羧胺三唑对角质形成细胞增殖与分化的影响及其作用机制的初步研究

发布时间：2018-03-13 11:20

本文选题：羧胺三唑　切入点：银屑病　出处：《北京协和医学院》2012年博士论文　论文类型：学位论文

【摘要】：研究背景银屑病(psoriasis)是一种常见的病情顽固且容易反复发作的慢性炎症性皮肤病。病程大多伴随终生,对人身心健康危害极大。其临床表现以红斑、丘疹、鳞屑为特点,主要组织病理学改变为：角质形成细胞(keratinocyte, KC)过度增殖,真皮炎性细胞浸润,真皮乳头部血管扩张。其独特的表皮棘层肥厚和角化不全,为典型的银屑病皮损特征,是由KC的过度增殖和异常分化造成的。银屑病的确切病因和发病机制尚未完全阐明,其发病机制中涉及到大量的细胞因子,其中肿瘤坏死因子TNF-α、白介素IL-12及IL-23等细胞因子是已明确的非常重要的免疫介导因子。迄今银屑病尚缺乏非常满意的治疗方法,传统的治疗药物毒副作用多,长期使用不良反应严重。而近年来针对细胞因子的抑制剂虽疗效肯定,但费用高昂,不良反应也不容忽视,长期使用的安全性尚不明确。因此,研发新型的疗效高、选择性高、不良反应小且少、成本低的抗银屑病药物意义尤为重大。羧胺三唑(carboxyamidotriazole, CAI)是一种人工合成的小分子化合物,多项研究证实为一种非细胞毒类的钙离子拮抗剂,在体内外模型中均表现出抑制多种肿瘤增殖和转移的作用,并能够抑制血管生成。本课题组的前期结果表明该药物有显著的抗急、慢性炎症作用,且能抑制TNF-a等多种促炎细胞因子的产生。综合以上背景,我们推测：羧胺三唑可能对银屑病有一定的治疗作用。为此,本课题主要针对羧胺三唑对角质形成细胞增殖与分化的影响展开研究,并对其作用机制进行初步探讨,为开发新型抗银屑病药物提供理论依据。研究方法 1.细胞增殖实验：采用台盼蓝染色细胞计数法和CCK-8法检测CAI对人角质形成细胞系HaCaT细胞及人胚胎皮肤成纤维细胞株ESF细胞的增殖影响。建立小鼠巨噬细胞样细胞株RAW264.7细胞与HaCaT细胞的共培养体系,采用CCK-8法观察巨噬细胞是否影响HaCaT细胞的增殖及CAI的抗增殖效应。 2.细胞凋亡实验：采用碘化丙啶染色流式细胞术与磷脂酰丝氨酸外翻分析法分别检测CAI对HaCaT细胞的凋亡及早期凋亡诱导作用。 3.细胞周期分布测定实验：采用碘化丙啶染色流式细胞术检测CAI对HaCaT细胞细胞周期分布的影响。 4.细胞分化检测实验：采用RT-PCR法及Western Blot法分别检测CAI对HaCaT细胞分化相关的多种分子标志mRNA(总苞蛋白、Ⅰ型谷氨酰胺转移酶、角蛋白10、兜甲蛋白、丝聚合蛋白、ΔNp63亚型)及蛋白(Ⅰ型谷氨酰胺转移酶、ΔNp63亚型)表达的影响。 5.动物模型实验：采用常用的抗银屑病药物筛选模型——鼠尾鳞片表皮模型检测CAI对颗粒层细胞生成的影响。通过初步的药效学评价,观察CAI是否有改善银屑病角化不全的作用。在上述模型中观察小鼠的一般状况(体重、排泄及精神状态),初步观察CAI的不良反应。参照国家2008版消毒技术规范中的皮肤刺激实验要求,检测自制CAI软膏对完整皮肤和破损皮肤的刺激强度。探讨CAI发展为局部外用制剂的初步可能性。研究结果 1.细胞增殖实验： 1.1台盼蓝染色细胞计数法结果显示10、20、40μM的CAI分别作用24、48、72h,均能显著抑制HaCaT细胞的增殖,且呈明显的浓度和时间依赖性。CCK-8法结果显示CAI(2.5、5、10、20、40μM)分别作用24、48、72h,均能抑制HaCaT细胞的增殖活力,且有明显的浓度和时间依赖性。两种检测方法结果一致。 1.2CAI(2.5、5、10、20、40μM)作用72h后,对HaCaT及ESF细胞的增殖活力影响结果显示,低浓度的CAI(2.5、5μM)对ESF细胞的增殖基本无影响,当CAI浓度≥10μM(10、20、40μM)后能显著抑制其增殖。而CAI作用HaCaT细胞72h后,即使2.5gM的低浓度CAI亦能显著抑制HaCaT细胞的增殖。表明与ESF细胞相比,低浓度的CAI(2.5、5、10μM)对HaCaT细胞有明显的选择性抑制作用。 1.3RAW264.7细胞经不同的药物诱导24h后,在光学显微镜下观察到经LPS(1μg/mL)诱导过的细胞大多伸出伪足,形态发生很大改变。