梓醇、左旋紫草素、丹皮酚对KGF诱导的HaCaT细胞过度增殖的影响

【作者】 王玉玲；

【导师】 白彦萍；

【作者基本信息】 北京中医药大学 ， 中医外科学， 2011， 硕士

【摘要】 文献综述部分：文献综述一：查阅近年来关于中药单体(方)及复方治疗银屑病的实验研究方面的文献,从中药单体(方)及复方对银屑病的实验动物模型、血液流变学及微循环、角质形成细胞(KC)、成纤维细胞(FB)、细胞因子、神经肽六个方面的影响进行分类、归纳和总结。以求对中医药治疗银屑病的实验研究有一个总体的认识。文献综述二：查阅近年来关于地黄、紫草、牡丹皮及其主要活性成分的研究性文献,对三者的来源、功效、化学成分(主要是梓醇、紫草素、丹皮酚)及药理研究概况作了综述。它们的药理活性都非常广泛,在银屑病治疗中也有很好的开发前景。实验研究部分：目的：研究生地、紫草、牡丹皮的主要单体成分梓醇、左旋紫草素、丹皮酚对KGF诱导的HaCaT细胞过度增殖的影响,探讨生地、紫草、牡丹皮治疗银屑病的有效成分和可能的作用机制。方法：1、选取HaCaT细胞株体外培养。2、CCK-8法检测不同浓度的梓醇、左旋紫草素、丹皮酚对KGF诱导的HaCaT细胞过度增殖和细胞生长曲线的影响。3、倒置显微镜下观察药物干预后HaCaT细胞形态学变化。4、流式细胞仪检测不同浓度的梓醇、左旋紫草素、丹皮酚对KGF诱导的HaCaT细胞过度增殖的细胞周期的影响。结果：1、CCK-8法的实验结果显示,KGF可明显促进HaCaT细胞增殖(P<0.05),其促增殖作用呈浓度依赖性,最佳浓度为10ng/ml。浓度高于或等于10-6mol/L的梓醇可促进KGF诱导的HaCaT细胞过度增殖效应(P<0.05),促增殖率呈浓度和时间依赖性,最佳浓度为10-4mol/L,随着作用时间延长,促增殖率反而降低；浓度高于或等于10-6mol/L的左旋紫草素和浓度高于或等于31.25 mg/L的丹皮酚均可抑制KGF诱导的HaCaT细胞过度增殖效应(P<0.05),生长抑制率呈浓度和时间依赖性,浓度越大,抑制性越强,随着作用时间延长,生长抑制率反而降低。2、细胞生长曲线显示：10ng/ml KGF作用下,HaCaT细胞生长加快,自培养1天后起各对应时间点细胞数均较无药对照组增加(P<0.01),细胞培养5天后,KGF组和无药对照组细胞生长速度又有接近的趋势。浓度高于或等于10-6mol/L的梓醇可促进KGF诱导的HaCaT细胞的生长加快,自培养1天后起,各对应时间点细胞数均较KGF对照组增加(P<0.05)；浓度高于或等于10-6mol/L的左旋紫草素和浓度高于或等于31.25 mg/L的丹皮酚均可抑制KGF诱导的HaCaT细胞的生长加快,自培养1天后起,各对应时间点细胞数均较KGF对照组减少(P<0.05)。3、倒置显微镜下HaCaT细胞形态学显示：KGF孔的细胞密度较无药对照孔的有所增加。梓醇干预孔的细胞密度较KGF对照孔更密集,左旋紫草素和丹皮酚干预孔的细胞密度较KGF对照孔的有所减少。细胞形态在各孔间无明显差异。4、细胞周期结果表明10ng/ml的KGF作用24小时后,进入S期的HaCaT细胞比率较无药对照组增加(P<0.01),同时,G0G1,期比率降低(P<0.01),G2M期比率升高(P<0.01)。浓度高于或等于10-6mol/L的梓醇可促进KGF诱导的HaCaT细胞的S期比率增加(P<0.05)、G0G1,期比率降低(P<0.01),促进作用呈浓度依赖性,最佳浓度是10-6mol/L；G2M期比率与KGF对照组无明显差异(P>0.05)。浓度高于或等于10-6mol/L的左旋紫草素和浓度高于或等于31.25 mg/L的丹皮酚均可抑制KGF诱导的HaCaT细胞的S期比率增加(P<0.01),浓度高于或等于10-mol/L的左旋紫草素和浓度高于或等于15.625 mg/L的丹皮酚均可抑制KGF诱导的HaCaT细胞的G0G1期比率降低(P<0.01),G2M期比率升高(P<0.01),抑制作用呈浓度依赖性,浓度越大,抑制作用越强。各组均未见凋亡峰。结论：1、KGF通过促进细胞进入S期和G2M期来诱导HaCaT细胞的过度增殖,可在体外模拟银屑病表皮KC过度增殖状态,KGF诱导的HaCaT细胞过度增殖模型是银屑病体外研究较理想的模型。2、左旋紫草素和丹皮酚通过阻滞HaCaT细胞向S期和G2M期转化,使细胞生长增殖停滞在G0G1，期来抑制KGF诱导的HaCaT细胞过度增殖效应,这是紫草和牡丹皮治疗银屑病的可能机制,其有效成分是左旋紫草素和丹皮酚。梓醇促进细胞进入S期,从而促进KGF诱导的HaCaT细胞过度增殖效应,不能通过抑制KC的增殖来治疗银屑病,生地治疗银屑病的有效成分和作用机制有待从其他角度进一步探讨。更多还原

