SERPINB1在银屑病中的表达及功能研究

发布时间：2019-05-19 14:59

【摘要】：研究背景银屑病是一种常见的慢性复发性炎症性皮肤病,给患者的身心带来极大的痛苦,长期以来都是皮肤病领域研究的重点和热点。银屑病的特征性病理改变是角质形成细胞的过度增殖和表皮内的中性粒细胞聚集,银屑病患者外周血中中性粒细胞计数也偏高。中性粒细胞是天然免疫的重要组成成分,还可通过多种途径在银屑病发生和病情进展中起着重要作用。其中中性粒细胞起作用的一种重要介质是中性粒细胞弹性蛋白酶(neutrophil elastase,NE)。已有的研究表明NE既有促进炎症反应的作用,又有抑制炎症反应的效应,NE在炎症反应中发挥十分复杂的调节作用。关于NE与银屑病的关系,已有研究表明NE在银屑病患者皮损、血清中有高表达且与病情平行,并有研究证实NE通过EGFR信号通路可刺激角质形成细胞增殖。NE抑制剂trappin-2、α1抗胰蛋白酶等在银屑病中均存在明显异常,且trappin-2可抑制角质形成细胞的增殖、降低IL-8、IL-6、ICAM-1等炎症增强因子的表达,NE及其抑制剂在银屑病中存在失衡。但尚无研究直接观察相应的NE抑制剂能否抑制NE引起的角质形成细胞增殖活跃。有研究表明低浓度NE能明显上调细胞转录和表达NE抑制剂elafin,高浓度的NE则使elafin表达减少。但关于NE及其抑制剂间的关系及其表达水平的调控缺乏进一步的深入研究。目前维生素D3类似物为治疗轻、中度银屑病最常用的外用药之一。关于其治疗机制的研究也一直是皮肤病领域的热点。在前期的课题研究中我们发现:骨化三醇作用于HaCaT后,卵清蛋白样丝氨酸蛋白酶抑制剂1(serine proteinase inhibitor,clade B,member 1,SERPINB1)(monocyte neutrophil elastase inhibitor,MNEI)蛋白的表达量升高,提示SERPINB1在骨化三醇治疗银屑病的过程中起着一定的作用。SERPINB1是一种快反应性针对中性粒细胞蛋白酶的抑制剂,可抑制NE及组织蛋白酶G(cat G)。目前关于SERPINB1在PMNs炎症性疾病中尤其急性肺损伤及肺绿脓杆菌感染等方面的研究比较深入:证实其在调节天然免疫反应中起重要的作用。但关于SERPINB1与银屑病的关系,尚无相关研究。本研究在前期课题的基础上,进一步验证骨化三醇作用于HaCaT后,SERPINB1在基因和蛋白质水平的变化,SERPINB1在银屑病皮损中的表达,并进一步研究SERPINB1对角质形成细胞增殖的影响,及其表达的调控,从而验证SERPINB1在银屑病治疗过程中的作用,对寻找银屑病新的治疗靶点具有一定的指导意义。第一部分骨化三醇对角质形成细胞SERPINB1表达的影响目的:研究骨化三醇对人永生化角质形成细胞株(HaCaT)SERPINB1 mRNA及蛋白水平的影响。方法:常规培养HaCaT细胞,细胞生长至80%融合时,加入新鲜配制的10-8mol/L的骨化三醇,阴性对照组加等量的无血清培养基,每种条件设置2个复孔,避光培养后,提取细胞总RNA及全蛋白。分别用RT-PCR、western-blot分析SERPINB1 mRNA及蛋白水平的含量,并设置不同的时间点观察药物对SERPINB1 mRNA的影响。结果:骨化三醇作用于HaCaT后较空白对照相比,SERPINB1 mRNA在作用4h后上调1.99倍,8h、12h、24h、48h时分别上调2.41、3.28、3.46及3.31倍,同0h比差别有统计学意义,而各时间组间差别无统计学意义。在蛋白质水平,24h及48h组较空白对照相比均有升高,差别有统计学意义。24h与48h组相比差别也有统计学意义。结论:骨化三醇能促进HaCaT中SERPINB1mRNA及蛋白质的表达。第二部分SERPINB1对HaCaT增殖的影响及NE对SERPINB1的调控目的:筛选有效地SERPINB1的siRNA,通过基因干扰研究SERPINB1对HaCaT细胞增殖的影响。并研究不同浓度NE对SERPINB1的调控。方法:通过RNA干扰,设计并合成针对SERPINB1的siRNA,共设计3对,采用Lipodectamin2000转染法转染HaCaT细胞,通过荧光标记的siRNA观察转染效率,通过RT-PCR法验证转染及干扰效率,挑选出最有效的siRNA即SERPINB1-homo-93,使用SERPINB1-homo-93干扰HaCaT,观察干扰前后细胞的MTT。另将不同浓度的NE作用于HaCaT,通过western-blot,观察SERPINB1量的变化。并观察不同浓度的NE及骨化三醇对HaCaT增殖的影响。结果:1. SERPINB1-homo-93可有效干扰HaCaT的SERPINB1,干扰效率达83%。2. NE在浓度1U/L-15U/L间较空白对照相比可促进细胞的增殖。在浓度为15U/L时其对细胞增殖的影响与空白对照相比无明显差别,在浓度为20U/L-50U/L间对细胞增殖起抑制作用。3.骨化三醇在10-9-10-6mol/L时均能抑制HaCaT增殖,剂量间无差别。4. SERPINB1有抑制增殖的活性;干扰后加入骨化三醇仍表现为增殖促进作用,骨化三醇的增殖抑制作用未能弥补干扰SERPINB1后对增殖的促进的效应。5.低浓度的NE 1、5、10、15U/L时可使得SERPINB1/GAPDH升高,在20U/L时与空白对照比无差别,之后随NE浓度升高蛋白质浓度下降,即NE浓度为33.3及50U/L时表现为对SERPINB1蛋白质浓度的抑制作用。结论:SERPINB1对HaCaT有增殖抑制作用,NE低浓度时促进增殖,高浓度时则抑制增殖;低浓度NE促进SERPINB1表达,高浓度时抑制SERPINB1表达。第三部分银屑病皮损中SERPINB1的表达目的:研究银屑病患者皮损较正常对照SERPINB1的变化。方法:收集重度寻常型银屑病患者的皮损及正常对照的皮肤标本,抽提组织的总蛋白,通过western-blot分析SERPINB1的量的差异。收集既往银屑病活检患者的蜡块,通过免疫组化法观察组织切片中SERPINB1的量的差异。结果:免疫组化染色切片中正常皮肤中SERPINB1在基底层有弱到中阳性的着色,银屑病皮损中SERPINB1弥漫性基底层、棘层中到强阳性着色。差别有统计学意义。新鲜组织SERPINB1/GAPDH在银屑病组较正常对照组低。结论:银屑病皮损石蜡切片免疫组化染色示SERPINB1的表达明显强于正常皮肤。而在新鲜组织中银屑病组的SERPINB1表达量低于正常对照。
[Abstract]:Study Background Psoriasis is a common chronic and recurrent inflammatory skin disease, which brings great pain to the body and mind of the patient, and has long been the focus of research in the field of skin diseases. The characteristic pathological change of psoriasis is the excessive proliferation of the keratinocytes and the aggregation of the neutrophils in the epidermis, and the neutrophil count in the peripheral blood of the patients with psoriasis is also High. Neutrophil is an important component of natural immunity, and can play an important role in the development of psoriasis and disease progression