纤溶酶原激活物抑制因子-1对肝星状细胞活化及其细胞外基质的影响
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摘要
研究目的肝纤维化已经成为严重危害人类健康的疾患,肝星状细胞活化是肝纤维化的中心环节,而肝纤维化形成关键是基质的产生。转化生长因子(transforming growth factor -β1,TGFβ1)已知是促进肝纤维化最强的细胞因子,它可促进肝星状细胞(hepatic stellate cell,HSC)活化和增殖,促进细胞外基质的产生。HSC通过产生细胞因子影响着肝纤维化的发生和发展。有研究表明HSC可表达纤溶酶原激活物抑制因子-1(plasminogen activator inhibitor type-1,PAI-1),参与肝纤维化的发生发展,TGFβ1可促进HSC PAI-1的表达。外源性PAI-1能否影响肝星状细胞TGFβ1表达,以及细胞外基质的合成目前尚不清楚。本实验通过体外培养肝星状细胞,研究外源性PAI-1对肝星状细胞活化及细胞外基质的影响。
研究方法人肝星状细胞LX-2贴壁细胞培养;MTT法测定PAI-1对LX-2细胞的最佳干预浓度;采用ELISA法测定细胞上清液中透明质酸(hyaluronic acid,HA)、TGFβ1、基质金属蛋白酶抑制抑制因子-1(tissue inhibitor of metalloproteinase,TIMP-1);免疫细胞化学法检测ɑ-平滑肌肌动蛋白(α-smooth muscle actin,ɑ-SMA)、基质金属蛋白酶-2(matrixmetallo proteinase-2 ,MMP-2)、TIMP-1、PAI-1、尿激酶型纤溶酶原激活物(urokinase plasminogen activator, uPA)蛋白表达;逆转录聚合酶链反应(reverse transcription ploymerase chain reaction, RT-PCR)检测TGFβ1及PAI-1mRNA的表达,并分析两者相关关系。
结果1、PAI-1对LX-2细胞增殖刺激作用明显,且PAI-1浓度为10ng/ml时刺激作用最为明显,A492为1.636±0.315(F=11.697, p<0.01)。
2、ɑ-SMA在LX-2胞浆中表达,核内无明显表达。PAI-1干预12h组蛋白表达量为(0.408±0.031)较对照组(0.301±0.015)增加明显(t=5.438, p<0.01)。
3、MMP-2、TIMP-1、PAI-1、uPA在LX-2胞浆中表达,核内无明显表达。PAI-1干预12h组MMP-2蛋白表达量为(0.245±0.028)较对照组(0.233±0.041)仅轻微增加(t=0.473, P>0.05);uPA 12h组为(0.303±0.052)较对照组(0.288±0.036)增加不明显(t=0.581, P>0.05);PAI-1 12h组为(0.331±0.045)较对照组(0.205±0.007)增加显著(t=4.857,P<0.01);TIMP-112h组为(0.412±0.031)较对照组(0.235±0.011)增加显著(t=9.511,P<0.01)。
4、PAI-1干预12h后TGFβ1 mRNA、PAI-1mRNA分别为(1.839±0.405)(,2.325±0.970),对照组分别为(1.090±0.123),(0.790±0.156),两者相比差异显著。且TGFβ1 mRNA与PAI-1 mRNA呈正相关(r=0.827,P<0.05)。
5、在细胞上清液中加入PAI-1 12h,24h,48h后(刺激组)TGFβ1蛋白表达量分别为(8.854±3.168)、(44.348±8.415)、(225.819±12.797)pg/ml ,对照组为(0.855±0.744)pg/ml ,干预组较阴性对照组增加显著(F=1566.752,P<0.01);HA分别为(2.577±0.801)、(18.177±7.141)、(50.473±4.903)ng/ml ,对照组(0.450±0.154 )ng/ml,干预组较阴性对照组增加显著(F=235.632,P<0.01);TIMP-1分别为(2.872±1.396),(20.409±11.821),(96.548±27.154)ng/ml,对照组(0.573±0.179)ng/ml,干预组较阴性对照组有统计学意义(F=67.359,P<0.05)。
结论PAI-1可刺激肝星状细胞活化,增加TGFβ1表达,增加细胞外基质合成,促进肝纤维化发生发展。
Objective Liver fibrosis has become the disease hazard severely the health of people,which the activity of hepatic stellate cell(HSC)is the center element and the development of extracellular matrix (ECM)is criticality.As is known, transforming growth factorβ1(TGFβ1) is the strongest cytokine to promote liver fibrosis.It not only can promote HSC’activation and proliferation,but also promote the development of ECM,which result in the development of liver fibrosis.Recently it has been found, HSC express the cytokines of plasminogen activator inhibitor-1(PAI-1)which can anticipate in the development of liver fibrosis,While TGFβ1 could induce the expression of PAI-1.But it has not been known that whether PAI-1 can influence the expression of TGFβ1 and development of ECM.So by means of culturing the HSC in vitro, we investigate the effect of PAI-1 on TGFβ1 and ECM.
