CsA和TNF-α对体外培养GFs增殖和胶原代谢的影响
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摘要
目的:研究环孢霉素A(CsA)和肿瘤坏死因子α(TNF-α)对体外培养人牙龈成纤维细胞(GFs)增殖和胶原合成的影响。探讨牙龈炎症与药物性牙龈增生的关系及CsA所致牙龈增生的相关机理。
方法:用原代培养的方法获取正常人牙龈成纤维细胞,体外培养、传代后,取生长良好的4～8代用于实验。按以下条件进行实验分组:A:空白对照组B_1:10ug/L CsA,B_2:50 ug/L CsA,B_3:250 ug/LCsA,B_4:1250 ug/L CsA,C:5ug/L TNF-α,D_1:10 ug/L CsA+5ug/LTNF-α,D_2:50ug/L CsA+5ug/L TNF-α,D_3:250ug/LCsA+5ug/LTNF-α,D_4:1250 ug/L CsA+5ug/L TNF-α。将条件培养液分别作用于牙龈成纤维细胞,培养3,5,7天后用MTT法测定细胞的增殖情况,并分别于第3,5,7天收集培养上清液,用羟脯氨酸法测定胶原合成情况。
结果:原代培养成功率为80%,细胞呈梭形,排列成条索状,漩涡状。细胞经CsA和TNF-α作用后形态无明显变化。不同浓度的CsA作用于成纤维细胞后,细胞的增殖受到抑制,A值下降,其中B_1、B_2、B_3组与对照组无显著差别,B4组与对照组相比差异具有统计学意义。5ug/L的TNF-α作用于成纤维细胞可以刺激细胞的增殖,A值与对照组相比显著升高(P＜0.01)。CsA和TNF-α共同作用于成纤维细胞后,D_1、D_2、D_3组A值较A组升高,但较C组降低。D4组细胞增殖显著增加,与C组相比差异具有统计学意义。
不同浓度的CsA作用于成纤维细胞后,上清液中胶原含量增加,且随着浓度的升高和培养时间延长,增加越明显。5ug/L的TNF-α作用于成纤维细胞后,上清液中胶原含量降低,与对照组相比差异具有统计学意义。CsA和TNF-α共同作用于成纤维细胞时,上清液中胶原含量增加,D_3、D_4组分别与B_3、B_4组相比,差异具有统计学意义,但D_1、D_2与B_1、B_2组相比,差异没有统计学意义。
结论:本实验通过体外培养人牙龈成纤维细胞,将不同浓度的CsA和TNF-α作用于细胞,观察细胞增殖和胶原合成的变化。结果表明:
1.CsA对成纤维细胞的增殖没有促进作用,高浓度时抑制细胞的增殖。CsA可以刺激细胞的胶原合成,这可能是CsA引起牙龈增生的主要机制。
2.高浓度CsA与TNF-α共同作用于成纤维细胞时,促进成纤维细胞的增殖。提示在一定浓度下CsA可能放大TNF-α刺激成纤维细胞增殖的效应。
3.一定浓度的CsA和TNF-α共同作用于成纤维细胞时,促进胶原含量的增加。提示TNF-α可能增加了成纤维细胞对CsA的敏感性。
Objective:Study the effect of cyclosporine A(CsA)and tumor necrosis factorα(TNF-α)on cell proliferation and collagen biosynthesis of cultured human gingival fibroblasts(GFs),to found the relationship between gingival inflammation and drug-induced gingival overgrowth,and the mechanisms of gingival overgrowth induced by CsA.
Methods:Human GFs were cultured in vitro using a method of tissue culture,then cells from the 4~(th)～8~(th)passage were used in experiment,cells were cultured and incubated with various concentrations of CsA and TNF-α(A:blank group,B_1:10 ug/L CsA,B_2:50 ug/L CsA,B_3:250 ug/L CsA,B_4:1250 ug/L CsA,C:5 ug/L TNF-α,D_1:10 ug/L CsA +5 ug/L TNF-α,D_2:50 ug/L CsA +5 ug/L TNF-α,D_3:250 ug/L CsA +5ug/L TNF-α,D_4:1250ug/L CsA +5 ug/L TNF-α)solution for 3,5,7days.MTT assay and hydroxyproline reagcuat were used to evaluate the cell proliferation and collagen production in the culture meidiun.