through a variety of ways. An important medium in which the neutrophil is active is a neutrophil elastase, N E). The existing studies have shown that NE has the effects of promoting the inflammatory reaction, and has the effect of inhibiting the inflammatory reaction, and the NE plays a very complicated role in the inflammatory reaction. The study of the relationship between NE and psoriasis has shown that NE is highly expressed in the skin of the patients with psoriasis and is parallel to the condition, and it has been shown that NE can stimulate the cutin shape through the EGFR signaling pathway. In that case of psoriasis, the proliferation of the keratinocytes, IL-8, IL-6, and ICAM-1 and the expression of NE and its inhibitor in psoriasis were decrease. There is an imbalance in the medium. However, there is no study to directly observe whether the corresponding NE inhibitor can inhibit the formation of the cutin caused by the NE. It is shown that the low concentration of NE can increase the transcription of the cells and the expression of the NE inhibitor elafin, and the high concentration of NE makes elafi n-expression is reduced. However, the relationship between NE and its inhibitor and the regulation of its expression level lack further The present vitamin D3 analog is the most effective in the treatment of light and moderate psoriasis. One of the most common forms of external use. The study on its treatment mechanism has also been In the study of the prophase, we found that calcitriol acts on HaCaT, and the egg-albumin-like serine protease inhibitor 1 (serine protein inhibitor, clade B, member 1, SERPINB1) (monocyte neutopia inhibisitor, MNEI) The expression of protein is increased, and it is suggested that SERPINB1 is in the process of treating psoriasis with calcitriol. SERPINB1 is a fast-reactive inhibitor of a neutrophil-protease inhibitor, which can inhibit the NE and the tissue protein. Enzyme G (cat G). At present, the study on SERPINB1 in the inflammatory diseases of PMNs, especially acute lung injury and the infection of pulmonary pseudomonas aeruginosa, has been in-depth: it is confirmed that it is in the regulation of natural immunity. It is important to play an important role, but with regard to the closure of SERPINB1 and psoriasis The results of this study, on the basis of the previous subject, further verified the changes of the level of SERPINB1 in the level of gene and protein, the expression of SERPINB1 in the skin of psoriasis, and the further study of SERPIN1 on the proliferation of keratinocytes. And the effect of SERPINB1 in the treatment of psoriasis is verified, and a new treatment for the treatment of psoriasis is carried out. The first part of calcitriol has a certain guiding significance, and the first part of calcitriol is formed in the cu@@ Objective: To study the effect of calcitriol on human immortalized keratinocytes (HaCaT) SER. Methods: In the conventional culture of HaCaT cells and the growth of cells to 80% fusion, fresh formulated 10-8 mol/ L calcitriol was added, and the negative control group was added with the same amount of serum-free medium. The total RNA and total protein of the cells were analyzed by RT-PCR and western-blot, and different time points were set. The effect of drug on SERPIN1 mRNA was observed. Results: Compared with the blank control after the action of calcitriol on HaCaT, the SERPINB1 