Methods The proliferation effect of PAI-1 on LX-2 was detected by Methyl thiazolyl tetrazolium(MTT).After incubation with PAI-1 12h,24h,48h,hyaluronic acid(HA), TGFβ1, tissue inhibitor of metalloproteinase -1(TIMP-1) in LX-2 supernatant was observed with ELISA,the protein expression level of smooth muscleɑ-actin(ɑ-SMA),matrix metalloproteinase-2(MMP-2), tissue inhibitor of metalloproteinase -1(TIMP-1), urokinase-type plasminogen activator(uPA)and PAI-1 were detected by immunocytochemistry and the level of PAI-1,TGFβ1 mRNA by reverse transcription polymerase chain reaction (RT-PCR).
Results (1)PAI-1 can induce activation and proliferation of LX-2 effectively,and the best concentration is 10ng/ml.
(2)The protein expression ofɑ-SMA is in the cytoplasm of LX-2. The expression in the PAI-1 treatment 12h group i(s0.408±0.031),negative control group i(s0.301±0.015). Compared to negative control group, the expression ofɑ-SMA increased significantly(t=5.438, p<0.01).
(3) The protein expression of MMP-2、TIMP-1、PAI-1、uPA is in the cytoplasm of LX-2. The expression of MMP-2 in the PAI-1 treatment 12h group is (0.245±0.028), negative control group is (0.233±0.041)Compared to negative control group, the expression of MMP-2 increased slightly(t=0.473, P>0.05); the expression of uPA in 12h group is (0.303±0.052), negative control group is (0.288±0.036).Compared to negative control group, the expression of PLAU increased slightly(t=0.581, P>0.05); the expression of PAI-1 in 12h group is (0.331±0.045), negative control group is (0.205±0.007).Compared to negative control group, the expression of PAI-1 increased significantly(t=4.857,P<0.01); the expression of TIMP-1 in 12h group is (0.412±0.031), negative control group is (0.235±0.011).Compared to negative control group, the expression of TIMP-1 increased significantly(t=9.511,P<0.01).
(4)With PAI-1 treated 12h,mRNA expression of TGFβ1 and PAI-1 seperately are (1.839±0.405),(2.325±0.970),compared to negative control groups (1.090±0.123),(0.790±0.156),the difference is significant(t=4.683、4.135,P<0.01). And there is positive relationship between PAI-1 and TGFβ1mRNA(r=0.827,P<0.05).
(5)After culured with PAI-1 12h、24h、48h , the expression of TGFβ1 in the cell supernatant seperately are (8.854±3.168)、(44.348±8.415)、(225.819±12.797)pg/ml, compared to negative control groups (0.855±0.744)pg/ml , the difference is significant (F=1566.752,P<0.01); the expression of HA seperately are (2.577±0.801)、(18.177±7.141)、(50.473±4.903)ng/ml, compared to negative control groups(0.450±0.154),the difference is significant(F=235.632,P<0.01); and the TIMP-1 seperately are (2.872±1.396),(20.409±11.821),(96.548±27.154)ng/ml, compared to negative control groups(0.573±0.179)ng/ml, the difference has statistic meaning.(F=67.359,P<0.05).
