【作者】 李慧；

【导师】 顾军；

【作者基本信息】 第二军医大学 ， 皮肤病与性病学， 2011， 博士

【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。更多还原

【Abstract】 As a kind of common, chronic, and recurrent inflammatory skin disease,psoriasis has a significant negative impact on patients’quality of life and also has been the focus for a long period in the area of skin diseases. Characteristical hispathological changes of psoriasis are the excessive proliferation of keratinocytes and accumulation of intraepidermal neutrophil. Neutrophils count is also high in the peripheral blood of patients with psoriasis. Neutrophils are important components of innate immunity, which play an important role in the generation and progression of psoriasis through a variety of ways. As an important mediator, NE (neutrophil elastase) promote the roles of neutrophils.Studies have shown that NE regulates inflammatory response in a complicated process because it can not only promote inflammation, but also inhibit the inflammation.About the relationship between NE and psoriasis, some studies had been shown that NE expressed highly in lesions and serum in patients with psoriasis, which match the severity of the disease. And some research had shown that NE could stimulate proliferation of keratinocytes through the EGFR signaling pathway. Trappin-2 andα1-antitrypsin,the inhibitor of NE, were significantly abnormal in psoriasis. Trappin-2 could also inhibit the proliferation of keratinocytes and reduce the expression of IL-8, IL-6, ICAM-1 and other inflammatory enhancement factors. But no studies have directly observed that NE inhibitors can inhibit the proliferation of keratinocytes induced by NE. Some research concluded that low concentrations of NE significantly increased transcription and expression of elafin, which is one kind of NE inhibitors, while high concentrations had the opposite effect. And now no further study about the relationship between NE and its inhibitors is presented.Vitamin D3 analogues are the first choice of local therapy for mild and moderate psoriasis. Research on its therapeutic mechanism has also been a hot area of skin disease. In my master’s research, it was found that SERPINB1 played a role in curing psoriasis by calcitriol because ovalbumin-like serine protease inhibitor 1 (serine proteinase inhibitor, clade B, member 1, SERPINB1) (monocyte neutrophil elastase inhibitor, MNEI) overexpressed after calcitriol was put in HaCaT. As a neutrophil protease inhibitor, SERPINB1 can inhibit NE and cathepsin G (cat G). Currently more studies had been done about curing PMNs inflammatory diseases, especially acute lung injury and pulmonary Pseudomonas aeruginosa infection by SERPINB1. It was confirmed that SERPINB1 played an important role in the regulation of immune responses. However, there is no research on the relationship between psoriasis and SERPINB1.Based on my master’s research, the first aim of this study is to further verify the differences of SERPINB1 in gene and protein level after calcitriol is given to HaCaT. And the second aim is to do some research about the impact of SERPINB1 on keratinocyte proliferation and differentiation so as to find the role SERPINB1 played in the process of treating psoriasis. This study may be necessary to find new therapeutic targets of psoriasis.PARTⅠObjective To study the effect of calcitriol on immortalized human keratinocyte cell line (HaCaT) SERPINB1 at the levels of mRNA and protein.Methods HaCaT cells were cultured with usual method. When 80% of cells got fused, fresh calcitriol on the concentration of 10-8mol/L was added while the negative control group was added with the same amount of serum-free DMEM. Two complex holes were set for each condition and then total cellular RNA and the whole protein were extracted after dark culture. Contents of SERPINB1 on mRNA and protein levels were analyzed with the methods of RT-PCR and western-blot, separately. And set the different time points to observe the impact of drugs on SERPINB1 mRNA.Results SERPINB1 mRNA was upregulated by 1.99 times in 4h-group compared with control group, and the at 8h, 12h,24h,48h SERPINB1 mRNA was separately upregulated by 2.41,3.28,3.46 and 3.31 times.There were statistical significance between every time group and control group. But there was no statistical significance between them.At the protein level, 24h-group and 48h-group were both upregulated when compared with control group and there were statistical significance. There was statistical significance between 24h-group and 48h-group.Conclusion Calcitriol could regulate SERPINB1 in HaCaT both at the mRNA and the protein levels.PARTⅡEffect of SERPINB1 on HaCaT in proliferation and the regulation of SERPINB1 Objective Select siRNAs interfering with SERPINB1 effectively. Discuss the effect of SERPINB1 on HaCaT cell proliferation by genetic interference, and study the differences of SERPINB1 when added different concentrations of NE.Methods Using RNA interference technology, design and synthesis the siRNAs to aim at SERPINB1, a total of 3 groups. HaCaT cells were transfected by Lipodectamin2000 and were observed on transfection efficiency by labeled siRNA. Use RT-PCR to Verify the efficiency of transfection and interference and then select the most effective siRNA (SERPINB1-homo-93) to interfere with HaCaT. MTT was observed before and after interfering. Different concentrations of NE also were joined into HaCaT,and then the quantity of SERPINB1 was studied by western-blot. At last, the effect of different concentration of NE and calcitriol on HaCaT was observed.Results 1. SERPINB1-homo-93 could interfere SERPINB1 of HaCaT effectively by 83%. 2. NE could promte cell proliferation at lower concentration at 1U/L to 5U/L compared with control group. At the concentration of 15U/L, the effect of NE on proliferation had no difference with control group, and at the level of 20U/L to 50U/L,NE could inhibit cell proliferation.3.Calcitriol could inhibit proliferation of HaCaT at the concentration of 10-9-10-6mol/L, but there were no statistical significance between different concentration groups.4. Cell proliferation was promoted when SERPINB1 was interfered. Calcitriol could not inhibit proliferation after SERPINB1 was interfered. 5. NE upregulated SERPINB1/GAPDH at lower concentration at 1、5、10、15U/L. There was on statistical significance compared with the control group with the concentration of 20U/L. And then, NE depressed the expression of SERPINB1.Conclusion SERPINB1 could inhibit the proliferation of HaCaT. NE could promote proliferation at lower concentration,but the effect was inhibition when the concentration was at a higher level. Lower concertration of NE could promote the expression of SERPINB1, higher concentration could inbibit its expression.PARTⅢThe expression of SERPINB1 in psoriatic lesionsObjective Study the difference of SERPINB1 between Lesions of psoriasis and normalMethods Collect severe skin lesions of psoriasis and normal skin to extract total protein from tissues and then analysis SERPINB1 differences using western-blot. Collect paraffins from previous biopsies of patients with psoriasis to investigate differences of SERPINB1 in tissue slices by immunohistochemistry.Result Immunohistochemical staining displayed that in the basal layer of normal skin SERPINB1 was positive with weak to moderate. But, in psoriatic lesions SERPINB1 staining was moderate to strong positive in the basal layer and spinous layer.There was statistically significance in difference. Total protein was extracted and the western-blot results showed that SERPINB1/GAPDH was lower in psoriasis group than health control. Conclusion The expression of SERPINB1 in paraffin sections of psoriatic lesions was significantly stronger than that in normal skin. In fresh tissue, expression of SERPINB1 was lower in psoriasis group than that in the control group.更多还原