【作者】 徐丽敏；

【导师】 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2007， 博士

【摘要】 本研究从研究银屑病皮损角质形成细胞过度增殖、新生血管和炎症细胞的浸润角度,采用免疫组织化学、抗体夹心酶联免疫吸附和流失细胞分析仪等方法,检测各类粘附分子和VEGF的表达。选择血管内皮细胞(HUVEC)原代培养和角质形成细胞的永生化细胞株HaCaT的基础上,以MTT比色法研究伊曲替酸对HUVEC和HaCaT增殖的调控作用,并通过流式细胞技术检测各个细胞周期的变化。依据中医辨证论治自组复方中药“七皮饮”治疗银屑病,采用抗体夹心酶联免疫吸附方法,检测治疗前后γ-IFN和VCAM-1。结果如下:采用免疫组织化学和抗体夹心酶联免疫吸附等方法,检测银屑病患者皮损组织和血清各类粘附分子和VEGF的表达,运用统计学手段分析实验结果,获得银屑病皮损组织中检测分子强弱排序为VCAM-1> PECAM-1> ICAM-1> VEGF> LFA-1> VLA-4> Mac-1。表明银屑病皮损真皮乳头毛细血管扩张、迂曲、通透性增加和血管内皮细胞增生与细胞因子密切相关程度。PECAM-1,可以作为银屑病皮损真皮乳头血管增生的敏感性标记。建立VCAM-1和ICAM-1判别诊治银屑病结果的敏感度和特异度实验室指标。本实验结果为治疗银屑病提供药物的作用靶点;为临床判别银屑病发生、发展和转归的判别指标,及用于研究治疗银屑病药物的药理作用的判别指标。采用免疫组织化学和流失细胞分析仪等方法,检测到进行期银屑病患者皮损CLA和E选择素的表达均呈强阳性,而非皮区和正常皮肤表达微量。证明银屑病皮损中有大量记忆的CLA+T细胞。检测到进展期银屑病患者外周血中CLA+ CD3+T、CLA+ CD4+T和CLA+ CD8+T细胞表达明显高于正常人和静止期患者。进一步表明进行期银屑病患者血循环中,有大量被激活的记忆CLA+ T细胞。这些记忆CLA+ T细胞不断定位向皮肤归巢,引发一系列细胞免疫异常,最终导致银屑病皮损发生。通过体外模拟感染刺激使记忆的CLA+T细胞增殖,更进一步证实CLA+T细胞是引起银屑病发生和加重的重要途径。选择阻断记忆CLA+ T细胞定位向皮肤归巢为药物作用靶点,将是开发治疗银屑病新药的又一有效途径。。以MTT比色法和流式细胞技术检测伊曲替酸对HUVEC和HaCaT增殖和细胞周期调控,结果发现伊曲替酸对内皮细胞的调控为先增殖后抑制的趋势,对角质形成细胞株HaCaT是直接抑制的作用,其作用都与药物浓度和作用时间相关。另外,伊曲替酸使内皮细胞的周期阻滞于G1期,而HaCaT的周期被阻滞于S期,对细胞周期的影响同样与药物浓度和作用时间相关。运用抗体夹心酶联免疫吸附检测复方中药“七皮饮”对银屑病患者外周血中的γ-IFN和VCAM-1的含量表达的作用。结果表现减少其表达量。表明“七皮饮”不仅抑制表皮角质形成细胞过度增殖,而且还可以抑制真皮微血管新生,降低血管的通透性。发现“七皮饮”具有抑制角质形成细胞增殖增值率,使细胞阻滞在G1期。其中的地骨皮具有促进细胞增殖,而桑白皮则抑制细胞增殖,显示了君药二者的一促一抑,相生相克。“七皮饮”即表现出抑制细胞的作用又不至于过分的抑制细胞的生长,从中西医结合的角度达到治疗的目的。进一步证明复方中药方具有多靶点、多层次的药理作用。更多还原

【Abstract】 At the point of keratinocytic proliferation, angiogenesis and invasion of inflammatory cell in psoriatic lesions, the adhesion molecules, VEGF, lymphocyte-associated antigen （CLA） in lesions and peripheral bloods from patients with psoriasis were detected by immunohistochemistory, Enzyme linked immunoabsorbent assay（ELISA） and flow cytometric analysis. we investigated the modulation of acitretin to the proliferation of endothelial cell keratinocyte line HaCaT by immunohistochemistory and MTT method; we measured the variation of acitretin to the cell cycle of endothelial cell and HaCaT with the flow cytometric method, According to the tradition Chinese medicine we composed a complex herbs of“QiPiYin”to treat psoriasis, then detected the content of VCAM-1 and IFN-γby Enzyme linked immunoabsorbent assay（ELISA） before and after treating. The results showed as the following:①By using statistical analysis , the results show that the intensity of each expression: VCAM-1> PECAM-1> ICAM-1> VEGF> LFA-1> VLA-4> Mac-1. PECAM-1 is the sensitive sign of the blood vessel proliferation of dermis layer in psoriatic lesions.②Increased VCAM-1 in serum set up the sensitive and special mark of laboratory to distinguish diagnosis and treatment of psoriatic.③The results shows that in psoriatic skin lesions the expression of CLA and its ligand E-selectin was strongly positive, that in peripheral blood CLA on CD4+、CD8+、CD3+T cell from progressive phase patients was much higher than that from static phase psoriatic patients and also higher than health controls. Compared with health controls, only the frequency of CLA+CD8+T cells was increased in static phase psoriatic patients.④HUVEC reacted to acitretin treatment with a double direction of modulation. Along with the gradually increased concentrations, the endothelial cell proliferation rates were enhanced earlier with a peak effect, and then were inhibited. HaCaT reacted with an obviously inhibitory effect. In a time-course study, the effect of acitretin on cell proliferation was notable gradually.⑤HUVEC and HaCaT cell cycle modulations to acitretin were different completely. The drug induced G1-phase arrest of endothelial cell, and S-phase arrest of HaCaT.⑥The heat blood type psoriasis showed to improve sing and reduce PASI score in lesions and decline sVCAM-1 and sIFN-γcontent in serum psoriasis patient by intervened Chinese traditional herbs—“QiPiYin”. Chinese Wolfberry Root-bark can promote the proliferation of HaCaT . White Mulberry Root-bark,Cortex Moutan,Areca Peel,Silktree Albizia Bark,Dictamni Cortex,Dried Tangerine Peel and“QiPiYin”can inhibit the proliferation of HaCaT. And with the increasing of concentration,the effect was enhanced.The inhibitive action of“QiPiYin”on HaCaT was the best. Further study by DNA flow cytometric analysis revealed that“QiPiYin”could alter the distribution of HaCaT cell cycle obviously. which shown that the cell percentage of HaCaT in G₀/G₁ phase increased and those in S phase decreased.更多还原