Conclusion This study suggests that PAI-1 can promote HSC activation and ECM development via some cytokines and enzymes, with emphasis on activation of latent TGFβ1 during the progress of liver fibrosis. Inhibiting the upregulation of PAI-1 during liver fibrosis may be an antifibrotic pathway worth exploiting.
研究方法人肝星状细胞LX-2贴壁细胞培养;MTT法测定PAI-1对LX-2细胞的最佳干预浓度;采用ELISA法测定细胞上清液中透明质酸(hyaluronic acid,HA)、TGFβ1、基质金属蛋白酶抑制抑制因子-1(tissue inhibitor of metalloproteinase,TIMP-1);免疫细胞化学法检测ɑ-平滑肌肌动蛋白(α-smooth muscle actin,ɑ-SMA)、基质金属蛋白酶-2(matrixmetallo proteinase-2 ,MMP-2)、TIMP-1、PAI-1、尿激酶型纤溶酶原激活物(urokinase plasminogen activator, uPA)蛋白表达;逆转录聚合酶链反应(reverse transcription ploymerase chain reaction, RT-PCR)检测TGFβ1及PAI-1mRNA的表达,并分析两者相关关系。
结果1、PAI-1对LX-2细胞增殖刺激作用明显,且PAI-1浓度为10ng/ml时刺激作用最为明显,A492为1.636±0.315(F=11.697, p<0.01)。
2、ɑ-SMA在LX-2胞浆中表达,核内无明显表达。PAI-1干预12h组蛋白表达量为(0.408±0.031)较对照组(0.301±0.015)增加明显(t=5.438, p<0.01)。
3、MMP-2、TIMP-1、PAI-1、uPA在LX-2胞浆中表达,核内无明显表达。PAI-1干预12h组MMP-2蛋白表达量为(0.245±0.028)较对照组(0.233±0.041)仅轻微增加(t=0.473, P>0.05);uPA 12h组为(0.303±0.052)较对照组(0.288±0.036)增加不明显(t=0.581, P>0.05);PAI-1 12h组为(0.331±0.045)较对照组(0.205±0.007)增加显著(t=4.857,P<0.01);TIMP-112h组为(0.412±0.031)较对照组(0.235±0.011)增加显著(t=9.511,P<0.01)。
4、PAI-1干预12h后TGFβ1 mRNA、PAI-1mRNA分别为(1.839±0.405)(,2.325±0.970),对照组分别为(1.090±0.123),(0.790±0.156),两者相比差异显著。且TGFβ1 mRNA与PAI-1 mRNA呈正相关(r=0.827,P<0.05)。
5、在细胞上清液中加入PAI-1 12h,24h,48h后(刺激组)TGFβ1蛋白表达量分别为(8.854±3.168)、(44.348±8.415)、(225.819±12.797)pg/ml ,对照组为(0.855±0.744)pg/ml ,干预组较阴性对照组增加显著(F=1566.752,P<0.01);HA分别为(2.577±0.801)、(18.177±7.141)、(50.473±4.903)ng/ml ,对照组(0.450±0.154 )ng/ml,干预组较阴性对照组增加显著(F=235.632,P<0.01);TIMP-1分别为(2.872±1.396),(20.409±11.821),(96.548±27.154)ng/ml,对照组(0.573±0.179)ng/ml,干预组较阴性对照组有统计学意义(F=67.359,P<0.05)。
结论PAI-1可刺激肝星状细胞活化,增加TGFβ1表达,增加细胞外基质合成,促进肝纤维化发生发展。
Objective Liver fibrosis has become the disease hazard severely the health of people,which the activity of hepatic stellate cell(HSC)is the center element and the development of extracellular matrix (ECM)is criticality.As is known, transforming growth factorβ1(TGFβ1) is the strongest cytokine to promote liver fibrosis.It not only can promote HSC’activation and proliferation,but also promote the development of ECM,which result in the development of liver fibrosis.Recently it has been found, HSC express the cytokines of plasminogen activator inhibitor-1(PAI-1)which can anticipate in the development of liver fibrosis,While TGFβ1 could induce the expression of PAI-1.But it has not been known that whether PAI-1 can influence the expression of TGFβ1 and development of ECM.So by means of culturing the HSC in vitro, we investigate the effect of PAI-1 on TGFβ1 and ECM.