mRNA was up to 1.99 times,8 h,12 h,24 h and 48 h, respectively, up to 2.41, 3.28, 3.46 and 3.31 times, and the same as 0 h. The difference was of statistical significance, and there was no statistical significance between the time groups. In the level of protein,24 h and 48 h, there was an increase in the level of protein,24 h and 48 h, and the difference was statistically significant. Conclusion: The calcitriol can be promoted. Expression of SERPINB1mRNA and Protein in HaCaT. Part II SERP The effect of INB1 on the proliferation of HaCaT and the control of the NE to SERPINB1: the siR of the effective SERPINB1 is selected. NA, by gene interference study SERPINB1 vs. Ha The effects of different concentrations of NE on SERPINB1 were studied. Methods: The siRNA of SERPINB1 was designed and synthesized by RNA interference,3 pairs were designed, and the HaCaT cells were transfected with the Lipofectamine 2000 transfection method. The transfection efficiency was observed by the siRNA of the fluorescent label. The efficiency of transfection and interference was verified by RT-PCR, and the most effective siRNA, SERPINB, was selected. 1-homo-93, using S ERPINB1-homoo-93 interferes with HaCaT to observe the MTT of cells before and after interference. HaCaT, by western-blot, SERP was observed INB1 The effects of NE and calcitriol on the proliferation of HaCaT were observed. -homoo-93 can effectively interfere with the SERPINB1 of HaCaT, and the interference efficiency 2. NE could promote the proliferation of cells in a concentration of 1 U/ L-15 U/ L. The effect of the concentration of 15 U/ L on the proliferation of cells was similar to that of the blank. In contrast, there was no significant difference in cell proliferation at a concentration of 20 U/ L-50 U/ L.3. Ossification The proliferation of HaCaT was inhibited by triol at 10-9-10-6 mol/ L. The inhibition of the proliferation of calcitriol could not compensate for the effect of interfering with SERPINB1 on proliferation.5. When NE 1,5,10,15 U/ L at low concentration, SERPINB1/ GAPDH could be increased, and the concentration of protein increased with the concentration of NE at 20 U/ L. The concentration of NE was 33.3 and 50U/ L. The inhibitory effect of SERPIN1 on the concentration of SERPIN1 protein was found. and the proliferation is inhibited; the low-concentration NE promotes the SERPINB; 1 Expression, inhibition of SERPINB1 expression at high concentrations. Part III silver Objective: To study the expression of SERPINB1 in psoriatic lesions. To damage the normal control skin specimen, the total protein of the tissue was extracted, and the SERPIN was analyzed by western-blot. The difference in the amount of B1 was collected. The difference in the amount of SERPINB1 in the tissue section was observed by immunohistochemistry using a wax block in a prior psoriatic biopsy patient. 涓璖ERPINB1 SERPINB1 was diffuse in the skin of the psoriatic lesions in the presence of weak to medium-positive staining in the basal layer In the base layer, the strong positive staining was found in the ratchet layer. The difference was statistically significant. The fresh tissue SERPINB1/ GAP DH was lower in the psoriatic group than in the normal control group. Conclusion: The paraffin section of psoriatic lesions
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R758.63

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