白介素37对转化生长因子β1诱导的大鼠肝星状细胞的影响
发布时间：2018-08-02 20:26
【摘要】：目的：观察重组人白细胞介素37（Interleukin37,IL-37）对转化生长因子β1（Transform Growth Factor-beta1,TGF-β1）诱导的活化大鼠肝星状细胞（rat hepatic stellate cell,HSC-T6）的增殖效应的影响，并观察其对HSC-T6表达的纤溶酶原激活抑制物1（PlasminogenActivator Inhibitor1,PAI-1）和平滑肌肌动蛋白α（Smooth MuscleActinα,SMA-α）表达的影响。初步探讨IL-37可能的抗肝纤维化作用及其机制。方法：1.不同细胞因子浓度培养基制备及实验分组：以改良杜氏伊格尔培养基（Dulbecco's Modified EagleMedium,DMEM）为基础液配制，实验共分为5组，即空白对照组：仅含DMEM培养基；实验组A：含TGF-β1（5ng/ml），不含有重组IL-37b；实验组B、实验组C及实验组D含有与A组相同浓度的TGF-β1，且含有浓度梯度逐渐升高的重组人IL-37b（10ng/ml,100ng/ml,200ng/ml）。2.检测HSC-T6增殖及目标蛋白表达：在各组不同细胞因子浓度作用下HSC-T6培养12h、24h、48h后，用四甲基偶氮唑盐比色法（MTT法）检测HSC增殖情况；细胞爬片后，继续药物作用24小时，用免疫组化法检测不同组培养基作用后SMA-α、PAI-1表达情况。3.统计学处理：检测相同时间下，不同药物浓度细胞增殖情况及SMA-α、PAI-1表达情况，采用单因素方差分析；B组、C组及D组间比较细胞增殖抑制率，采用两因素方差分析，以P0.05为差异有统计学意义。采用SPSS19.0统计软件进行数据分析。结果：1.对HSC-T6增殖抑制作用：在重组人IL-37b与TGF-β1共培养12h、24h、48h后，B组、C组、D组HSC-T6吸光度均低于A组，P值均小于0.05，差异有统计学意义，表明不同浓度重组人IL-37b在12h、24h、48h均有对活化的大鼠肝星状细胞增殖的抑制作用。在不同时间点的组内比较提示，在24h各组抑制作用明显，且不同浓度的抑制率不同，为浓度越高，抑制率越明显，P值0.001，有统计学差异；在12h不同药物浓度组抑制率均低，且各组间抑制率差异无统计学意义，而在48h不同药物组的抑制率均较高，且各组间抑制率差异无统计学意义，P值0.05，无明显统计学差异。2. IL-37对活化的HSC-T6表达SMA-α的影响：在IL-37b干预组的B组、C组、D组SMA-α表达量可较无IL-37b的A组干预组明显减少，且存在有剂量效应关系，IL-37b浓度越高对SMA-α表达抑制作用越明显，有统计学意义（P0.05）。3.IL-37对活化的HSC-T6表达PAI-1的影响：在IL-37b干预组的B组、C组、D组PAI-1表达量可较无IL-37b的A组干预组明显减少，且存在有剂量效应关系，IL-37b浓度越高对PAI-1表达抑制作用越明显，有统计学意义（P0.05）。结论：1.重组人IL-37b可抑制TGF-β1活化的HSC-T6增殖，且在作用24h有剂量效应关系，浓度越高对细胞增殖抑制作用越明显，而在12h及48h时无明显剂量效应关系；2.重组人IL-37b可抑制TGF-β1活化的HSC-T6表达SMA-α及PAI-1，在作用24h有剂量效应关系，，浓度越高的IL-37b抑制作用越明显。总之本实验研究表明IL-37可能通过抑制HSC-T6细胞增殖及抑制其表达SMA-α、PAI-1发挥抗肝纤维化作用。
[Abstract]:Aim: to investigate the effect of recombinant human interleukin-37 (IL-37) on the proliferation of activated rat hepatic stellate cells (rat hepatic stellate cell line HSC-T6) induced by transforming growth factor 尾 1 (Transform Growth Factor-beta 1 (TGF- 尾 1). The expression of PlasminogenActivator inhibitor 1 (PAI-1) and smooth muscle actin 伪 (Smooth MuscleActin 伪 (SMA- 伪) were observed. To explore the possible anti-hepatic fibrosis effect of IL-37 and its mechanism. Method 1: 1. Preparation and grouping of different cytokine concentration medium: the modified Duchenne medium (Dulbecco's Modified Eagle Media was prepared as the base solution. The experiment was divided into five groups: blank control group: only DMEM medium, experimental group A: TGF- 尾 1 (5ng/ml), no recombinant IL-37b, the control group: TGF- 尾 1 (5ng/ml), the control group: TGF- 尾 1 (TGF- 尾 1), no recombinant IL-37b; Experimental group B, experimental group C and experimental group D contained TGF- 尾 1 of the same concentration as group A, and the recombinant human IL-37b (10 ng / ml / ml 100 ng / ml / ml). 2. Detection of HSC-T6 proliferation and expression of target protein: after HSC-T6 was cultured for 24 h or 48 h under different cytokine concentrations, the proliferation of HSC was detected by MTT assay, and the cell climbing tablet was used for 24 hours. Immunohistochemical method was used to detect the expression of SMA- 伪 -PAI-1 in different culture medium. Statistical analysis: cell proliferation and SMA- 伪 PAI-1 expression in different drug concentrations were detected at the same time. Univariate analysis of variance (ANOVA) was used to compare the inhibition rate of cell proliferation between group C and group D, and two factors ANOVA was used. P0.05 as the difference was statistically significant. SPSS19.0 statistical software was used to analyze the data. The result is 1: 1. Inhibition of HSC-T6 proliferation: after co-culture of recombinant human IL-37b and TGF- 尾 1 for 24 h or 48 h, the absorbance of HSC-T6 in group C was lower than that in group A (P < 0.05), and the difference was statistically significant. The results