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Mechanism of Effects of Recombinant Human Endostatin on Fibroblast-like Synoviocyte in Rats with Adjuvant Arthritis

Author: HuangXueYing
Tutor: ChenFeiHu
School: Anhui Medical University,
Course: Pharmacology
Keywords: Adjuvant arthritis Fibroblast-like synovial cells Recombinant human endostatin Cell cycle Apoptosis Gene Expression Calcium
CLC: R96
Type: PhD thesis
Year: 2008
Downloads: 154
Quote: 0
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Abstract


Rheumatoid arthritis (rheumatoid arthritis, RA) is a chronic autoimmune disease, the average worldwide prevalence of about 1%, of the prevalence of about 4 ‰; prominent clinical manifestations of joint swelling, pain , stiffness and deformity; There is no effective treatment, the aim of the current treatment is to relieve symptoms, prevent joint damage and loss of function, improve quality of life. The pathogenesis of RA is unknown, its main pathological features excessive proliferation of fibroblast-like synovial cells (fibroblast-like synoviocytes, FLS), inflammatory cell infiltration and neovascularization; proliferation of synovial tissue constitute pannus erosion of articular cartilage and bone cause joint destruction, deformity and loss of function. Therefore, synovial hyperplasia plays an important role in the development of rheumatoid synovitis. Recent studies have shown that cell proliferation and cell death are closely related to RA FLS proliferation and death perhaps there is an imbalance, which led to the synovial hyperplasia. Therefore, inhibition of RA FLS proliferation, induced apoptosis of synovial hyperplasia reducing effective strategy for the treatment of RA. Freund's complete adjuvant (Freund's complete adjuvant, FCA) induced adjuvant arthritis (adjuvantarthritis, AA) is an experimental arthritis model to similar joint pathological changes and cellular and humoral immunity with RA, can be used as evaluation RA treatment of the test system. Endostatin (endostatin) is a potent endogenous angiogenesis inhibitor, molecular weight of about 20kD degradation product of collagen Ⅹ Ⅷ, its anti-angiogenic activity of the mechanism may be specific inhibition of vascular endothelial cell adhesion, migration, and proliferation and induce apoptosis. Recent studies have confirmed that endostatin not only by inhibition of endothelial cell function (ie inhibition of tumor angiogenesis), but also play the anti-tumor effect by direct inhibition of tumor cell migration, proliferation and induce apoptosis. FLS RA pathology change the ultimate target cells and their proliferation activity, having the characteristics of the tumor transformed cells, RA synovial tissue similar limitations invasion and the growth of tumors, suggesting that the pathway can be used for the treatment of RA of the treatment of neoplastic diseases, including apoptosis-inducing factor and anti-angiogenic factors. Endostatin research focused on the anti-tumor activity and mechanism of the impact at home and abroad, such as chronic inflammatory vascular generated few reports of other pathologic angiogenesis; FLS target, to explore the role and mechanism of endostatin on RA has not been reported. Our previous studies have shown that abdominal subcutaneous injection of recombinant human endostatin (recombinant human endostatin, rhEndostatin; (1.25,2.5,5.0 mg · kg -1 · d -1 , × 7d) rat AA treatment, significantly reduced the AA rat paw edema degree of to reduce synovial tissue vascular endothelial (cell) growth factors (vascular endothelial growth factor, VEGF) expression, reduce synovial microvascular density ( microvessel density, MVD). confirmed by further studies, AA FLS rhEndostatin at (3.125,6.25,12.5,25,50 μg / ml) can directly inhibit proliferation and induce apoptosis, but the specific mechanism of action is unclear. research is on this basis on further explore the the potential mechanisms rhEndostain AA FLS effect, to provide experimental evidence for RA treatment and to find its new target for therapy. the the: ① to observe rhEndostatin AA FLS cell cycle; ② observed rhEndostatin AA FLS cell cycle related genes p53 of p21, CDK 4 , of cyclinD 1 PCNAmRNA and CyclinD 1 , PCNA in protein expression; ③ of observation rhEndostatin on the AA FLS apoptosis genes fas, bcl-2, c-fos and c-jun mRNA and c-Jun, NFκB, Caspase-3 protein expression; of ④ observed rhEndostatin on AA FLS cytoplasmic free calcium (ionized free calcium of Ca ). explore AA FLS proliferation rhEndostatin inhibition, molecular and ionic mechanisms that promote apoptosis, and provide experimental evidence for treatment of RA drugs and looking for its treatment of the new target method: ① AA model in rats: male SD rats, weighing 140-160g, Experimental Animal Center of Anhui Medical University, after toe intradermal injection the FCA preparation AA rat model. ② synovial tissue preparation: The rats were randomly divided into normal group, the AA model group, rhEndostatin (2.5mg/kg) treatment group and methotrexate (methotrexate, MTX; (1mg/kg) treatment of the control group; days 10-16 after three sets after modeling, rhEndostatin treatment of rats