【作者】 范玉；

【导师】 杜锡贤；

【作者基本信息】 山东中医药大学 ， 中医外科学， 2013， 博士

【摘要】 目的：揭示中药清热利湿饮对HaCaT细胞增殖抑制、诱导凋亡及表达IL-6mRNA、IL-8mRNA、IL-10mRNA的作用机制，为清热利湿饮治疗寻常型银屑病提供科学理论依据。方法：以角质形成细胞HaCaT细胞为研究对象，进行传代培养，分别采用MTT法、流式细胞仪技术、RT-PCR技术及ELISA技术观察中药清热利湿饮提取液对经TNF-α诱导活化的HaCaT细胞增殖抑制作用、诱导凋亡作用、对细胞周期的影响、表达IL-6mRNA、IL-8mRNA和IL-10mRNA和分泌IL-8和IL-10的影响。结果：1.TNF-α对HaCaT细胞增殖效应的影响TNF-α对HaCaT细胞作用24h、48h和72h，对HaCaT细胞有一定的增殖效应，在TNF-α作用72h浓度为2.5×105u/L时与无TNF-α细胞对照相比，其增殖率为14.11%，本研究选择TNF-α2.5×105u/L作为对HaCaT细胞的诱导剂量。2.三种中药提取液对HaCaT细胞增殖抑制效应的比较清热利湿饮全方①、拆方②和拆方③提取液在不同浓度（3.1g/L-100g/L）和不同时相（24h、48h和72h）对经TNF-α诱导的HaCaT细胞有一定的增殖抑制效应。清热利湿饮全方①与拆方②和拆方③提取液相比，不同药物浓度对HaCaT细胞增殖的抑制效应明显高于后二者，差异均具有统计学意义（P＜0.05），本研究选择清热利湿饮提取液对经TNF-α诱导活化的HaCaT细胞的增殖抑制效应做进一步探讨。3.不同浓度的清热利湿饮对TNF-α诱导的HaCaT细胞的增殖抑制效应的影响药物作用48h与MTX阳性对照相比，清热利湿饮在12.5g/L时对HaCaT细胞的抑制率为40%，高于MTX（38.76%），在25g/L～100g/L三个浓度，其抑制效应与MTX相比明显升高，差异显著（P＜0.05或P＜0.01）。4.清热利湿饮对HaCaT细胞凋亡的影响HaCaT细胞经浓度分别为12.5g/L、10g/L、7.5g/L和5g/L清热利湿饮提取液作用48h后细胞凋亡总数分别为45.75%、44.94%、36.26%和34.43%，TNF-α诱导活化无药对照组仅为24.65%，前者高于后者，差异显著（P＜0.05和P＜0.01）。5.清热利湿饮对HaCaT细胞周期的影响MTX作用于HaCaT细胞48h，G1期细胞为65.94%，正常对照和TNF-α对照组分别为49.01%和50.73%，而S期前者为30.38%，MTX将细胞阻滞于G1期。清热利湿饮在12.5g/L和15g/L时，G1期细胞与TNF-α组相比细胞明显减少（38.06%和25.04%），S期细胞明显增加（56.98%和67.90%），MTX将HaCaT细胞阻滞于G1期，而清热利湿饮提取液则将其阻滞于S期。6.清热利湿饮对HaCaT细胞表达IL-6mRNA、IL-8mRNA和IL-10mRNA的影响清热利湿饮对经TNF-α诱导活化的HaCaT细胞产生的炎性细胞因子IL-6mRNA和IL-8mRNA的表达有下调作用，在10g/L和12.5g/L时对IL-10mRNA的表达量增强较为明显，与TNF-α诱导活化组相比，差异有显著性（P＜0.05和P＜0.01）。7.清热利湿饮对HaCaT细胞分泌IL-8和IL-10的影响清热利湿饮对经TNF-α诱导活化的HaCaT细胞可降低IL-8的分泌，但对IL-10的影响不明显。结论：中药清热利湿饮对HaCaT细胞增殖有明显的抑制作用，且在一定浓度范围内其抑制率呈明显的时间和剂量依赖性；作用于HaCaT细胞后均可诱导其凋亡；干扰HaCaT细胞周期，将细胞阻滞于S期；下调IL-6mRNA、IL-8mRNA的表达，IL-10mRNA的表达随药物浓度的升高而增强；可降低IL-8的分泌，但对IL-10的影响不明显。提示中药清热利湿饮治疗银屑病的机制可能与此有关。更多还原

