SERPINB1在銀屑病中的表達及功能研究
發(fā)布時間：2019-05-19 14:59
【摘要】：研究背景 銀屑病是一種常見的慢性復發(fā)性炎癥性皮膚病,給患者的身心帶來極大的痛苦,長期以來都是皮膚病領域研究的重點和熱點。銀屑病的特征性病理改變是角質(zhì)形成細胞的過度增殖和表皮內(nèi)的中性粒細胞聚集,銀屑病患者外周血中中性粒細胞計數(shù)也偏高。中性粒細胞是天然免疫的重要組成成分,還可通過多種途徑在銀屑病發(fā)生和病情進展中起著重要作用。其中中性粒細胞起作用的一種重要介質(zhì)是中性粒細胞彈性蛋白酶(neutrophil elastase,NE)。已有的研究表明NE既有促進炎癥反應的作用,又有抑制炎癥反應的效應,NE在炎癥反應中發(fā)揮十分復雜的調(diào)節(jié)作用。 關(guān)于NE與銀屑病的關(guān)系,已有研究表明NE在銀屑病患者皮損、血清中有高表達且與病情平行,并有研究證實NE通過EGFR信號通路可刺激角質(zhì)形成細胞增殖。NE抑制劑trappin-2、α1抗胰蛋白酶等在銀屑病中均存在明顯異常,且trappin-2可抑制角質(zhì)形成細胞的增殖、降低IL-8、IL-6、ICAM-1等炎癥增強因子的表達,NE及其抑制劑在銀屑病中存在失衡。但尚無研究直接觀察相應的NE抑制劑能否抑制NE引起的角質(zhì)形成細胞增殖活躍。有研究表明低濃度NE能明顯上調(diào)細胞轉(zhuǎn)錄和表達NE抑制劑elafin,高濃度的NE則使elafin表達減少。但關(guān)于NE及其抑制劑間的關(guān)系及其表達水平的調(diào)控缺乏進一步的深入研究。 目前維生素D3類似物為治療輕、中度銀屑病最常用的外用藥之一。關(guān)于其治療機制的研究也一直是皮膚病領域的熱點。在前期的課題研究中我們發(fā)現(xiàn):骨化三醇作用于HaCaT后,卵清蛋白樣絲氨酸蛋白酶抑制劑1(serine proteinase inhibitor,clade B,member 1,SERPINB1)(monocyte neutrophil elastase inhibitor,MNEI)蛋白的表達量升高,提示SERPINB1在骨化三醇治療銀屑病的過程中起著一定的作用。SERPINB1是一種快反應性針對中性粒細胞蛋白酶的抑制劑,可抑制NE及組織蛋白酶G(cat G)。目前關(guān)于SERPINB1在PMNs炎癥性疾病中尤其急性肺損傷及肺綠膿桿菌感染等方面的研究比較深入:證實其在調(diào)節(jié)天然免疫反應中起重要的作用。但關(guān)于SERPINB1與銀屑病的關(guān)系,尚無相關(guān)研究。 本研究在前期課題的基礎上,進一步驗證骨化三醇作用于HaCaT后,SERPINB1在基因和蛋白質(zhì)水平的變化,SERPINB1在銀屑病皮損中的表達,并進一步研究SERPINB1對角質(zhì)形成細胞增殖的影響,及其表達的調(diào)控,從而驗證SERPINB1在銀屑病治療過程中的作用,對尋找銀屑病新的治療靶點具有一定的指導意義。 第一部分骨化三醇對角質(zhì)形成細胞SERPINB1表達的影響 目的:研究骨化三醇對人永生化角質(zhì)形成細胞株(HaCaT)SERPINB1 mRNA及蛋白水平的影響。 方法:常規(guī)培養(yǎng)HaCaT細胞,細胞生長至80%融合時,加入新鮮配制的10-8mol/L的骨化三醇,陰性對照組加等量的無血清培養(yǎng)基,每種條件設置2個復孔,避光培養(yǎng)后,提取細胞總RNA及全蛋白。分別用RT-PCR、western-blot分析SERPINB1 mRNA及蛋白水平的含量,并設置不同的時間點觀察藥物對SERPINB1 mRNA的影響。 結(jié)果:骨化三醇作用于HaCaT后較空白對照相比,SERPINB1 mRNA在作用4h后上調(diào)1.99倍,8h、12h、24h、48h時分別上調(diào)2.41、3.28、3.46及3.31倍,同0h比差別有統(tǒng)計學意義,而各時間組間差別無統(tǒng)計學意義。