showed that different concentrations of recombinant human IL-37b could inhibit the proliferation of activated rat hepatic stellate cells at 12h or 24h or 48h. The results of comparison at different time points showed that the inhibition rate of each group was obvious at 24 h, and the inhibition rate of different concentration was different. The higher the concentration, the more obvious the inhibition rate was (P value 0.001), the inhibition rate of different drug concentration group was lower at 12 h, and the inhibition rate of different drug concentration group was lower than that of control group at 12 h. There was no significant difference in inhibition rate among groups, but the inhibition rate was higher in different drug groups at 48 h, and there was no significant difference in inhibition rate among groups (P = 0.05, P < 0.05), and there was no significant difference in inhibition rate between groups (P = 0.05, P < 0.05). The effect of IL-37 on the expression of SMA- 伪 in activated HSC-T6: the expression of SMA- 伪 in group B and C of IL-37b intervention group was significantly lower than that in group A without IL-37b, and the higher the concentration of IL-37b in IL-37b intervention group was, the more obvious the inhibitory effect was on SMA- 伪 expression. There was significant difference (P0.05) .3.The effect of IL-37 on the expression of PAI-1 in activated HSC-T6: the expression of PAI-1 in group B and C of IL-37b intervention group was significantly lower than that in group A without IL-37b, and the higher the concentration of IL-37b in IL-37b intervention group was, the more obvious the inhibitory effect of IL-37b on PAI-1 expression was. There was statistical significance (P0.05). Conclusion 1. Recombinant human IL-37b could inhibit the proliferation of HSC-T6 activated by TGF- 尾 1, and had a dose-effect relationship at 24 h. The higher the concentration was, the more obvious the inhibitory effect was on cell proliferation, but there was no significant dose-effect relationship at 12h and 48h. Recombinant human IL-37b could inhibit the expression of SMA- 伪 and PAI-1in HSC-T6 activated by TGF- 尾 _ 1. There was a dose-effect relationship between them at 24 h. The higher the concentration of IL-37b was, the more obvious the inhibitory effect was. In conclusion, this study suggests that IL-37 may inhibit the proliferation of HSC-T6 cells and inhibit the expression of SMA- 伪 -PAI-1.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.2
本文编号：2160627
[Abstract]:Aim: to investigate the effect of recombinant human interleukin-37 (IL-37) on the proliferation of activated rat hepatic stellate cells (rat hepatic stellate cell line HSC-T6) induced by transforming growth factor 尾 1 (Transform Growth Factor-beta 1 (TGF- 尾 1). The expression of PlasminogenActivator inhibitor 1 (PAI-1) and smooth muscle actin 伪 (Smooth MuscleActin 伪 (SMA- 伪) were observed. To explore the possible anti-hepatic fibrosis effect of IL-37 and its mechanism. Method 1: 1. Preparation and grouping of different cytokine concentration medium: the modified Duchenne medium (Dulbecco's Modified Eagle Media was prepared as the base solution. The experiment was divided into five groups: blank control group: only DMEM medium, experimental group A: TGF- 尾 1 (5ng/ml), no recombinant IL-37b, the control group: TGF- 尾 1 (5ng/ml), the control group: TGF- 尾 1 (TGF- 尾 1), no recombinant IL-37b; Experimental group B, experimental group C and experimental group D contained TGF- 尾 1 of the same concentration as group A, and the recombinant human IL-37b (10 ng / ml / ml 100 ng / ml / ml). 