白介素37對轉(zhuǎn)化生長因子β1誘導(dǎo)的大鼠肝星狀細胞的影響
發(fā)布時間：2018-08-02 20:26
【摘要】：目的：觀察重組人白細胞介素37（Interleukin37,IL-37）對轉(zhuǎn)化生長因子β1（Transform Growth Factor-beta1,TGF-β1）誘導(dǎo)的活化大鼠肝星狀細胞（rat hepatic stellate cell,HSC-T6）的增殖效應(yīng)的影響，并觀察其對HSC-T6表達的纖溶酶原激活抑制物1（PlasminogenActivator Inhibitor1,PAI-1）和平滑肌肌動蛋白α（Smooth MuscleActinα,SMA-α）表達的影響。初步探討IL-37可能的抗肝纖維化作用及其機制。方法：1.不同細胞因子濃度培養(yǎng)基制備及實驗分組：以改良杜氏伊格爾培養(yǎng)基（Dulbecco's Modified EagleMedium,DMEM）為基礎(chǔ)液配制，實驗共分為5組，即空白對照組：僅含DMEM培養(yǎng)基；實驗組A：含TGF-β1（5ng/ml），不含有重組IL-37b；實驗組B、實驗組C及實驗組D含有與A組相同濃度的TGF-β1，且含有濃度梯度逐漸升高的重組人IL-37b（10ng/ml,100ng/ml,200ng/ml）。2.檢測HSC-T6增殖及目標蛋白表達：在各組不同細胞因子濃度作用下HSC-T6培養(yǎng)12h、24h、48h后，用四甲基偶氮唑鹽比色法（MTT法）檢測HSC增殖情況；細胞爬片后，繼續(xù)藥物作用24小時，用免疫組化法檢測不同組培養(yǎng)基作用后SMA-α、PAI-1表達情況。3.統(tǒng)計學(xué)處理：檢測相同時間下，不同藥物濃度細胞增殖情況及SMA-α、PAI-1表達情況，采用單因素方差分析；B組、C組及D組間比較細胞增殖抑制率，采用兩因素方差分析，以P0.05為差異有統(tǒng)計學(xué)意義。采用SPSS19.0統(tǒng)計軟件進行數(shù)據(jù)分析。結(jié)果：1.對HSC-T6增殖抑制作用：在重組人IL-37b與TGF-β1共培養(yǎng)12h、24h、48h后，B組、C組、D組HSC-T6吸光度均低于A組，P值均小于0.05，差異有統(tǒng)計學(xué)意義，表明不同濃度重組人IL-37b在12h、24h、48h均有對活化的大鼠肝星狀細胞增殖的抑制作用。在不同時間點的組內(nèi)比較提示，在24h各組抑制作用明顯，且不同濃度的抑制率不同，為濃度越高，抑制率越明顯，P值0.001，有統(tǒng)計學(xué)差異；在12h不同藥物濃度組抑制率均低，且各組間抑制率差異無統(tǒng)計學(xué)意義，而在48h不同藥物組的抑制率均較高，且各組間抑制率差異無統(tǒng)計學(xué)意義，P值0.05，無明顯統(tǒng)計學(xué)差異。2. IL-37對活化的HSC-T6表達SMA-α的影響：在IL-37b干預(yù)組的B組、C組、D組SMA-α表達量可較無IL-37b的A組干預(yù)組明顯減少，且存在有劑量效應(yīng)關(guān)系，IL-37b濃度越高對SMA-α表達抑制作用越明顯，有統(tǒng)計學(xué)意義（P0.05）。3.IL-37對活化的HSC-T6表達PAI-1的影響：在IL-37b干預(yù)組的B組、C組、D組PAI-1表達量可較無IL-37b的A組干預(yù)組明顯減少，且存在有劑量效應(yīng)關(guān)系，IL-37b濃度越高對PAI-1表達抑制作用越明顯，有統(tǒng)計學(xué)意義（P0.05）。結(jié)論：1.重組人IL-37b可抑制TGF-β1活化的HSC-T6增殖，且在作用24h有劑量效應(yīng)關(guān)系，濃度越高對細胞增殖抑制作用越明顯，而在12h及48h時無明顯劑量效應(yīng)關(guān)系；2.重組人IL-37b可抑制TGF-β1活化的HSC-T6表達SMA-α及PAI-1，在作用24h有劑量效應(yīng)關(guān)系，，濃度越高的IL-37b抑制作用越明顯�？傊緦嶒炑芯勘砻鱅L-37可能通過抑制HSC-T6細胞增殖及抑制其表達SMA-α、PAI-1發(fā)揮抗肝纖維化作用。
[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 偽 (Smooth MuscleActin 偽 (SMA- 偽) 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- 偽 -PAI-1 in different culture medium. Statistical analysis: cell proliferation and SMA- 偽 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- 偽 in activated HSC-T6: the expression of SMA- 偽 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- 偽 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- 偽 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- 偽 -PAI-1.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.2
本文編號：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 偽 (Smooth MuscleActin 偽 (SMA- 偽) 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- 偽 -PAI-1 in different culture medium. Statistical analysis: cell proliferation and SMA- 偽 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- 偽 in activated HSC-T6: the expression of SMA- 偽 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- 偽 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- 偽 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- 偽 -PAI-1.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.2
【參考文獻】
相關(guān)期刊論文 前2條
1 鄭素軍;邢欣悅;韓源平;武聚山;王世美;張瑩;劉梅;陳煜;劉霜;段鐘平;;TGF-β1對大鼠HSC-T6細胞增殖、細胞周期和膠原分泌的影響[J];實用肝臟病雜志;2012年04期
2 黃瑛;吉慶偉;曾秋棠;;新型抗炎因子白介素-37與動脈粥樣硬化[J];心血管病學(xué)進展;2013年03期
本文編號：2160627
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