Methods The proliferation effect of PAI-1 on LX-2 was detected by Methyl thiazolyl tetrazolium(MTT).After incubation with PAI-1 12h,24h,48h,hyaluronic acid(HA), TGFβ1, tissue inhibitor of metalloproteinase -1(TIMP-1) in LX-2 supernatant was observed with ELISA,the protein expression level of smooth muscleɑ-actin(ɑ-SMA),matrix metalloproteinase-2(MMP-2), tissue inhibitor of metalloproteinase -1(TIMP-1), urokinase-type plasminogen activator(uPA)and PAI-1 were detected by immunocytochemistry and the level of PAI-1,TGFβ1 mRNA by reverse transcription polymerase chain reaction (RT-PCR).
Results (1)PAI-1 can induce activation and proliferation of LX-2 effectively,and the best concentration is 10ng/ml.
(2)The protein expression ofɑ-SMA is in the cytoplasm of LX-2. The expression in the PAI-1 treatment 12h group i(s0.408±0.031),negative control group i(s0.301±0.015). Compared to negative control group, the expression ofɑ-SMA increased significantly(t=5.438, p<0.01).
(3) The protein expression of MMP-2、TIMP-1、PAI-1、uPA is in the cytoplasm of LX-2. The expression of MMP-2 in the PAI-1 treatment 12h group is (0.245±0.028), negative control group is (0.233±0.041)Compared to negative control group, the expression of MMP-2 increased slightly(t=0.473, P>0.05); the expression of uPA in 12h group is (0.303±0.052), negative control group is (0.288±0.036).Compared to negative control group, the expression of PLAU increased slightly(t=0.581, P>0.05); the expression of PAI-1 in 12h group is (0.331±0.045), negative control group is (0.205±0.007).Compared to negative control group, the expression of PAI-1 increased significantly(t=4.857,P<0.01); the expression of TIMP-1 in 12h group is (0.412±0.031), negative control group is (0.235±0.011).Compared to negative control group, the expression of TIMP-1 increased significantly(t=9.511,P<0.01).
(4)With PAI-1 treated 12h,mRNA expression of TGFβ1 and PAI-1 seperately are (1.839±0.405),(2.325±0.970),compared to negative control groups (1.090±0.123),(0.790±0.156),the difference is significant(t=4.683、4.135,P<0.01). And there is positive relationship between PAI-1 and TGFβ1mRNA(r=0.827,P<0.05).
(5)After culured with PAI-1 12h、24h、48h , the expression of TGFβ1 in the cell supernatant seperately are (8.854±3.168)、(44.348±8.415)、(225.819±12.797)pg/ml, compared to negative control groups (0.855±0.744)pg/ml , the difference is significant (F=1566.752,P<0.01); the expression of HA seperately are (2.577±0.801)、(18.177±7.141)、(50.473±4.903)ng/ml, compared to negative control groups(0.450±0.154),the difference is significant(F=235.632,P<0.01); and the TIMP-1 seperately are (2.872±1.396),(20.409±11.821),(96.548±27.154)ng/ml, compared to negative control groups(0.573±0.179)ng/ml, the difference has statistic meaning.(F=67.359,P<0.05).
Conclusion This study suggests that PAI-1 can promote HSC activation and ECM development via some cytokines and enzymes, with emphasis on activation of latent TGFβ1 during the progress of liver fibrosis. Inhibiting the upregulation of PAI-1 during liver fibrosis may be an antifibrotic pathway worth exploiting.
引文
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