CAI和LPS共同孵育组只有少数细胞伸出很短的伪足。提示LPS能诱导RAW264.7细胞的活化,而CAI则能部分阻断LPS对RAW264.7细胞的激活。 LPS直接作用于HaCaT细胞,并不影响其增殖。RAW264.7细胞无论是先经LPS诱导24h后再与HaCaT细胞共培养,亦或是在与HaCaT细胞共培养体系中经LPS持续诱导,都不直接影响HaCaT细胞的增殖。并且LPS以及经LPS诱导的RAW264.7细胞均不直接影响CAI对HaCaT细胞的抗增殖效应。 2.细胞凋亡实验：碘化丙啶染色法流式细胞术检测凋亡结果显示,不同浓度的CAI(5、20、40μM)分别作用24、48、72h后,只有高浓度的CAI(40μM)对HaCaT细胞有显著的凋亡诱导作用,且有一定的时间依赖性；而低浓度的CAI(≤20μM)对HaCaT细胞的凋亡诱导作用不明显。磷脂酰丝氨酸外翻分析法检测早期凋亡结果显示,不同浓度的CAI(20、40μM)分别作用24、48h后,只有40μM CAI能显著诱导HaCaT细胞的早期凋亡和晚期凋亡,而20μM CAI仅在作用24h时显示对HaCaT细胞的早期凋亡有诱导作用。两种检测方法结果基本一致。 3.细胞周期分布测定实验：碘化丙啶染色法流式细胞术检测细胞周期分布结果显示,不同浓度的CAI(5、20、40μM)分别作用24、48、72h后,随着CAI浓度的提高,HaCaT细胞在Go/G1期的比例明显升高,而S期比例显著下降,具有显著性统计学差异。表明CAI对HaCaT细胞的细胞周期有明显的阻滞作用,并主要将细胞阻滞在Go/G1期,且呈现明显的浓度依赖性,时间依赖性关系不明显。 4.细胞分化检测实验： RT-PCR结果显示, CAI(20μM)作用12h后,能显著降低HaCaT细胞中分化相关的分子标志Ⅰ型谷氨酰胺转移酶、总苞蛋白、兜甲蛋白和ANp63亚型的mRNA表达水平,具有显著性统计学差异(P0.05或P0.01)。Western Blot结果显示,40μMCAI作用48h后,⒈型谷氨酰胺转移酶蛋白的表达水平明显降低,有显著性统计学差异(P0.01)。而ΔNp63蛋白的表达水平没有改变。提示CAI有调节细胞分化、改善异常分化作用,可能能改善角化异常。 5.动物模型实验： 5.1鼠尾鳞片表皮模型的表皮切片在光学显微镜下观察发现,生理盐水组和PEG组颗粒层细胞少,有缺失。阳性药物甲氨蝶呤组和CAI高剂量(30、40mg/kg)给药组颗粒层细胞较多,比较完整。表明阳性药物甲氨蝶呤和CAI高剂量给药可以促进小鼠尾部鳞片中颗粒层细胞的生成。统计结果显示,阳性药物甲氨蝶呤组含颗粒层的鳞片数百分率与生理盐水对照组比较明显上升,具有显著性统计学差异(n=12,P0.001)。CAI30、40mg/kg组与PEG组比较明显上升,有显著性统计学差异(n=12,P0.01)。表明CAI能够促进鼠尾鳞状上皮中颗粒细胞的生成,并呈现-定的剂量依赖性,提示CAI能改善角化不全。 5.2鼠尾鳞片表皮模型中观察小鼠的一般状况,CAI组小鼠均有不同程度的精神萎靡及腹泻症状,且呈明显的剂量依赖性关系,同一个体的腹泻症状一周后有所减轻。从第10天开始,40mg/kg CAI剂量组的体重与PEG组比较,有显著性差异(n=12,P0.05)。初步验证了CAI有中枢抑制及消化道的不良反应。自制CAI软膏的皮肤刺激试验中,一次完整皮肤刺激试验和多次完整皮肤刺激试验(即每天涂抹1次CAI软膏,连续涂抹14d)结果显示,4只豚鼠皮肤均无红斑和水肿形成,刺激指数为0,刺激强度级别属无刺激性。一次破损皮肤刺激试验的结果显示,只有1只豚鼠的背部皮肤在去除CAI1h后出现勉强可见的红斑和水肿,取其中最高皮肤刺激指数0.5,评定刺激强度级别属轻刺激性。表明CAI外用涂抹对完整皮肤无刺激性,对破损皮肤有轻刺激性。提示CAI发展为局部外用制剂的初步可行性。研究结论 1.羧胺三唑能够显著抑制HaCaT细胞的增殖,该抗增殖效应可能是通过诱导HaCaT细胞Go/G1期周期阻滞及细胞凋亡来实现。且该效应具有一定细胞选择性。巨噬细胞不直接影响羧胺三唑对HaCaT细胞的抗增殖效应。 2.根据羧胺三唑能够抑制HaCaT细胞增殖、诱导细胞周期阻滞、诱导细胞凋亡、改善异常分化、改善角化不全的结果,我们推测,羧胺三唑可能对银屑病有一定的潜在应用价值。
[Abstract]:Research background
