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尼古丁对大鼠气道平滑肌细胞内钙离子浓度及膜瞬时受体电位通道mRNA表达的影响

Effects of nicotine on intracellular Ca2+ concentration and transient receptor potential channels mRNA expression in rat airway smooth muscle cells

摘要:

目的 观察尼古丁刺激大鼠气道平滑肌细胞(ASMC)后细胞内基础钙离子浓度的改变及平滑肌细胞膜瞬时受体电位通道(TRPC1,TPRC6)mRNA表达水平变化.方法 不同浓度尼古丁[O(对照组)、10、20、50、100μg/L]刺激大鼠气道平滑肌细胞,作用24及48 h后,采用Incyte细胞内钙浓度检测系统观察细胞内钙离子浓度的变化.提取细胞总RNA,反转录成cDNA,实时定量PCR方法检测细胞TRPC1和TRPC6 mRNA表达量变化.结果 20、50、100μg/L的尼古丁刺激大鼠气道平滑肌细24和48 h后,细胞内基础钙离子浓度均较对照组明显升高[(117.99±19.39)、(122.89±17.91)、(124.70±17.93)nmol/L比(85.85±12.60)nmol/L;(142.07±18.99)、(162.27±19.91)、(207.30±26.56)nmol/L比(98.04±2.39)nmol/L,均P<0.05].而细胞中TRPC1和TPRC6 mRNA相对浓度也均较对照组显著升高(P<0.05),其中,100μg/L尼古丁刺激气道平滑肌细胞48 h后,细胞内基础钙离子浓度和TRPC6 mRNA相对表达量均较刺激24 h后的明显升高(P<0.05).结论 尼古丁可能通过上调大鼠气道平滑肌细胞膜瞬时受体电位通道TRPC1和TPRC6的表达而导致大鼠气道平滑肌细胞内基础钙离子浓度增加.

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abstracts:

Objective To study the changes of intracellular Ca2+ concentration and transient receptor potential channels 1 and 6(TRPC1,TPRC6)mRNA expression in rat airway smooth muscle cells (ASMC) after nicotine intervention.Methods Various concentrations of nicotine(10 μg/L,20 μg,L,50μg/L,100μg/L)were used to stimulate rat airway smooth muscle cells.The intracellular Ca2+ concentrations after nicotine intervention for 24 and 48 hours were measured by Incyte system.The total RNA was extracted from rat ASMC.and reversely transcribed into cDNA.The real-time PCR method was used to analyze the changes in TRPC1 and TRPC6 mRNA expression.Results The groups treated with different concentrations of nicotine(20 μg/L,50μg/L,100 μg/L)for 24 or 48 hours showed increases in intracellular Ca2+concentration when compared with control[(117.99±19.39),(122.89±17.91),(124.70±17.93)nmol/L vs (85.85±12.60)nmol/L;(142.07±18.99),(162.27±19.91),(207.30±26.56)nmol/L vs(98.04±2.39)nmol/L;P<0.05].In addition,relative expressions of TRPC1 and TPRC6 mRNA in rat ASMC also increased significantly compared with controls(P<0.05).The levels of intracellular Ca2+ concentration and TRPC6mRNA were significantly higher with 48 h nicotine(100 μg/L,L)intervention than as found with 24 h nicotineintervention.Conclusion Nicotine may increase the intracellular Ca2+ concentration by up-regulating the expression of TRPC1 and TPRC6 in rat ASMC.

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