate the CIA mice spleen cell proliferation, ELISA assay mPDL1-hlg the CIA mice therapy denatured calf type II collagen stimulation of red blood cells, spleen cells express IL-17 and IL-23. ⑦ all data mean ± standard deviation, the difference between the two sets of data saliency detection U test. SPSS13.0 statistical analysis software processing data, p lt; 0.05 significant difference. Results ① The flow cytometry results show: mPDL1-hIg with activated normal mouse spleen cells to erythrocytes combination; in our experimental conditions, the binding rate is about 43.62%. ② proof of flow cytometry: mPDLl-hIg suppressed the denatured calf collagen type II stimulated proliferation of spleen cells of mice immunized with collagen type II derived from calf to erythrocytes; ELISA test results show that: mPDLl-hIg inhibit denaturation Bovine collagen type II stimulated spleen cells derived from the calf to erythrocytes of the mice immunized with collagen type II expression of IL-17 and IL-23. ③ mPDL1-hIg treatment can significantly reduce the the calf mice immunized with collagen type II clinical score, incidence not affected (incidence rate of the experimental group and the control group are 6/7 (86%)). The derived from mPDL1-hIg treatment of mouse and human the the HE staining IgG treated mice paw organization, mPDL1-hIg treatment can reduce joint pathological changes. These results demonstrate, mPDL1-hIg treatment can inhibit CIA aggravated Tip: pathological mechanisms of PD-1-PDL pathway involving CIA. ④ LISA test results show that the: mPDL1-hlg treatment can down-regulate the expression of IL-17 and IL-23 in CIA mice serum. ⑤ flow cytometry show: mPDL1-hlg denatured calf type II collagen stimulation therapy can be suppressed CIA mice to erythrocytes spleen cell proliferation. ELISA results showed that the denatured calf collagen type II CIA mice stimulated spleen cells to erythrocytes: mPDLl-the hIg treatment can be suppressed the expression of IL-17 and IL-23. These results suggest that: mPDLl-hlg treatment to through inhibition of cell proliferation and IL-17 and IL-23 expression to improve the CIA. The conclusion mPDL1-hIg can significantly improve the CIA, manifested in lower clinical scores, reduce joint pathological changes and lowered serum IL-17 and IL-23 expression. mPDL1-hIg through the inhibition of cell proliferation and of IL-17 and IL-23 expression and alleviate the CIA.

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