Psoriasis (psoriasis) is a common chronic inflammatory skin disease was stubborn and easy to attack again. The course is usually accompanied by a lifetime, great harm to human health. The clinical manifestations of erythema, papules, scales as the main features, histopathological changes: keratinocytes (keratinocyte, KC) proliferation. Dermal inflammatory cell infiltration, dermal papilla vascular dilatation. Its unique epidermal acanthosis and hyperkeratosis, as the typical feature of psoriasis, is caused by excessive proliferation and abnormal differentiation of KC. The exact etiology and pathogenesis of psoriasis has not been fully elucidated, involving a large number of cytokines in the pathogenesis of TNF-, tumor necrosis factor alpha, interleukin IL-12 and IL-23 cytokine mediated immune is very important. So far has a clear guiding factor of psoriasis treatment method is lack of very satisfied, traditional The treatment of drug side effects, long-term use of serious adverse reactions. In recent years, inhibitors targeting cytokines although certainly effective, but expensive, adverse reactions can not be ignored, the safety of long-term use is not clear. Therefore, the development of a new kind of high efficacy, high selectivity, and less adverse reactions, anti psoriasis a medicine low cost is particularly important.
Three carboxyamido triazole (carboxyamidotriazole, CAI) is a kind of small molecule synthetic compounds, a number of studies have confirmed a non cytotoxic calcium antagonist, in vitro and in vivo models showed inhibition of proliferation and metastasis of many kinds of tumors, which can inhibit the angiogenesis of the early results of our research group. Show that the drug has significant anti acute, chronic inflammation, and a variety of proinflammatory cytokines inhibit TNF-a production.
