本文選題：沙利度胺 + 銀屑病�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文

【摘要】：背景 在20世紀(jì)50年代,沙利度胺曾作為一種鎮(zhèn)靜劑而廣泛用于孕吐的治療,隨后卻因其致畸作用而退市。但近年來,沙利度胺因其能降低患者體內(nèi)TNF-a水平及抑制血管生成的作用而被FDA批準(zhǔn)用于麻風(fēng)結(jié)節(jié)性紅斑及多發(fā)性骨髓瘤的治療。此外,研究者也陸續(xù)發(fā)現(xiàn),沙利度胺還對多種經(jīng)常規(guī)治療方法無效的皮膚病有良好的治療作用,但其治療這些疾病的作用機(jī)制尚不明確。因沙利度胺具有免疫調(diào)節(jié)作用,很早就有學(xué)者推測沙利度胺可以用于銀屑病的治療。故在前期的臨床實(shí)踐中,我們采用沙利度胺治療了部分患者后,發(fā)現(xiàn)沙利度胺對銀屑病有一定的治療作用。研究發(fā)現(xiàn)樹突狀細(xì)胞及其分泌的IL-12和IL23、淋巴細(xì)胞及其分泌的IL-17和IL22,角質(zhì)形成細(xì)胞及其分泌的TNF-a及VEGF在銀屑病的發(fā)病機(jī)制中起著十分關(guān)鍵的作用。目前已有較多關(guān)于沙利度胺對樹突狀細(xì)胞及淋巴細(xì)胞影響的實(shí)驗(yàn)研究,而沙利度胺對角質(zhì)形成細(xì)胞作用的研究則較為罕見,故有必要進(jìn)一步研究沙利度胺對角質(zhì)形成細(xì)胞的作用。 目的 TNF-a是銀屑病中核心的促炎癥細(xì)胞因子之一,角質(zhì)形成細(xì)胞過度增殖及真皮乳頭層毛細(xì)血管增生是銀屑病皮損中最為典型的組織病理學(xué)表現(xiàn)。TNF-a與角質(zhì)形成細(xì)胞過度增殖在銀屑病中所起的關(guān)鍵作用已被目前廣泛用于重度銀屑病治療的TNF-a拮抗劑及甲氨蝶呤的顯著療效所證實(shí)；在動物模型中,抗血管生成藥也可以顯著改善銀屑病樣皮損。故我們擬通過研究沙利度胺對角質(zhì)形成細(xì)胞活性的影響及其對角質(zhì)形成細(xì)胞VEGF和TNF-a表達(dá)的影響,以進(jìn)一步了解沙利度胺在皮膚疾病治療中的作用機(jī)制,并為沙利度胺用于銀屑病的治療提供實(shí)驗(yàn)基礎(chǔ)。 方法 1)體外培養(yǎng)HaCaT細(xì)胞。 2)WST-l法檢測不同濃度沙利度胺對HaCaT細(xì)胞活性的影響。 3)通過引物設(shè)計(jì)及驗(yàn)證和調(diào)整反應(yīng)體系擴(kuò)增效率使實(shí)時(shí)定量PCR反應(yīng)體系滿足通過2-ΔΔCt法分析相對基因表達(dá)差異的條件。 4)實(shí)時(shí)定量PCR法測定不同時(shí)間點(diǎn)沙利度胺對HaCaT細(xì)胞合成VEGF與TNF-α mRNA的影響。 5)實(shí)時(shí)定量PCR法測定不同濃度沙利度胺對HaCaT細(xì)胞合成VEGF與TNF-α mRNA的影響。 5) ELISA法測定不同濃度沙利度胺對HaCaT細(xì)胞分泌VEGF與TNF-a蛋白的影響。 結(jié)果 1)當(dāng)沙利度胺濃度≤100nM時(shí),對HaCaT細(xì)胞增殖無影響；當(dāng)沙利度胺濃度為1000nM時(shí),HaCaT細(xì)胞活性受到抑制,約為對照組的90.8%；沙利度胺濃度≥1000nM時(shí),其抑制HaCaT細(xì)胞活性的作用呈濃度依賴模式。 2)在3小時(shí)、6小時(shí)、9小時(shí)、12小時(shí)4個(gè)時(shí)間點(diǎn)中,當(dāng)沙利度胺作用HaCaT細(xì)胞3小時(shí)后,VEGF及TNF-a mRNA表達(dá)水平分別為對照組51.0%及53.1%,此時(shí)沙利度胺對其合成VEGF及TNF-a mRNA的抑制作用最為明顯。 3)0.01nM沙利度胺作用HaCaT細(xì)胞3小時(shí)后,VEGF mRNA與蛋白表達(dá)水平分別為對照組的63.4%及92.3%；當(dāng)濃度0.01nM時(shí),沙利度胺對HaCaT細(xì)胞VEGF mRNA及蛋白的表達(dá)有抑制作用,其作用呈現(xiàn)濃度依賴模式。 4)沙利度胺濃度≤0.01nM時(shí),對HaCaT細(xì)胞合成TNF-a mRNA及蛋白無抑制作用；0.1nM沙利度胺作用HaCaT細(xì)胞3小時(shí)后, TNF-a mRNA與蛋白表達(dá)水平分別為對照組的58.7%及61.7%；當(dāng)濃度0.1nM時(shí),沙利度胺對HaCaT細(xì)胞TNF-a mRNA的表達(dá)有抑制作用,其抑制程度與濃度無關(guān)；同時(shí),沙利度胺對HaCaT細(xì)胞TNF-a蛋白的表達(dá)有抑制作用,其作用呈濃度依賴模式。 結(jié)論 1)沙利度胺濃度≥1000nM時(shí)可抑制HaCaT細(xì)胞的活性。 2)沙利度胺在mRNA與蛋白水平上均可抑制HaCaT細(xì)胞VEGF與TNF-a的表達(dá)。 3)沙利度胺可能因其抑制角質(zhì)形成細(xì)胞活性及減少VEGF和TNF-a分泌的作用而作為一種銀屑病治療藥物,應(yīng)對沙利度胺的作用機(jī)制及臨床應(yīng)用進(jìn)行進(jìn)一步的探討。
[Abstract]:background
In 1950s, thalidomide as a sedative and widely used for morning sickness treatment, but because of its teratogenic effects and delisting. But in recent years, thalidomide because it can reduce the levels of TNF-a in patients and angiogenesis inhibition is FDA approved for the treatment of leprosy erythema and nodules of multiple myeloma in addition. The researchers also found that, in succession, ineffective treatment of thalidomide but also for a variety of regular skin diseases have a good therapeutic effect, but the mechanism of the treatment of these diseases is not clear. Because thalidomide has immunomodulatory effects, early scholars have speculated that thalidomide can be used for the treatment of psoriasis. So in the early clinical practice, we use of thalidomide in the treatment of some patients, found that thalidomide has certain therapeutic effect on psoriasis. The study found that dendritic cells and the secretion of IL- 12 and IL23, IL-17 and IL22 lymphocytes and secretion of keratinocytes, TNF-a cells and VEGF cells and its secretion plays a key role in the pathogenesis of psoriasis. There are many experimental studies on effects of thalidomide on dendritic cells and lymphocytes, and the study of thalidomide effect on the cuticle forming cells are relatively rare, so it is the need for further study of thalidomide cells forming effects on keratinocytes.
objective