Result:The success rate of primary culture of human gingival fibroblasts was up to 80%,the cells were spindle-shaped,arranged in trabs or swirls.The cells had no morphological changes after exposure. When exposure to CsA of different concentration,the proliferation of fibroblasts was inhibited.A value decreased,group B_1,B_2,B_3 had no significant difference with control group,while the A value of group B_4 was significantly higher than control group(P＜0.01).Fibroblast proliferation was significantly increased while cultured with 5ug/L TNF-α.A value increased(P＜0.01).When exposure to CsA+TNF-α,A value of group D_1、D_2、D_3 were much higher than group A,but were lower than group C.Cell proliferation in group D_4 was significantly increased,the difference with group C had statistical significance.
When exposure to CsA of different concentration,collagen content in supernatant was increased,and the growth rate was positively correlated with CsA concentrations and time.Collagen content was decreased When exposure to 5ug/L TNF-α,and the difference with control group had statistical significance.When exposure to CsA+TNF-α,Collagen content in group D_3、D_4 was increased significantly compared to group B_3、B_4(P＜0.01),but there were no significant differences group D_1 and B_1,D_2 and B_2.
Conclusion:This study was to investigate the effect of CsA and TNF-αon cell proliferation and collagen biosynthesis of cultured human GFs.The results showed that:
1.CsA did not simulate the cell proliferation,but high-concentration CsA inhibited cell proliferation.CsA had an enhancement effect on the collagen synthesis.This may be the main mechanism by which CsA induced gingival hyperplasia.
2.High-concentration CsA+TNF-αcan enhance the fibroblast proliferation,which suggests that CsA in certain concentration have amplification effect on TNF-αto stimulate fibroblast proliferation.
3.High-concentration CsA+TNF-αcan enhance collagen synthesis, which suggests that fibroblasts are more sensitive when exposed to TNF-α.
方法:用原代培养的方法获取正常人牙龈成纤维细胞,体外培养、传代后,取生长良好的4～8代用于实验。按以下条件进行实验分组:A:空白对照组B_1:10ug/L CsA,B_2:50 ug/L CsA,B_3:250 ug/LCsA,B_4:1250 ug/L CsA,C:5ug/L TNF-α,D_1:10 ug/L CsA+5ug/LTNF-α,D_2:50ug/L CsA+5ug/L TNF-α,D_3:250ug/LCsA+5ug/LTNF-α,D_4:1250 ug/L CsA+5ug/L TNF-α。将条件培养液分别作用于牙龈成纤维细胞,培养3,5,7天后用MTT法测定细胞的增殖情况,并分别于第3,5,7天收集培养上清液,用羟脯氨酸法测定胶原合成情况。
结果:原代培养成功率为80%,细胞呈梭形,排列成条索状,漩涡状。细胞经CsA和TNF-α作用后形态无明显变化。不同浓度的CsA作用于成纤维细胞后,细胞的增殖受到抑制,A值下降,其中B_1、B_2、B_3组与对照组无显著差别,B4组与对照组相比差异具有统计学意义。5ug/L的TNF-α作用于成纤维细胞可以刺激细胞的增殖,A值与对照组相比显著升高(P＜0.01)。CsA和TNF-α共同作用于成纤维细胞后,D_1、D_2、D_3组A值较A组升高,但较C组降低。D4组细胞增殖显著增加,与C组相比差异具有统计学意义。
不同浓度的CsA作用于成纤维细胞后,上清液中胶原含量增加,且随着浓度的升高和培养时间延长,增加越明显。5ug/L的TNF-α作用于成纤维细胞后,上清液中胶原含量降低,与对照组相比差异具有统计学意义。CsA和TNF-α共同作用于成纤维细胞时,上清液中胶原含量增加,D_3、D_4组分别与B_3、B_4组相比,差异具有统计学意义,但D_1、D_2与B_1、B_2组相比,差异没有统计学意义。
结论:本实验通过体外培养人牙龈成纤维细胞,将不同浓度的CsA和TNF-α作用于细胞,观察细胞增殖和胶原合成的变化。结果表明:
1.CsA对成纤维细胞的增殖没有促进作用,高浓度时抑制细胞的增殖。CsA可以刺激细胞的胶原合成,这可能是CsA引起牙龈增生的主要机制。
2.高浓度CsA与TNF-α共同作用于成纤维细胞时,促进成纤维细胞的增殖。提示在一定浓度下CsA可能放大TNF-α刺激成纤维细胞增殖的效应。