Based on the above background, we hypothesized that carboxyamido triazole three may be useful for treating psoriasis. Therefore, this study focuses on the influence of carboxyamido triazole three formation of cell proliferation and differentiation of keratinocytes was investigated, and to explore its mechanism, provide a theoretical basis for the development of new anti psoriasis drug.
research method
1. cell proliferation experiment:
Trypan blue staining, cell counting and CCK-8 were used to detect the effect of CAI on the proliferation of human keratinocyte line HaCaT cells and human embryonic fibroblast cell line ESF cells.
A co culture system of mouse macrophage like cell line RAW264.7 cells and HaCaT cells was established. CCK-8 method was used to observe whether macrophages affect the proliferation of HaCaT cells and the anti proliferative effect of CAI.
2. cell apoptosis experiment: using propidium iodide staining flow cytometry and phosphatidyl serine valgus analysis method, respectively
The effect of CAI on apoptosis and early apoptosis induced by HaCaT cells was detected.
3. cell cycle distribution test: the effect of CAI on the cell cycle distribution of HaCaT cells was detected by flow cytometry with propidium iodide staining.
4. cell differentiation assay: to detect CAI on the differentiation of HaCaT cells of various molecular markers related to mRNA using RT-PCR method and Western Blot method (involucre protein, type I transglutaminase, keratin 10, loricrin, filaggrin, Np63 subtype) and protein (type I transglutaminase, Delta Np63 the expression of subtypes).
5. animal model experiment:
A commonly used psoriasis drug screening model, rat tail flake epidermis model, was used to detect the effect of CAI on granulosa cell formation. Preliminary pharmacodynamic evaluation was carried out to observe whether CAI could improve psoriatic keratinization.
In the above model, the general state of the mice (weight, excretion and mental state) was observed, and the adverse reaction of CAI was observed.
Referring to the skin irritation test requirements in the disinfection technology specification of national 2008 edition, the irritation intensity of homemade CAI ointment on intact skin and damaged skin was detected, and the possibility of developing CAI as topical external preparation was discussed.
Research results
1. cell proliferation experiment:
1.1 trypan blue staining and cell counting method showed that 10,20,40 M CAI respectively. 24,48,72h can significantly inhibit the proliferation of HaCaT cells, and showed a time and concentration dependent.CCK-8 assay showed that CAI (2.5,5,10,20,40 M) respectively. 24,48,72h can inhibit HaCaT cell proliferation activity, and has obvious concentration and time dependence. The results of the two detection methods.
1.2CAI (2.5,5,10,20,40 M) 72h, HaCaT and ESF on the proliferation activity of the cells showed that low concentration of CAI (2.5,5 M) on the proliferation of ESF cells has no effect when the CAI concentration is 10 M (10,20,40 M) could significantly inhibit the proliferation of CAI and HaCaT fine. 72h cell, even low concentration of CAI 2.5gM can significantly inhibit the proliferation of HaCaT cells. Compared with ESF cells, the low concentration of CAI (2.5,5,10 M) significantly inhibited the selectivity of HaCaT cells.
1.3RAW264.7 cells were treated with different drugs induced by 24h, were observed under light microscope after LPS (1 g/mL) induced by the cell mostly stretched out pseudopodia, style change greatly.CAI and LPS incubation group only a few cells extended short pseudopodia. That activation of LPS can induce RAW264.7 cells, whereas CAI partially blocking the activation of LPS on RAW264.7 cells.
The effect of LPS on HaCaT cells, did not affect the proliferation of.RAW264.7 cells induced by LPS and whether it is the first 24h after co cultured with HaCaT cells or in co cultured with HaCaT cells in LPS induced by continuous system, do not directly affect the proliferation of HaCaT cells. And LPS and LPS induced by RAW264.7 cells do not directly affect the anti proliferative effect of CAI on HaCaT cells.
2. cell apoptosis experiment:
Flow cytometry results showed that propidium iodide staining, different concentrations of CAI (5,20,40 M) respectively. After 24,48,72h, only the high concentration of CAI (40 M) were induced apoptosis of HaCaT cells, and there is a certain time dependence; while low concentrations (less than 20 CAI M) on the apoptosis of HaCaT cells induced by the effect is not obvious.
