本文選題：羧胺三唑　切入點(diǎn)：銀屑病　出處：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景 銀屑病(psoriasis)是一種常見的病情頑固且容易反復(fù)發(fā)作的慢性炎癥性皮膚病。病程大多伴隨終生,對(duì)人身心健康危害極大。其臨床表現(xiàn)以紅斑、丘疹、鱗屑為特點(diǎn),主要組織病理學(xué)改變?yōu)椋航琴|(zhì)形成細(xì)胞(keratinocyte, KC)過度增殖,真皮炎性細(xì)胞浸潤(rùn),真皮乳頭部血管擴(kuò)張。其獨(dú)特的表皮棘層肥厚和角化不全,為典型的銀屑病皮損特征,是由KC的過度增殖和異常分化造成的。銀屑病的確切病因和發(fā)病機(jī)制尚未完全闡明,其發(fā)病機(jī)制中涉及到大量的細(xì)胞因子,其中腫瘤壞死因子TNF-α、白介素IL-12及IL-23等細(xì)胞因子是已明確的非常重要的免疫介導(dǎo)因子。迄今銀屑病尚缺乏非常滿意的治療方法,傳統(tǒng)的治療藥物毒副作用多,長(zhǎng)期使用不良反應(yīng)嚴(yán)重。而近年來針對(duì)細(xì)胞因子的抑制劑雖療效肯定,但費(fèi)用高昂,不良反應(yīng)也不容忽視,長(zhǎng)期使用的安全性尚不明確。因此,研發(fā)新型的療效高、選擇性高、不良反應(yīng)小且少、成本低的抗銀屑病藥物意義尤為重大。 羧胺三唑(carboxyamidotriazole, CAI)是一種人工合成的小分子化合物,多項(xiàng)研究證實(shí)為一種非細(xì)胞毒類的鈣離子拮抗劑,在體內(nèi)外模型中均表現(xiàn)出抑制多種腫瘤增殖和轉(zhuǎn)移的作用,并能夠抑制血管生成。本課題組的前期結(jié)果表明該藥物有顯著的抗急、慢性炎癥作用,且能抑制TNF-a等多種促炎細(xì)胞因子的產(chǎn)生。 綜合以上背景,我們推測(cè)：羧胺三唑可能對(duì)銀屑病有一定的治療作用。為此,本課題主要針對(duì)羧胺三唑?qū)琴|(zhì)形成細(xì)胞增殖與分化的影響展開研究,并對(duì)其作用機(jī)制進(jìn)行初步探討,為開發(fā)新型抗銀屑病藥物提供理論依據(jù)。 研究方法 1.細(xì)胞增殖實(shí)驗(yàn)： 采用臺(tái)盼藍(lán)染色細(xì)胞計(jì)數(shù)法和CCK-8法檢測(cè)CAI對(duì)人角質(zhì)形成細(xì)胞系HaCaT細(xì)胞及人胚胎皮膚成纖維細(xì)胞株ESF細(xì)胞的增殖影響。 建立小鼠巨噬細(xì)胞樣細(xì)胞株RAW264.7細(xì)胞與HaCaT細(xì)胞的共培養(yǎng)體系,采用CCK-8法觀察巨噬細(xì)胞是否影響HaCaT細(xì)胞的增殖及CAI的抗增殖效應(yīng)。 2.細(xì)胞凋亡實(shí)驗(yàn)：采用碘化丙啶染色流式細(xì)胞術(shù)與磷脂酰絲氨酸外翻分析法分別 檢測(cè)CAI對(duì)HaCaT細(xì)胞的凋亡及早期凋亡誘導(dǎo)作用。 3.細(xì)胞周期分布測(cè)定實(shí)驗(yàn)：采用碘化丙啶染色流式細(xì)胞術(shù)檢測(cè)CAI對(duì)HaCaT細(xì)胞細(xì)胞周期分布的影響。 4.細(xì)胞分化檢測(cè)實(shí)驗(yàn)：采用RT-PCR法及Western Blot法分別檢測(cè)CAI對(duì)HaCaT細(xì)胞分化相關(guān)的多種分子標(biāo)志mRNA(總苞蛋白、Ⅰ型谷氨酰胺轉(zhuǎn)移酶、角蛋白10、兜甲蛋白、絲聚合蛋白、ΔNp63亞型)及蛋白(Ⅰ型谷氨酰胺轉(zhuǎn)移酶、ΔNp63亞型)表達(dá)的影響。 5.動(dòng)物模型實(shí)驗(yàn)： 采用常用的抗銀屑病藥物篩選模型——鼠尾鱗片表皮模型檢測(cè)CAI對(duì)顆粒層細(xì)胞生成的影響。通過初步的藥效學(xué)評(píng)價(jià),觀察CAI是否有改善銀屑病角化不全的作用。 在上述模型中觀察小鼠的一般狀況(體重、排泄及精神狀態(tài)),初步觀察CAI的不良反應(yīng)。 