【Abstract】 This dissertation was composed of two parts:the literature review and the experimental research.The part of literature review:Literature review one:To form a general understanding about the experimental researches on the treatment of psoriasis with traditional Chinese medicine, the literatures about the experimental researches about the treatment of psoriasis with traditional Chinese herbs and their monomers or compounds in. the recent years were reviewed. They were assorted into six parts, which were the influences from traditional Chinese herbs and their monomers or compounds to the experimental animal model of psoriasis, hemorheology and microcirculation, keratinocyte(KC), fibroblast (FB), cell factor and neuropeptide, to induce and summarize.Literature review two:The literatures about the researches of Rehmannia glutinosa Libosch, Radix Arnebiae and Cortex Moutan and their mainly active ingredients in the recent years were reviewed. And the source, efficacy, chemical compositions that mainly composed of catalpol, shikonin and paeonol, and the overview of pharmacological studies of the three were summarized. Their pharmacological activities are extremely extensive. They also have good development prospects for the treatments of psoriasis.The part of experimental research:.Objective:To study the effective ingredients and the potential mechanisms of action in the treatment of psoriasis with Rehmannia glutinosa Libosch, Radix Arnebiae and Cortex Moutan, the effects to the proliferation of HaCaT cells induced by KGF caused by catalpol,1-shikonin and paeonol, which are the main monomers of the three, are researched.Methods:1. HaCaT cell strain is selected and cultivated in vitro.2. The effects to the proliferation and the cell growth curve of KGF induced HaCaT cells caused by catalpol,1-shikonin and paeonol are detected by CCK-8 assay. 3. The changes of the form of HaCaT cells after drug intervention are observed under a upside down microscope.4. The effects to the cell cycle of KGF induced HaCaT cells proliferation caused by catalpol,1-shikonin and paeonol are detected by a flow cytometry.Results:1. The experimental results of CCK-8 assay showed that after the treatment with KGF, the HaCaT cells proliferation was increased significantly in a dose dependent manner (P<0.05), of which the best concentration is 10ng/ml. The proliferation of HaCaT cells induced by KGF was enhanced by Catalpol at more than or equal to 10-6mol/L concentrat ion (P<0.05). The promotion of prol iferation rate of Catalpol was showed in a dose and time dependent manner, and the best concentration is 10-4mol/L. With the acting time extended, the promotion of proliferation rate of Catalpol was reduced. The proliferation of HaCaT cells induced by KGF was inhibited by 1-shikonin at more than or equal to 10-6mol/L concentration (P<0.05) as well as paeonol at more than or equal to 31.25 mg/L concentration(P<0.05). The inhibition of proliferation rate of 1-shikonin and paeonol was showed in a dose and time dependent manner. As the concentration increased, the inhibiting effect was enhanced. With the acting time extended, the inhibition of proliferation rate of 1-shikonin and paeonol was reduced.2. The cell growth curve showed that HaCaT cells in the treatment with 10ng/ml KGF growed more rapidly compared with control group after the first day of treatment (P<0.01), and after the fifth day of treatment, the growth rate of KGF group showed trend towards control group. The up-regulated growth rate induced by KGF was increased by Catalpol at more than or equal to 10-6mol/L concentration after the first day(P<0.05). And The up-regulated growth rate induced by KGF was depressed by 1-shikonin at more than or equal to 10-6mol/L concentration and paeonol at more than or equal to 31.25 mg/L concentration after the first day(P<0.05).3. The form of HaCaT cells under a upside down microscope showed that the cell density increased and was more intensive in the KGF hole compared with the control hole. The cell density in the hole treated with Catalpol was more intensive than that in the KGF hole. The cell density in the holes treated with 1-shikonin and paeonol were less intensive than that in the KGF hole. There was no significant differences of the HaCaT cells form among each holes.4. The results of the cell cycle showed that after incubated with 10ng/ml KGF for 24 hours, the ratio of S phase and G2M phase of HaCaT cells was increased compared with the control group(P<0.01), and the ratio of G0G1 phase was decreased(P<0.01). The increased S phase and the decreased G0G1 phase induced by KGF was enhanced by Catalpol at more than or equal to 10-6mol/L concentration in a dose dependent manner (P<0.05 and P<0.01), and the best concentration is 10-4mol/L. The ratio of G2M phase had no significant difference campared with KGF group(P>0.05). The increased S phase phase induced by KGF was inhibited by 1-shikonin at more than or equal to 10-6mol/L concentration(P<0.01) as well as paeonol at more than or equal to 31.25 mg/L concentration (P<0.01). The increased G2M phase and the decreased G0G1 phase induced by KGF was inhibited by 1-shikonin at more than or equal to 10-7mol/L concentration (P<0.01) as well as paeonol at more than or equal to 15.625 mg/L concentration(P<0.01). The inhibiting effect of 1-shikonin and paeonol was in a dose dependent manner. As the concentration increased, the inhibiting effect was enhanced. There is no apoptotic peak could be seen in each group.Conclusion:1. The proliferation of HaCaT cells could be induced by KGF by means of increasing the S phase and G2M phase cells. It could simulate in vitro the over proliferative state of epidermal KC in psoriasis. The proliferative model of HaCaT cells induced by KGF is a ideal model for research in vitro about psoriasis.2. The proliferation of HaCaT cells induced by KGF could be inhibited by 1-shikonin and paeonol by means of preventing the transformation of HaCaT cells into S phase and G2M phase, and the growth as well as the proliferation of HaCaT cells would be at a standstill in G0G1 phase, which might be the mechanisms of action in the treatment of psoriasis with Radix Arnebiae and CortexMoutan, and the the effective ingredients might be 1-shikonin and paeonol. The proliferation of HaCaT cells induced by KGF could be enhanced by Catalpol by means of increasing the number of HaCaT cells into S phase, and it could not treat psoriasis by means of inhibiting the proliferation of KC. The effective ingredient and the mechanisms of action in the treatment of psoriasis with Catalpol would be further explored from other angle.更多还原