TRPC1与硝苯地平及环孢素A诱导的牙龈增生的相关性研究
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摘要
目的探讨经典瞬时受体电位通道1(TRPC1)与硝苯地平(Nif)及环孢素A(CsA)单独及联合诱导的牙龈增生的相关性。方法一定浓度的Nif和CsA单独及联合作用于体外培养的人牙龈成纤维细胞(HGF),1、3、5、7 d后,CCK8法检测药物作用下HGF增殖活性的变化,实时荧光定量PCR和Western blot分别检测各组细胞TRPC1、增殖细胞核抗原(PCNA)、凋亡标记分子B淋巴细胞瘤-2(Bcl-2) mRNA和蛋白表达水平的变化及差异,免疫细胞化学染色法检测各组细胞TRPC1的表达变化及差异情况。结果 CCK8结果显示,Nif、CsA和Nif+CsA均可促进HGF的增殖;实时荧光定量PCR和Western blot结果表明,Nif和CsA单独及联合作用于HGF后均可显著上调Bcl-2和TRPC1的表达,但PCNA的表达并没有显著变化;免疫细胞化学染色法结果提示,TRPC1在Nif和CsA单独及联合刺激的细胞中其表达量明显升高。结论 HGF在Nif和CsA单独及联合作用下,其TRPC1的表达量均明显上升,提示TRPC1可能在药物性牙龈增生的发病过程中发挥一定的作用。
Objective To investigate the correlation between classical transient receptor potential channel 1(TRPC1) and gingival overgrowth iuduced by nifedipine(Nif) and cyclosporin A(CsA) alone or in combination.Methods The certain concentration of Nif and CsA was used alone or in combination to stimulate human gingival fibroblasts(HGF) in vitro. After 1 day, 3 days, 5 days, and 7 days, CCK8 method was used to detect the proliferation of HGF. The mRNA and protein expression of TRPC1, proliferating cell nuclear antigen(PCNA) and apoptosis marker molecule B-cell lymphoma-2(Bcl-2) were detected by Real-Time qPCR and Western blot.The expression of TRPC1 in each group was detected by ICC method.Results CCK8 results showed that Nif and CsA alone or in combination could promote the proliferation of HGF;Real-Time qPCR and Western blot results showed that Nif and CsA alone or in combination could up-regulated the expression of Bcl-2 and TRPC1, but not PCNA. ICC results suggested that the expression of TRPC1 in HGF stimulated by Nif and CsA alone or in combination was significantly increased.Conclusion The expression of TRPC1 is increased by Nif and CsA alone and in combination, suggesting that TRPC1 may play a role in the pathogenesis of drug-induced gingival overgrowth.
Objective To investigate the correlation between classical transient receptor potential channel 1(TRPC1) and gingival overgrowth iuduced by nifedipine(Nif) and cyclosporin A(CsA) alone or in combination.Methods The certain concentration of Nif and CsA was used alone or in combination to stimulate human gingival fibroblasts(HGF) in vitro. After 1 day, 3 days, 5 days, and 7 days, CCK8 method was used to detect the proliferation of HGF. The mRNA and protein expression of TRPC1, proliferating cell nuclear antigen(PCNA) and apoptosis marker molecule B-cell lymphoma-2(Bcl-2) were detected by Real-Time qPCR and Western blot.The expression of TRPC1 in each group was detected by ICC method.Results CCK8 results showed that Nif and CsA alone or in combination could promote the proliferation of HGF;Real-Time qPCR and Western blot results showed that Nif and CsA alone or in combination could up-regulated the expression of Bcl-2 and TRPC1, but not PCNA. ICC results suggested that the expression of TRPC1 in HGF stimulated by Nif and CsA alone or in combination was significantly increased.Conclusion The expression of TRPC1 is increased by Nif and CsA alone and in combination, suggesting that TRPC1 may play a role in the pathogenesis of drug-induced gingival overgrowth.
引文
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[8] 王艳春.低浓度硝苯地平诱导人牙龈上皮细胞bcl-2,bax及P53表达的实验研究[D].昆明:昆明医科大学口腔医学院,2014.
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[10] 文海燕.硝苯地平对人牙龈上皮细胞增殖和凋亡的影响[D].上海:上海交通大学口腔医学院,2009.
[11] Strubing C,Krapivinsky G,Krapivinsky L,et al.TRPC1 and TRPC5 form a novel cation channel in mammalian brain[J].Neuron,2001,29(3):645-55.
[12] Yang J,Cai W,Lu X,et al.RNA-sequencing analyses demonstrate the involvement of canonicaltransient receptor potential channels in rat tooth germ development[J].Front Physiol,2017,8:455.
[13] Ambudkar I S.TRPC1:a core component of store-operated calcium channels[J].Biochem Soc Trans,2007,35(Pt 1):96-100.
[2] Pamuk F,Cetinkaya B O,Gulbahar M Y,et al.Effects of Tacrolimus and Nifedipine,Alone or in Combination,on Gingival Tissues[J].J Periodontol,2013,84(11):1673-82.
[3] Butler R T,Kalkwarf K L,Kaldahl W B,et al.Drug-induced gingival hyperplasia:phenytoin,cyclosporine,and nifedipine[J].J Am Dent Assoc,1987,114(1):56-60.
[4] Arora P D,Silvestri L,Ganss B,et al.Mechanism of cyclosporin-induced inhibition of intracellular collagen degradation[J].J biol chem,2001,276(17):14100-9.
[5] Cai S,Fatherazi S,Presland R B,et al.Evidence that TRPC1 contributes to calcium-induced differentiation of human keratinocytes[J].Pflugers Arch,2006,452(1):43-52.
[6] Hattori T,Wang P L.Elevation of cytosolic calcium level triggers calcium antagonist-induced gingival overgrowth[J].Eur J Pharmacol,2008,583(1):37-9.
[7] Cotrim P,Martelli-Junior H,Graner E,et al.Cyclosporin a induces proliferation in humangingival fibroblasts via induction of transforming growth factor-β1[J].J Periodontol,2003,74(11):1625-33.
[8] 王艳春.低浓度硝苯地平诱导人牙龈上皮细胞bcl-2,bax及P53表达的实验研究[D].昆明:昆明医科大学口腔医学院,2014.
[9] Takeuchi R,Matsumoto H,Akimoto Y,et al.Reduction in lipopolysaccharide induced apoptosis of fibroblasts obtained from a patient with gingival overgrowth during nifedipine-treatment[J].Arch Oral Biol,2011,56(10):1073-80
[10] 文海燕.硝苯地平对人牙龈上皮细胞增殖和凋亡的影响[D].上海:上海交通大学口腔医学院,2009.
[11] Strubing C,Krapivinsky G,Krapivinsky L,et al.TRPC1 and TRPC5 form a novel cation channel in mammalian brain[J].Neuron,2001,29(3):645-55.
[12] Yang J,Cai W,Lu X,et al.RNA-sequencing analyses demonstrate the involvement of canonicaltransient receptor potential channels in rat tooth germ development[J].Front Physiol,2017,8:455.
[13] Ambudkar I S.TRPC1:a core component of store-operated calcium channels[J].Biochem Soc Trans,2007,35(Pt 1):96-100.