在蛋白質(zhì)水平,24h及48h組較空白對照相比均有升高,差別有統(tǒng)計學意義。24h與48h組相比差別也有統(tǒng)計學意義。 結(jié)論:骨化三醇能促進HaCaT中SERPINB1mRNA及蛋白質(zhì)的表達。 第二部分SERPINB1對HaCaT增殖的影響及NE對SERPINB1的調(diào)控 目的:篩選有效地SERPINB1的siRNA,通過基因干擾研究SERPINB1對HaCaT細胞增殖的影響。并研究不同濃度NE對SERPINB1的調(diào)控。 方法:通過RNA干擾,設計并合成針對SERPINB1的siRNA,共設計3對,采用Lipodectamin2000轉(zhuǎn)染法轉(zhuǎn)染HaCaT細胞,通過熒光標記的siRNA觀察轉(zhuǎn)染效率,通過RT-PCR法驗證轉(zhuǎn)染及干擾效率,挑選出最有效的siRNA即SERPINB1-homo-93,使用SERPINB1-homo-93干擾HaCaT,觀察干擾前后細胞的MTT。另將不同濃度的NE作用于HaCaT,通過western-blot,觀察SERPINB1量的變化。并觀察不同濃度的NE及骨化三醇對HaCaT增殖的影響。 結(jié)果:1. SERPINB1-homo-93可有效干擾HaCaT的SERPINB1,干擾效率達83%。2. NE在濃度1U/L-15U/L間較空白對照相比可促進細胞的增殖。在濃度為15U/L時其對細胞增殖的影響與空白對照相比無明顯差別,在濃度為20U/L-50U/L間對細胞增殖起抑制作用。3.骨化三醇在10-9-10-6mol/L時均能抑制HaCaT增殖,劑量間無差別。4. SERPINB1有抑制增殖的活性;干擾后加入骨化三醇仍表現(xiàn)為增殖促進作用,骨化三醇的增殖抑制作用未能彌補干擾SERPINB1后對增殖的促進的效應。5.低濃度的NE 1、5、10、15U/L時可使得SERPINB1/GAPDH升高,在20U/L時與空白對照比無差別,之后隨NE濃度升高蛋白質(zhì)濃度下降,即NE濃度為33.3及50U/L時表現(xiàn)為對SERPINB1蛋白質(zhì)濃度的抑制作用。 結(jié)論:SERPINB1對HaCaT有增殖抑制作用,NE低濃度時促進增殖,高濃度時則抑制增殖;低濃度NE促進SERPINB1表達,高濃度時抑制SERPINB1表達。 第三部分銀屑病皮損中SERPINB1的表達 目的:研究銀屑病患者皮損較正常對照SERPINB1的變化。 方法:收集重度尋常型銀屑病患者的皮損及正常對照的皮膚標本,抽提組織的總蛋白,通過western-blot分析SERPINB1的量的差異。收集既往銀屑病活檢患者的蠟塊,通過免疫組化法觀察組織切片中SERPINB1的量的差異。 結(jié)果:免疫組化染色切片中正常皮膚中SERPINB1在基底層有弱到中陽性的著色,銀屑病皮損中SERPINB1彌漫性基底層、棘層中到強陽性著色。差別有統(tǒng)計學意義。新鮮組織SERPINB1/GAPDH在銀屑病組較正常對照組低。 結(jié)論:銀屑病皮損石蠟切片免疫組化染色示SERPINB1的表達明顯強于正常皮膚。而在新鮮組織中銀屑病組的SERPINB1表達量低于正常對照。
[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
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R758.63
[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
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R758.63
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