參照國(guó)家2008版消毒技術(shù)規(guī)范中的皮膚刺激實(shí)驗(yàn)要求,檢測(cè)自制CAI軟膏對(duì)完整皮膚和破損皮膚的刺激強(qiáng)度。探討CAI發(fā)展為局部外用制劑的初步可能性。 研究結(jié)果 1.細(xì)胞增殖實(shí)驗(yàn)： 1.1臺(tái)盼藍(lán)染色細(xì)胞計(jì)數(shù)法結(jié)果顯示10、20、40μM的CAI分別作用24、48、72h,均能顯著抑制HaCaT細(xì)胞的增殖,且呈明顯的濃度和時(shí)間依賴性。CCK-8法結(jié)果顯示CAI(2.5、5、10、20、40μM)分別作用24、48、72h,均能抑制HaCaT細(xì)胞的增殖活力,且有明顯的濃度和時(shí)間依賴性。兩種檢測(cè)方法結(jié)果一致。 1.2CAI(2.5、5、10、20、40μM)作用72h后,對(duì)HaCaT及ESF細(xì)胞的增殖活力影響結(jié)果顯示,低濃度的CAI(2.5、5μM)對(duì)ESF細(xì)胞的增殖基本無影響,當(dāng)CAI濃度≥10μM(10、20、40μM)后能顯著抑制其增殖。而CAI作用HaCaT細(xì)胞72h后,即使2.5gM的低濃度CAI亦能顯著抑制HaCaT細(xì)胞的增殖。表明與ESF細(xì)胞相比,低濃度的CAI(2.5、5、10μM)對(duì)HaCaT細(xì)胞有明顯的選擇性抑制作用。 1.3RAW264.7細(xì)胞經(jīng)不同的藥物誘導(dǎo)24h后,在光學(xué)顯微鏡下觀察到經(jīng)LPS(1μg/mL)誘導(dǎo)過的細(xì)胞大多伸出偽足,形態(tài)發(fā)生很大改變。CAI和LPS共同孵育組只有少數(shù)細(xì)胞伸出很短的偽足。提示LPS能誘導(dǎo)RAW264.7細(xì)胞的活化,而CAI則能部分阻斷LPS對(duì)RAW264.7細(xì)胞的激活。 LPS直接作用于HaCaT細(xì)胞,并不影響其增殖。RAW264.7細(xì)胞無論是先經(jīng)LPS誘導(dǎo)24h后再與HaCaT細(xì)胞共培養(yǎng),亦或是在與HaCaT細(xì)胞共培養(yǎng)體系中經(jīng)LPS持續(xù)誘導(dǎo),都不直接影響HaCaT細(xì)胞的增殖。并且LPS以及經(jīng)LPS誘導(dǎo)的RAW264.7細(xì)胞均不直接影響CAI對(duì)HaCaT細(xì)胞的抗增殖效應(yīng)。 2.細(xì)胞凋亡實(shí)驗(yàn)： 碘化丙啶染色法流式細(xì)胞術(shù)檢測(cè)凋亡結(jié)果顯示,不同濃度的CAI(5、20、40μM)分別作用24、48、72h后,只有高濃度的CAI(40μM)對(duì)HaCaT細(xì)胞有顯著的凋亡誘導(dǎo)作用,且有一定的時(shí)間依賴性；而低濃度的CAI(≤20μM)對(duì)HaCaT細(xì)胞的凋亡誘導(dǎo)作用不明顯。 磷脂酰絲氨酸外翻分析法檢測(cè)早期凋亡結(jié)果顯示,不同濃度的CAI(20、40μM)分別作用24、48h后,只有40μM CAI能顯著誘導(dǎo)HaCaT細(xì)胞的早期凋亡和晚期凋亡,而20μM CAI僅在作用24h時(shí)顯示對(duì)HaCaT細(xì)胞的早期凋亡有誘導(dǎo)作用。兩種檢測(cè)方法結(jié)果基本一致。 3.細(xì)胞周期分布測(cè)定實(shí)驗(yàn)： 碘化丙啶染色法流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期分布結(jié)果顯示,不同濃度的CAI(5、20、40μM)分別作用24、48、72h后,隨著CAI濃度的提高,HaCaT細(xì)胞在Go/G1期的比例明顯升高,而S期比例顯著下降,具有顯著性統(tǒng)計(jì)學(xué)差異。表明CAI對(duì)HaCaT細(xì)胞的細(xì)胞周期有明顯的阻滯作用,并主要將細(xì)胞阻滯在Go/G1期,且呈現(xiàn)明顯的濃度依賴性,時(shí)間依賴性關(guān)系不明顯。 