甲泼尼龙对鼻息肉中自噬相关基因LC3-B和P62表达影响的研究
发布时间：2018-08-16 09:19
【摘要】：背景:慢性鼻-鼻窦炎伴鼻息肉(Chronic Rhinosinusitis with Nasal Polyps,CRSwNP)是鼻窦及鼻腔粘膜的慢性炎症性疾病,其特点是在鼻粘膜慢性炎症的基础上伴发高度水肿的炎性组织。CRSwNP在人群中的发病率为1%～4%,是鼻科学长期关注的焦点问题之一。目前CRSwNP被认为是多种因素导致的炎症反应,包括解剖异常、感染和非感染性炎症、免疫、遗传因素等。但这些都只是假说,其病因及发病机制并不完全清楚,以至于对于鼻息肉的分型、治疗及减少复发原则方面存在争议,目前有研究表明鼻息肉中自噬水平降低[1,2],自噬参与了鼻息肉的发生。细胞自噬(autophagy)是真核生物细胞中广泛存在的生物现象,其通过多种酶实现自身代谢及细胞器的更新,贯穿于细胞生长发育及病理生理过程,对维持细胞稳态有重要意义,其中LC3-B及P62是两种自噬相关基因。自噬与肿瘤、感染、免疫疾病、退行性病变、炎症性疾病等有关,而且近年来对于靶向自噬的研究也成为热点,期待通过调节细胞自噬水平达到治疗疾病的目的。目前有研究证明调节自噬水平是治疗肿瘤、神经退行性病变、免疫疾病以及炎症性疾病是一种有效途径。鼻息肉的治疗主要包括药物治疗和手术治疗,其中糖皮质激素的治疗可贯穿于手术治疗的前后,局部鼻喷或口服激素可缩小息肉的体积并延缓息肉的生长速度,被形象的称为"药物性鼻息肉切除"[3],但是口服激素对鼻息肉患者自噬水平是否有影响还有待进一步研究。目的:本课题从组织学入手,检测口服糖皮质激素(甲泼尼龙)治疗前后慢性鼻-鼻窦炎伴鼻息肉患者病变组织中自噬相关LC3-B、P62基因及蛋白表达水平的变化情况,分析激素对鼻息肉中自噬水平表达的影响,探讨自噬及其在鼻息肉中的治疗潜力,以探讨甲泼尼龙治疗鼻息肉的药理机制。方法:收集2015.04-2016.04于山东大学齐鲁医院经病理确诊的CRSwNP患者38例(男20例,女18例,年龄27～55岁,平均43.3岁),诊断标准参照2012年昆明诊疗指南[4],服药前于鼻内镜下钳取中鼻道鼻息肉组织为NP(-GC)组,然后给予患者口美卓乐激素治疗15天(甲泼尼龙,前10天20mg/qd,后5天8mg/qd)后,再次钳取同一侧的息肉组织为NP(+GC)组。选取我科同时期确诊的鼻中隔偏曲患者20例(男11例,女9例,年龄23～52岁,平均40.2岁),行鼻内镜手术时,于镜下钳取的中鼻甲黏膜作对照组。各组患者之间的年龄构成差异(P=0.318)及性别构成差异(P=0.460)之间无统计学意义。所有患者术前1月均未口服或鼻喷抗组胺类及抗生素类等药物,排除肿瘤、免疫缺陷等全身性疾病,排除鼻窦单纯囊性变、真菌性鼻窦炎等。本研究经山东大学齐鲁医院医院伦理委员会审核并批准,研究的目的及方法均已向患者及家属说明,均签署参与本研究的知情同意书。本实验采用HE染色检测鼻息肉组织的一般形态特征和炎性细胞的浸润情况;应用免疫组化S-P法检测自噬相关LC3-B、P62蛋白表达情况;采用实时定量反转录聚合酶链反应(quantitativereal-timePCR,RT-PCR)技术检测自噬相关LC3-B、P62基因mRNA表达的情况。采用SPSS21.0软件进行统计学分析,P0.05为差异具有统计学意义。结果:1、HE染色结果:服用甲泼尼龙治疗前的CRSwNP患者息肉组织粘膜表面被覆假复层纤毛柱状上皮,粘膜下见结缔组织呈明显疏松状态,细胞高度水肿,可见组织间隙内腺体增大明显伴增生,血管管腔增宽。与之形成明显对比,经甲泼尼龙治疗后患者鼻息肉组织可见明显好转,主要表现为上皮水肿减弱,嗜酸性粒细胞及中性粒细胞浸润均可见大幅度减少。2、免疫组化染色结果①各组间LC3-B蛋白的表达差异:在对照组、NP(-GC)及NP(+GC)高表达率为分别为 75.00%、13.10%和73.68%。LC3-B在NP(-GC)与NP(+GC)之间(P0.05)、在对照组与NP(-GC)之间(P0.05),差异有统计学意义;②各组间P62蛋白的表达差异:在对照组、NP(-GC)及NP(+GC)高表达率为分别为 20.00%、71.05%和 23.68%。P62 在 NP(-GC)与 NP(+GC)之间(P0.05),在对照组与NP(-GC)之间(P0.05),差异有统计学意义。3、RT-PCR检测自噬相关基因mRNA水平及变化:①LC3-B mRNA 的表达情况:NP(-GC)组为 1.009±0.4583,NP(+GC)组为 2.685±0.5284,对照组为 3.291±0.8650。两两比较:LC3-B 在 NP(-GC)与NP(+GC)之间(P0.05)、在对照组与NP(-GC)之间差异有统计学意义(P0.05)。②P62mRNA 的表达情况:P62mRNA:NP(-GC)组为 2.2175±0.4974,NP(+GC)组为 0.4617±0.1367,对照组为 0.4775±0.3270。两两比较:P62在NP(-GC)与NP(+GC)之间(P0.05),在对照组与NP(-GC)之间差异有统计学意义(P0.05)。结论:1、鼻息肉组织自噬相关LC3-B基因及蛋白较正常对照组表达下调,P62表达上调,说明鼻息肉组织自噬水平降低,自噬可能参与了鼻息的发生;2、甲泼尼龙作用后,息肉组织中自噬相关LC3-B基因及蛋白表达上调,P62表达下调,自噬水平升高,说明甲泼尼龙能够通过上调息肉组织的自噬水平来治疗鼻息肉。表明自噬是激素干预的靶点,并且针对自噬的治疗策略可能为治疗鼻息肉提供新的治疗方法。
[Abstract]:BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the nasal sinuses and nasal mucosa. It is characterized by a high degree of edema on the basis of chronic inflammation of the nasal mucosa. The incidence of CRSwNP in the population ranges from 1% to 4%, which has long been the focus of attention in rhinology. At present, CRSwNP is considered to be an inflammatory response caused by many factors, including anatomical abnormalities, infectious and non-infectious inflammation, immune, genetic factors, etc. But these are only hypotheses. The etiology and pathogenesis of CRSwNP are not completely clear, so there is controversy about the classification, treatment and the principle of reducing recurrence of nasal polyps. Autophagy is a ubiquitous biological phenomenon in eukaryotic cells. Autophagy is a metabolic and organelle renewal mechanism that occurs throughout cell growth, development and pathophysiology. It is important to maintain cell homeostasis. LC3-B and P62 are two autophagy-related genes.Autophagy is associated with tumors, infections, immune diseases, degenerative diseases, inflammatory diseases and so on.In recent years, the research on targeting autophagy has become a hot spot, expecting to achieve the purpose of treating diseases by regulating the level of autophagy.Currently, studies have proved that regulating the level of autophagy is the treatment of tumors, God. The treatment of