Phosphatidylserine analysis method for early detection of apoptosis showed that different concentrations of CAI (20,40 M) respectively. After 24,48h, early and late apoptosis only 40 M CAI could significantly induce HaCaT cells, while 20 M CAI only in the role of the 24h showed early apoptosis of HaCaT cells induced by. Two detection methods were basically the same.
3. cell cycle distribution test:
Flow cytometry results showed that the distribution of propidium iodide staining, different concentrations of CAI (5,20,40 M 24,48,72h) respectively, with the increase of the concentration of CAI, HaCaT cells significantly increased in the proportion of Go/G1 phase, and S phase was significantly decreased, with significant statistical difference. That block effect of CAI on the cell cycle of HaCaT cells, and arrest cells in Go/G1 phase, and the obvious concentration dependent, time dependent relationship is not obvious.
4. cell differentiation test:
RT-PCR results showed that CAI (20 M) after 12h treatment can significantly reduce the HaCaT cell differentiation related markers of transglutaminase, involucre protein, loricrin and ANp63 subtype mRNA expression, with significant statistical differences (P0.05 or P0.01).Western Blot results showed that 40 MCAI after 48h, the type of transglutaminase enzyme protein expression level decreased significantly, there was significant difference (P0.01). The expression of delta Np63 protein has not changed. Suggesting that CAI regulates cell differentiation, improve abnormal differentiation, can improve the abnormal keratosis.
5. animal model experiment:
The 5.1 mouse tail epidermis model skin were observed under the light microscope, normal saline group and PEG group of granulosa cells, are missing. The positive drug group and CAI high dose methotrexate (30,40mg/kg) treatment group granulosa cells are relatively complete. Show the positive drug methotrexate and high dose of CAI can promote the formation of granular layer cells of mouse tail scales.
The statistical results showed that the percentage of the number of scales with saline positive drug methotrexate group particle containing layer was significantly increased compared with control group, significant difference (n=12, P0.001).CAI30,40mg/kg group and PEG group increased significantly, there was significant difference (n= 12, P0.01). The results indicated that CAI can promote the formation of granular cells of rat tail squamous epithelium, and showed certain dose dependent, suggesting that CAI can improve parakeratosis.
Observe the general situation of the 5.2 mouse tail scaled epidermis model, CAI mice were depressed and the symptoms of diarrhea, and a significant dose dependent relationship, diarrhea symptoms of the same individual after a week has been reduced. From the beginning of the tenth day, 40mg/kg dose of CAI body weight compared with the PEG group, a significant differences (n=12, P0.05). A preliminary validation of the CAI adverse reaction of central inhibition and digestive tract.
Homemade CAI ointment on the skin irritation test, a complete skin irritation test and skin irritation test (repeated every 1 times smear CAI ointment, continuous smear 14d) showed that 4 guinea pigs showed no skin erythema and edema formation, stimulation index was 0, the stimulus intensity level no stimulation. Once damaged skin irritation test showed that only 1 of the guinea pig dorsal skin barely visible erythema and edema in the removal of CAI1h, the highest skin irritation index 0.5, assessment of stimulus intensity level is a light stimulus. External application showed that CAI had no irritation to the intact skin, light irritation on the damaged skin. CAI the development for the preliminary feasibility of topical preparations.
research conclusion
1. three carboxyamido triazole can significantly inhibit the proliferation of HaCaT cells, the anti proliferation effect could be achieved by inducing HaCaT cell cycle arrest and apoptosis of Go/G1 cells. And this effect has certain selectivity. Macrophages do not directly affect the anti proliferative effects of carboxyamido Triazole on HaCaT three cells.
2., carboxyamines three azole can inhibit the proliferation of HaCaT cells, induce cell cycle arrest, induce cell apoptosis, improve abnormal differentiation and improve the results of keratinization. We speculate that carboxyamine three azole may have potential application in psoriasis.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R758.63

【共引文献】