4.細(xì)胞分化檢測(cè)實(shí)驗(yàn)： RT-PCR結(jié)果顯示, CAI(20μM)作用12h后,能顯著降低HaCaT細(xì)胞中分化相關(guān)的分子標(biāo)志Ⅰ型谷氨酰胺轉(zhuǎn)移酶、總苞蛋白、兜甲蛋白和ANp63亞型的mRNA表達(dá)水平,具有顯著性統(tǒng)計(jì)學(xué)差異(P0.05或P0.01)。Western Blot結(jié)果顯示,40μMCAI作用48h后,⒈型谷氨酰胺轉(zhuǎn)移酶蛋白的表達(dá)水平明顯降低,有顯著性統(tǒng)計(jì)學(xué)差異(P0.01)。而ΔNp63蛋白的表達(dá)水平?jīng)]有改變。提示CAI有調(diào)節(jié)細(xì)胞分化、改善異常分化作用,可能能改善角化異常。 5.動(dòng)物模型實(shí)驗(yàn)： 5.1鼠尾鱗片表皮模型的表皮切片在光學(xué)顯微鏡下觀察發(fā)現(xiàn),生理鹽水組和PEG組顆粒層細(xì)胞少,有缺失。陽性藥物甲氨蝶呤組和CAI高劑量(30、40mg/kg)給藥組顆粒層細(xì)胞較多,比較完整。表明陽性藥物甲氨蝶呤和CAI高劑量給藥可以促進(jìn)小鼠尾部鱗片中顆粒層細(xì)胞的生成。 統(tǒng)計(jì)結(jié)果顯示,陽性藥物甲氨蝶呤組含顆粒層的鱗片數(shù)百分率與生理鹽水對(duì)照組比較明顯上升,具有顯著性統(tǒng)計(jì)學(xué)差異(n=12,P0.001)。CAI30、40mg/kg組與PEG組比較明顯上升,有顯著性統(tǒng)計(jì)學(xué)差異(n=12,P0.01)。表明CAI能夠促進(jìn)鼠尾鱗狀上皮中顆粒細(xì)胞的生成,并呈現(xiàn)-定的劑量依賴性,提示CAI能改善角化不全。 5.2鼠尾鱗片表皮模型中觀察小鼠的一般狀況,CAI組小鼠均有不同程度的精神萎靡及腹瀉癥狀,且呈明顯的劑量依賴性關(guān)系,同一個(gè)體的腹瀉癥狀一周后有所減輕。從第10天開始,40mg/kg CAI劑量組的體重與PEG組比較,有顯著性差異(n=12,P0.05)。初步驗(yàn)證了CAI有中樞抑制及消化道的不良反應(yīng)。 自制CAI軟膏的皮膚刺激試驗(yàn)中,一次完整皮膚刺激試驗(yàn)和多次完整皮膚刺激試驗(yàn)(即每天涂抹1次CAI軟膏,連續(xù)涂抹14d)結(jié)果顯示,4只豚鼠皮膚均無紅斑和水腫形成,刺激指數(shù)為0,刺激強(qiáng)度級(jí)別屬無刺激性。一次破損皮膚刺激試驗(yàn)的結(jié)果顯示,只有1只豚鼠的背部皮膚在去除CAI1h后出現(xiàn)勉強(qiáng)可見的紅斑和水腫,取其中最高皮膚刺激指數(shù)0.5,評(píng)定刺激強(qiáng)度級(jí)別屬輕刺激性。表明CAI外用涂抹對(duì)完整皮膚無刺激性,對(duì)破損皮膚有輕刺激性。提示CAI發(fā)展為局部外用制劑的初步可行性。 研究結(jié)論 1.羧胺三唑能夠顯著抑制HaCaT細(xì)胞的增殖,該抗增殖效應(yīng)可能是通過誘導(dǎo)HaCaT細(xì)胞Go/G1期周期阻滯及細(xì)胞凋亡來實(shí)現(xiàn)。且該效應(yīng)具有一定細(xì)胞選擇性。巨噬細(xì)胞不直接影響羧胺三唑?qū)aCaT細(xì)胞的抗增殖效應(yīng)。 2.根據(jù)羧胺三唑能夠抑制HaCaT細(xì)胞增殖、誘導(dǎo)細(xì)胞周期阻滯、誘導(dǎo)細(xì)胞凋亡、改善異常分化、改善角化不全的結(jié)果,我們推測(cè),羧胺三唑可能對(duì)銀屑病有一定的潛在應(yīng)用價(jià)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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R758.63

【共引文獻(xiàn)】

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