TNF-a is one of the pro inflammatory cytokines in psoriasis core, keratinocyte proliferation and capillary hyperplasia and papillary dermis in psoriatic lesions is the most typical histopathological manifestations of.TNF-a and hyperproliferation of keratinocytes in psoriasis plays key role has been widely used in the treatment of severe psoriasis curative effect of TNF-a antagonist agent and methotrexate proved; in animal models, anti angiogenic drugs can significantly improve psoriasis like lesions. We intend to form a cell activity of keratinocytes by studying the effects of thalidomide on keratinocyte influence the expression of VEGF and TNF-a, in order to further understand the mechanism of action of thalidomide in the treatment of skin diseases, and to provide the experimental basis for the treatment of psoriasis for thalidomide.
Method
1) HaCaT cells were cultured in vitro.
2) the effect of different concentrations of thalidomide on the activity of HaCaT cells was detected by WST-l method.
3) through primer design, verification and adjustment of amplification efficiency of the reaction system, the real-time quantitative PCR reaction system can meet the requirement of analyzing the relative gene expression difference by 2- Delta Ct method.
4) real-time quantitative PCR assay was used to determine the effect of thalidomide at different time points on the synthesis of VEGF and TNF- alpha mRNA in HaCaT cells.
5) the effects of different concentrations of thalidomide on the synthesis of VEGF and TNF- alpha mRNA in HaCaT cells were measured by real-time quantitative PCR.
5) the effects of different concentrations of thalidomide on the secretion of VEGF and TNF-a protein in HaCaT cells were measured by ELISA.
Result
1) when the concentration of thalidomide is less than or equal to 100nM, had no effect on the proliferation of HaCaT cells; thalidomide when the concentration of 1000nM, the activity of HaCaT cells was inhibited, approximately 90.8% of the control group; thalidomide concentration higher than 1000nM, the inhibition of HaCaT cell activity in a concentration dependent mode.
2) in 3 hours, 6 hours, 9 hours, 12 hours, 4 time points, when thalidomide acted on HaCaT cells for 3 hours, the expression levels of VEGF and TNF-a mRNA were 51% and 53.1% in the control group, respectively. The inhibitory effect of thalidomide on the synthesis of VEGF and TNF-a mRNA was the most obvious.
3) after 3 hours of thalidomide treatment, the expression level of VEGF mRNA and protein was 63.4% and 92.3% in the control group, respectively. When the concentration of 0.01nM was 0.01nM, thalidomide could inhibit the expression of VEGF mRNA and protein in HaCaT cells, and its effect was HaCaT concentration dependent mode.
4) thalidomide concentration is less than or equal to 0.01nM, had no inhibitory effect on TNF-a synthesis of HaCaT cells and mRNA protein; 0.1nM thalidomide HaCaT cells after 3 hours, TNF-a mRNA and protein expression levels were 58.7% and 61.7% in control group; when the concentration of 0.1nM, the expression of thalidomide on HaCaT cells TNF-a mRNA was inhibited, the inhibition and the degree of concentration independent; at the same time, the expression of thalidomide on HaCaT cells TNF-a protein was inhibited, the effect was concentration dependent.
conclusion
1) thalidomide concentration greater than or equal to 1000nM can inhibit the activity of HaCaT cells.
2) the expression of VEGF and TNF-a in HaCaT cells was inhibited by thalidomide at the level of mRNA and protein.
3) thalidomide may be a psoriasis therapeutic agent because it inhibits keratinocyte activity and reduces the secretion of VEGF and TNF-a, and further discusses the mechanism and clinical application of thalidomide.

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

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李峰;晉紅中;王寶璽;;北京協(xié)和醫(yī)院銀屑病住院患者中代謝綜合征的患病率調(diào)查[J];中國醫(yī)學(xué)科學(xué)院學(xué)報(bào);2010年05期

，

本文編號：1758661