沙利度胺对角质形成细胞分泌VEGF及TNF-α影响的研究

发布时间：2018-04-16 11:18

本文选题：沙利度胺 + 银屑病　；参考：《北京协和医学院》2012年博士论文

【摘要】：背景在20世纪50年代,沙利度胺曾作为一种镇静剂而广泛用于孕吐的治疗,随后却因其致畸作用而退市。但近年来,沙利度胺因其能降低患者体内TNF-a水平及抑制血管生成的作用而被FDA批准用于麻风结节性红斑及多发性骨髓瘤的治疗。此外,研究者也陆续发现,沙利度胺还对多种经常规治疗方法无效的皮肤病有良好的治疗作用,但其治疗这些疾病的作用机制尚不明确。因沙利度胺具有免疫调节作用,很早就有学者推测沙利度胺可以用于银屑病的治疗。故在前期的临床实践中,我们采用沙利度胺治疗了部分患者后,发现沙利度胺对银屑病有一定的治疗作用。研究发现树突状细胞及其分泌的IL-12和IL23、淋巴细胞及其分泌的IL-17和IL22,角质形成细胞及其分泌的TNF-a及VEGF在银屑病的发病机制中起着十分关键的作用。目前已有较多关于沙利度胺对树突状细胞及淋巴细胞影响的实验研究,而沙利度胺对角质形成细胞作用的研究则较为罕见,故有必要进一步研究沙利度胺对角质形成细胞的作用。目的 TNF-a是银屑病中核心的促炎症细胞因子之一,角质形成细胞过度增殖及真皮乳头层毛细血管增生是银屑病皮损中最为典型的组织病理学表现。TNF-a与角质形成细胞过度增殖在银屑病中所起的关键作用已被目前广泛用于重度银屑病治疗的TNF-a拮抗剂及甲氨蝶呤的显著疗效所证实；在动物模型中,抗血管生成药也可以显著改善银屑病样皮损。故我们拟通过研究沙利度胺对角质形成细胞活性的影响及其对角质形成细胞VEGF和TNF-a表达的影响,以进一步了解沙利度胺在皮肤疾病治疗中的作用机制,并为沙利度胺用于银屑病的治疗提供实验基础。方法 1)体外培养HaCaT细胞。 2)WST-l法检测不同浓度沙利度胺对HaCaT细胞活性的影响。 3)通过引物设计及验证和调整反应体系扩增效率使实时定量PCR反应体系满足通过2-ΔΔCt法分析相对基因表达差异的条件。 4)实时定量PCR法测定不同时间点沙利度胺对HaCaT细胞合成VEGF与TNF-α mRNA的影响。 5)实时定量PCR法测定不同浓度沙利度胺对HaCaT细胞合成VEGF与TNF-α mRNA的影响。 5) ELISA法测定不同浓度沙利度胺对HaCaT细胞分泌VEGF与TNF-a蛋白的影响。结果 1)当沙利度胺浓度≤100nM时,对HaCaT细胞增殖无影响；当沙利度胺浓度为1000nM时,HaCaT细胞活性受到抑制,约为对照组的90.8%；沙利度胺浓度≥1000nM时,其抑制HaCaT细胞活性的作用呈浓度依赖模式。 2)在3小时、6小时、9小时、12小时4个时间点中,当沙利度胺作用HaCaT细胞3小时后,VEGF及TNF-a mRNA表达水平分别为对照组51.0%及53.1%,此时沙利度胺对其合成VEGF及TNF-a mRNA的抑制作用最为明显。 3)0.01nM沙利度胺作用HaCaT细胞3小时后,VEGF mRNA与蛋白表达水平分别为对照组的63.4%及92.3%；当浓度0.01nM时,沙利度胺对HaCaT细胞VEGF mRNA及蛋白的表达有抑制作用,其作用呈现浓度依赖模式。 4)沙利度胺浓度≤0.01nM时,对HaCaT细胞合成TNF-a mRNA及蛋白无抑制作用；0.1nM沙利度胺作用HaCaT细胞3小时后, TNF-a mRNA与蛋白表达水平分别为对照组的58.7%及61.7%；当浓度0.1nM时,沙利度胺对HaCaT细胞TNF-a mRNA的表达有抑制作用,其抑制程度与浓度无关；同时,沙利度胺对HaCaT细胞TNF-a蛋白的表达有抑制作用,其作用呈浓度依赖模式。结论 1)沙利度胺浓度≥1000nM时可抑制HaCaT细胞的活性。 2)沙利度胺在mRNA与蛋白水平上均可抑制HaCaT细胞VEGF与TNF-a的表达。 3)沙利度胺可能因其抑制角质形成细胞活性及减少VEGF和TNF-a分泌的作用而作为一种银屑病治疗药物,应对沙利度胺的作用机制及临床应用进行进一步的探讨。
[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.

【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

【参考文献】