3.一定浓度的CsA和TNF-α共同作用于成纤维细胞时,促进胶原含量的增加。提示TNF-α可能增加了成纤维细胞对CsA的敏感性。
Objective:Study the effect of cyclosporine A(CsA)and tumor necrosis factorα(TNF-α)on cell proliferation and collagen biosynthesis of cultured human gingival fibroblasts(GFs),to found the relationship between gingival inflammation and drug-induced gingival overgrowth,and the mechanisms of gingival overgrowth induced by CsA.
Methods:Human GFs were cultured in vitro using a method of tissue culture,then cells from the 4~(th)～8~(th)passage were used in experiment,cells were cultured and incubated with various concentrations of CsA and TNF-α(A:blank group,B_1:10 ug/L CsA,B_2:50 ug/L CsA,B_3:250 ug/L CsA,B_4:1250 ug/L CsA,C:5 ug/L TNF-α,D_1:10 ug/L CsA +5 ug/L TNF-α,D_2:50 ug/L CsA +5 ug/L TNF-α,D_3:250 ug/L CsA +5ug/L TNF-α,D_4:1250ug/L CsA +5 ug/L TNF-α)solution for 3,5,7days.MTT assay and hydroxyproline reagcuat were used to evaluate the cell proliferation and collagen production in the culture meidiun.
Result:The success rate of primary culture of human gingival fibroblasts was up to 80%,the cells were spindle-shaped,arranged in trabs or swirls.The cells had no morphological changes after exposure. When exposure to CsA of different concentration,the proliferation of fibroblasts was inhibited.A value decreased,group B_1,B_2,B_3 had no significant difference with control group,while the A value of group B_4 was significantly higher than control group(P＜0.01).Fibroblast proliferation was significantly increased while cultured with 5ug/L TNF-α.A value increased(P＜0.01).When exposure to CsA+TNF-α,A value of group D_1、D_2、D_3 were much higher than group A,but were lower than group C.Cell proliferation in group D_4 was significantly increased,the difference with group C had statistical significance.
When exposure to CsA of different concentration,collagen content in supernatant was increased,and the growth rate was positively correlated with CsA concentrations and time.Collagen content was decreased When exposure to 5ug/L TNF-α,and the difference with control group had statistical significance.When exposure to CsA+TNF-α,Collagen content in group D_3、D_4 was increased significantly compared to group B_3、B_4(P＜0.01),but there were no significant differences group D_1 and B_1,D_2 and B_2.
Conclusion:This study was to investigate the effect of CsA and TNF-αon cell proliferation and collagen biosynthesis of cultured human GFs.The results showed that:
1.CsA did not simulate the cell proliferation,but high-concentration CsA inhibited cell proliferation.CsA had an enhancement effect on the collagen synthesis.This may be the main mechanism by which CsA induced gingival hyperplasia.
2.High-concentration CsA+TNF-αcan enhance the fibroblast proliferation,which suggests that CsA in certain concentration have amplification effect on TNF-αto stimulate fibroblast proliferation.
3.High-concentration CsA+TNF-αcan enhance collagen synthesis, which suggests that fibroblasts are more sensitive when exposed to TNF-α.
引文
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