2. Detection of HSC-T6 proliferation and expression of target protein: after HSC-T6 was cultured for 24 h or 48 h under different cytokine concentrations, the proliferation of HSC was detected by MTT assay, and the cell climbing tablet was used for 24 hours. Immunohistochemical method was used to detect the expression of SMA- 伪 -PAI-1 in different culture medium. Statistical analysis: cell proliferation and SMA- 伪 PAI-1 expression in different drug concentrations were detected at the same time. Univariate analysis of variance (ANOVA) was used to compare the inhibition rate of cell proliferation between group C and group D, and two factors ANOVA was used. P0.05 as the difference was statistically significant. SPSS19.0 statistical software was used to analyze the data. The result is 1: 1. Inhibition of HSC-T6 proliferation: after co-culture of recombinant human IL-37b and TGF- 尾 1 for 24 h or 48 h, the absorbance of HSC-T6 in group C was lower than that in group A (P < 0.05), and the difference was statistically significant. The results showed that different concentrations of recombinant human IL-37b could inhibit the proliferation of activated rat hepatic stellate cells at 12h or 24h or 48h. The results of comparison at different time points showed that the inhibition rate of each group was obvious at 24 h, and the inhibition rate of different concentration was different. The higher the concentration, the more obvious the inhibition rate was (P value 0.001), the inhibition rate of different drug concentration group was lower at 12 h, and the inhibition rate of different drug concentration group was lower than that of control group at 12 h. There was no significant difference in inhibition rate among groups, but the inhibition rate was higher in different drug groups at 48 h, and there was no significant difference in inhibition rate among groups (P = 0.05, P < 0.05), and there was no significant difference in inhibition rate between groups (P = 0.05, P < 0.05). The effect of IL-37 on the expression of SMA- 伪 in activated HSC-T6: the expression of SMA- 伪 in group B and C of IL-37b intervention group was significantly lower than that in group A without IL-37b, and the higher the concentration of IL-37b in IL-37b intervention group was, the more obvious the inhibitory effect was on SMA- 伪 expression. There was significant difference (P0.05) .3.The effect of IL-37 on the expression of PAI-1 in activated HSC-T6: the expression of PAI-1 in group B and C of IL-37b intervention group was significantly lower than that in group A without IL-37b, and the higher the concentration of IL-37b in IL-37b intervention group was, the more obvious the inhibitory effect of IL-37b on PAI-1 expression was. There was statistical significance (P0.05). Conclusion 1. Recombinant human IL-37b could inhibit the proliferation of HSC-T6 activated by TGF- 尾 1, and had a dose-effect relationship at 24 h. The higher the concentration was, the more obvious the inhibitory effect was on cell proliferation, but there was no significant dose-effect relationship at 12h and 48h. Recombinant human IL-37b could inhibit the expression of SMA- 伪 and PAI-1in HSC-T6 activated by TGF- 尾 _ 1. There was a dose-effect relationship between them at 24 h. The higher the concentration of IL-37b was, the more obvious the inhibitory effect was. In conclusion, this study suggests that IL-37 may inhibit the proliferation of HSC-T6 cells and inhibit the expression of SMA- 伪 -PAI-1.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.2
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相关期刊论文 前2条
1 郑素军;邢欣悦;韩源平;武聚山;王世美;张莹;刘梅;陈煜;刘霜;段钟平;;TGF-β1对大鼠HSC-T6细胞增殖、细胞周期和胶原分泌的影响[J];实用肝脏病杂志;2012年04期
2 黄瑛;吉庆伟;曾秋棠;;新型抗炎因子白介素-37与动脉粥样硬化[J];心血管病学进展;2013年03期
本文编号：2160627
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