injected subcutaneously rhEndostatin (2.5 -1 · d mg · kg -1 ), the MTX group rats were injected subcutaneously MTX (1 mg / kg twice weekly), AA model group and normal rats given isodose water for injection; rats were sacrificed on day 28, remove the tissue of the synovial layer. ③ FLS preparation: application of the normal and AA of fresh rat knee synovial tissue for cell culture experiments using cultured synovial cell passage 2, AA in synovial cells of vascular cell adhesion molecule -1 (vascular cell adhesion molecule-1, VCAM-1) expression situation identified FLS. ④ FLS cell cycle detection detected by flow cytometry (flow cytometry, FCM): passage 1 normal and AA rats synovial cells were seeded in 6-well plates the AA in synovial cells divided AA model group rhEndostatin (50μg/ml) treatment group, MTX (1μg/ml) treatment group, serum-free culture in RPMI-1640 culture solution cultured 24h, cell cycle synchronization, followed replaced with RPMI-1640 culture medium containing 20% ??serum, and accordingly to join rhEndostatin (final concentration 50μg/ml) and MTX (final concentration 1μg/ml) cells were collected, the the FCM detection cell cycle. ⑤ cell cycle-related genetic testing, continue to cultivate 48h: were extracted from rats synovial tissue RNA, proteins; real-time quantitative PCR assay of p53, p21, of cyclinD the 1 < / sub>, PCNA, CDK 4 mRNA expression of PCNA, CyclinD 1 protein; Western Blot detection (6) of apoptosis-related genetic testing: real-time PCR method to detect the rats synovial tissue expression of fas, bcl-2, c-fos and c-jun mRNA expression; Western Blot detection of c-Jun of NFκB, Caspase-3 protein expression. the ⑦ AA FLS cytoplasmic Ca 2 detection: Fluo-3/AM as Ca 2 indicator, the final concentration of 50μg/ml rh-Endostatin perfused AA FLS, at the same time using a confocal laser scanning microscope (confocal laser scanningmicroscope, CLSM) dynamic observation record AA FLS Ca 2 the liquid and Ca 2 solution the cytoplasm of Ca 2 changes in fluorescence intensity (fluorescence intensity, FI), the the detection rhEndostatin of AA FLS cytoplasmic Ca 2 concentration (cytosolic free in calcium concentration [Ca 2 ] i ), impact results the: ① rhEndostatin on rat FLS cell cycle: FCM detection and analysis of the results showed that the the the normal rats FLS cell cycle mainly stay in the G 0 / G 1 of 68.3% fewer S phase and G 2 / M phase cells were 23.65%, 6.32%; compared with the normal group, the AA model rats FLS G 1 of significantly reduced the proportion of about 10.0%, most of the cells into the S phase, 65.6%, the G 2 / M phase cells account for nearly 19.5%; join rhEndostatin (50μg/ml), G 1 phase cells by the AA model group, 10.0% to 74.1%, while the S phase and G significantly reduced 2 / M phase cell proportion, 12.6% and 4.15%, respectively. ② rhEndostatin on AA in synovial tissue in cell cycle-related gene expression: real-time quantitative PCR results, rhEndostatin (2.5 mg / kg) significantly reduced the AA in synovial tissue of p53, of p21 of cyclinD 1 < / sub> and PCNA mRNA expression levels (P <0.01), increased CDK 4 mRNA expression (P <0.01). confirmed by Western Blot, rhEndostatin treatment group rat the PCNA and CyclinD synovial tissue < sub> 1 protein expression than the AA model group was significantly lower (P <0.01). ③ rhEndostatin apoptosis-related genes in the AA synovium: real-time quantitative PCR analysis showed that, rhEndostatin (2.5mg / kg) significantly increased the AA rats synovial tissue expression of fas, bcl-2, c-fos and c-jun mRNA expression levels (P <0.01). further confirmed by Western Blot, the application rhEndostatin after treatment, rats synovial organization 43kD and 48kD c-Jun protein of Caspase-3 p20 expression level than AA model group significantly significantly improve (P <0.01); but of NFκB of p65 expression than the AA model group increase was not obvious (P> 0.05) ④ rhEndostatin of AA FLS [ Ca 2 ] i impact: CLSM test results show that, when the cells in the the Ca 2 solution rhEndostatin (50μg/ml) can not caused the the AA the FLS cytoplasmic Ca 2 the Fi of change. When the extracellular buffer to have Ca 2 solution, final concentration of the 50μg/ml rhEndostatin perfusion AA FLS allows extracellular quality of of Ca 2 FI increased dramatically, reached the peak, then the fluorescence began to weaken, FI slow decline over time. Conclusions: ① rhEndostatin inhibit AA FLS proliferation and its cause AA FLS cell cycle G of 1 of block on the basis of the molecular biology may be rhEndostatin inhibit AA FLS CyclinD 1 and PCNA expression to promote AA FLS apoptosis is not dependent on the classical pathway of P53-P21-CDK-Cyclin. ② rhEndostatin their increased AA FLS fas, c-fos, c-jun, caspase-3 gene expression and activation of the apoptotic process does not depend on bcl-2 expression decreased, can not be bcl-2 expression and NFkB activation inhibited. ③ rhEndostatin can cause AA FLS extracellular Ca 2 influx of calcium homeostasis is an early event of rhEndostatin evoked AAFLS damage, intracellular calcium overload might as AA FLS apoptosis initiating an important link.

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