【Abstract】 Objective: To reveal the mechanism of the inhibition of the proliferation, theinductive apoptosis and the expression of IL-6、IL-8and IL-10,which is of traditionalChinese medicinal Qing Re Li Shi Yin on HaCat cells, and provide scientific basis for thetreatment of Qing Re Li Shi Yin on psoriasis vulgaris.Methods: To take the KeratinocytesHaCaT cell line as the research object to subculture, to observe the proliferation inhibitioneffect of Qing Re Li Shi Yin extracts on HaCaT cell cultured in vitro by MTT method; toobserve of the inductive apoptosis of Qing Re Li Shi Yin on HaCaT cells by flowcytometry; and to examine the effect of expression of IL-6mRNA、IL-8mRNA andIL-10mRNA by HaCaT cell lines through ELISA and real-time fluorescence quantitativePCR method.Result:1.Effect of TNF-α on proliferation of HaCaT cells TNF-αaffected on HaCaT cells for24h,48h and72h,and had the proliferation effect on HaCaTcells. Compared the effect of TNF-α on48h concentration which is2.5×10~5u/L with noTNF-α cells control group, its proliferation rate was14.11%. the research chose TNF-α2.5×10~5u/L as the induction dose of HaCaT cells.2. Comparison of inhibitory effect onthe proliferation of HaCaT cells in the liquid extracted from three kinds of traditionalChinese medicine Qing Re Li Shi Yin’s whole party①extract、split party②extract andsplit party③extract have a certain inhibitory effect on the proliferation of HaCaT cellsinduced by TNF-α at different concentrations (3.1g/L-100g/L) and different time (24h,48h and72h). compared with split party②extract and split party③extract, inhibitoryeffect of different concentration on proliferation of HaCaT cells in whole party①extractwas significantly higher than that of the latter two, and the difference was statisticallysignificant (P <0.05). This study researched that Qing Re Li Shi Yin extract had proliferation inhibitory effect on HaCaT cells induced by TNF-α further.3. Influenceof Qing Re Li Shi Yin with different concentration on the proliferation inhibitoryeffect of HaCaT cell induced by TNF-α. Compared drug effects for48h with MTXpositive controls, when Qing Re Li Shi Yin was on12.5g/L, the inhibition rate to HaCaTcell was40%, which was higher than that of MTX (38.76%). In the three concentration25g/L-100g/L, its inhibitory effect significantly increased and had evident difference (P <0.05or P <0.01) compared with MTX.4.Effect of Qing Re Li Shi Yin on the apoptosisof HaCaT cells After Qing Re Li Shi Yin extract with concentration of12.5g/L,10g/L,7.5g/L and5g/L effected HaCaT cells for48h, total number of cell apoptosis was45.75%,44.94%,41.26%and34.43%respectively, and TNF-α activation no drug control group (P<0.05or P <0.01).Obviously higher than no drug control group activated by TNF-α, itwas only24.65%, and the former is higher than the latter, difference was evident as well (P<0.05and P <0.01).5. Effect of Qing Re Li Shi Yin on cycle of HaCaT cells WhenMTX affected HaCaT cells for48h, cell in G1phase was65.94%, normal control groupand TNF-αcontrol group were49.01%and50.73%respectively. The former in S phasewas30.38%, and MTX arrested cells in G1phase. When Qing Re Li Shi Yin were12.5g/Land15g/L, cells in G1phase was reduced evidently (38.06%and25.04%) and cells in Sphase increased significantly (56.98%and67.90%), compared with cells in TNF-α group.HaCaT cells were arrested in G1phase by MTX, and yet arrested in S phase by Qing Re LiShi Yin extract.6. Effect of Qing Re Li Shi Yin extract on HaCaT cells expression ofIL-6mRNA, IL-8mRNA and IL-10mRNA Expression of Qing Re Li Shi Yin oninflammatory cytokines IL-6mRNA and IL-8mRNA produced by HaCaT cells which wereactivated by TNF-α induced. The expression of IL-10mRNA in10g/L and12.5g/Lincreased obviously, and compared to TNF-α activation group, there was significantdifference (P <0.05and P <0.01).7.Effect of Qing Re Li Shi Yin extract on HaCaTcells secretion of IL-8and IL-10.Qing Re Li Shi Yin can reduce IL-8produced by thesecretion of HaCaT cells which were activated by TNF-α induced., but no obvious effecton IL-10.Conclusion: Chinese traditional medical Qing Re Li Shi Yin has obvious inhibitoryeffect on the proliferation of HaCaT cells, and the inhibition rate has time-and-dosedependent manner in a certain range of concentration. The effect on HaCaT cells caninduce the apoptosis of HaCaT cell; the cycle of HaCaT cells are interfered, in which cells are blocked in S phase; expression of IL-6mRNA, IL-8mRNA is weakened, and expressionof IL-10mRNA is enhanced with the increase of drug concentration, with which maybe theanti-psoriatic mechanism of Chinese traditional medical Qing Re Li Shi Yin hasrelationship.更多还原