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The Effects of Interfering Ca2+ on TNF-α Induced Rat Lung Fibroblast Proliferation

Author: XiDanDan
Tutor: LiSuPing
School: Shanxi Medical
Course: Occupational and Environmental Health
Keywords: Tumor necrosis factor -α Nimodipine Protein kinase A Central - adenosine triphosphate G_q protein Ca2
CLC: R135.2
Type: Master's thesis
Year: 2010
Downloads: 44
Quote: 0
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Abstract


Objective: To investigate the Ca2 antagonist nimodipine (Nim) rat lung fibroblast proliferation, induction of tumor necrosis factor-alpha (TNF-a) provide a scientific basis for the mechanism of pneumoconiosis. Methods: SD rats suckling rat lung fibroblast cells in primary culture and subculture spread to 4-5 generations, with different concentrations of TNF-α stimulate fibroblasts to cultivate a certain time, fibroblasts using Western blot analysis Ca2 content fibroblast cell Gq protein expression; using flow cytometry. TNF-α combined with different concentrations of calcium antagonist nimodipine (Nim) stimulates fibroblasts to cultivate a certain time, the content of hydroxyproline in the fiber cells detected using the chloramine T method; using enzyme-linked immunosorbent assay (ELISA ) Determination of fibroblast inner ring - adenosine monophosphate (cAMP) content fibroblast protein kinase A (PKA); fluorescent immunohistochemical techniques to detect the expression. Results: Western blot results show: When different concentrations of the role of TNF-α in the rat lung fibroblasts in 4h point in time, with increasing doses of TNF-α, Gq protein expression levels in each experimental group was significantly liter high, and the differences between groups were significant (P lt; 0.05). In the 24h time point, the Gq protein expression levels in each experimental group than in the control group was significantly higher (P lt; 0.05). 20ng/ml TNF-α group Gq protein expression compared 10ng/ml and 5ng/mlTNF-a, group, the difference was statistically significant (P lt; 0.05). 4h low 10ng/ml and 20ng/mlTNF-a of Gq protein expression, the difference was significant (P lt; 0.05). 2. Intracellular free Ca2 results show: when the role of the different concentrations of TNF-a in fibroblasts, the fluorescence intensity of each experimental group (F) values ??significantly higher than those in the control group (P lt; 0.05) 10ng/mlTNF-a Group fluorescence intensity (F) value compared 5ng/ml and 20ng/mlTNF-a group, the difference was significant (P lt; 0.05). Hydroxyproline content results: at different time points, compared with the blank control group, the hydroxyproline content of each experimental group were significantly higher (P lt; 0.05). With the increase in the duration of action, hydroxyproline content was significantly higher (P <0.05) .40μmol/LNim TNF-a group and 80μmol/LNim TNF-a group hydroxyproline content than TNF-a group (P lt; 0.05). To combine different Nim group in 4h time point, TNF-a, reduce as Nim dose increase in hydroxyproline content of each experimental group, and the differences between groups were significant (P lt; 0.05). Hydroxyproline content in 24h time point, 40μmol/LNim TNF-α group than 20μmol / the L NimTNF-a group (P lt; 0.05). 4.cAMP content results show: at different time points, compared with the blank control group, the cAMP content of each experimental group were significantly higher (P lt; 0.05). 40gmol/LNim TNF-a group and cAMP content of TNF-a 80μmol/LNim group than individually TNF-a group and TNF-a 20gmol/LNim group, the differences were statistically significant (P lt; 0.05). 5.PKA results show: 4h time point, 40gmol/LNim TNF-a group and 80gmol/LNim TNF-a mean optical density value compared to the average optical density 80μmol/LNim TNF-a separate TNF-a group compared 20gmol / LNim TNF-a group, the differences were statistically significant (P lt; 0.05). In the 24h time point 80μmol/Lnim TNF-a group of average optical density value than the separate TNF-a group, 20μmol/LNim TNF-a group 40μmol/LNim TNF-a group, the difference was significant resistance (P lt; 0.05) . Conclusion: 1.TNF-a rat lung into Gq protein expression in fibroblasts and expression of Ca2 role in promoting and possibly through the the Gq-Ca2 signal transduction pathway affects proliferation of fibroblasts. 2 in the role of TNF-a in the rat lung fiber, calcium antagonist nimodipine can make hydroxyproline decreased expression of cAMP and PKA expression was increased, suggesting that calcium antagonist nimodipine may be via the cAMP-PKA pathway affects proliferation of rat lung fibroblasts.

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CLC: > Medicine, health > Preventive Medicine,Health > Industrial Hygiene and > Occupational disease prevention > Production of dust and pneumoconiosis
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