nasal polyps mainly includes drug therapy and surgical treatment. Glucocorticoid therapy can be used before and after surgery. Local nasal spraying or oral administration of glucocorticoids can reduce the size of polyps and slow down the growth of polyps. It is known as "nasal polyps". Objective: To detect the expression of autophagy-related LC3-B, P62 gene and protein in the tissues of patients with chronic rhinosinusitis and nasal polyps before and after oral glucocorticoid (methylprednisolone) therapy. Methods: Thirty-eight patients (20 males and 18 females, aged 27-5 years) with CRSwNP diagnosed by pathology in Qilu Hospital of Shandong University from April 2015 to April 2016 were collected. Five years old, mean 43.3 years old. According to the Kunming Diagnostic and Therapeutic Guidelines of 2012 [4], the middle nasal meatus and nasal polyp tissues were forcefully taken under nasal endoscope before taking the medicine as NP (-GC) group. Then the patients were given praziquantel for 15 days (20 mg/qd before methylprednisolone, 8 mg/qd after the first 10 days, and NP (+GC) group again. Twenty patients (11 males and 9 females, aged 23-52, with an average age of 40.2 years) with nasal septum deviation were diagnosed at the first stage. The middle turbinate mucosa was clamped under the endoscope as the control group. There was no significant difference in age composition (P = 0.318) and sex composition (P = 0.460) between the two groups. Spraying antihistamines and antibiotics, excluding systemic diseases such as tumor and immunodeficiency, excluding simple cystic degeneration of sinuses, fungal sinusitis and so on. This study was examined and approved by the ethics committee of Qilu Hospital of Shandong University. The purpose and methods of the study have been explained to the patients and their families. All the participants signed the same information. In this study, HE staining was used to detect the general morphological characteristics of nasal polyps and the infiltration of inflammatory cells; immunohistochemical S-P method was used to detect the expression of autophagy-related LC3-B, P62 protein; real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the autophagy-related LC3-B, P62 gene mRNA table. Results: 1. HE staining results: Before taking methylprednisolone treatment, the surface of the polyp tissue was covered with pseudostratified ciliated columnar epithelium, the connective tissue was obviously loose, the cells were highly edematous, and the interstitial space was visible. The enlargement of internal glands was accompanied by hyperplasia and the enlargement of vascular lumen. In contrast, the nasal polyps of the patients treated with methylprednisolone showed a marked improvement. The epithelial edema was weakened and the infiltration of eosinophils and neutrophils was significantly reduced. Differences: In the control group, the high expression rates of NP (-GC) and NP (+GC) were 75.00%, 13.10% and 73.68%, respectively. LC3-B was significantly different between NP (-GC) and NP (+GC) (P 0.05), and between the control group and NP (-GC) (P 0.05). The difference of P62 protein expression among the control group was significant. In the control group, the high expression rates of NP (-GC) and NP (+GC) were 20.00%, 71.05% and 23.68%, respectively. P62 was significantly different between NP (-GC) and NP (+GC) (P 0.05), and between the control group and NP (-GC) (P 0.05). The levels and changes of autophagy-related gene mRNA were detected by RT-PCR. The expression of LC3-B mRNA was 1.009 (-GC) in NP (-GC) group, 2.685 (+GC) in NP (+GC) group and 3.291 (-GC) in NP (-GC) group, respectively. There was significant difference between P62 mRNA expression and NP (+ GC) in the control group and NP (- GC) (P 0.05). The expression of P62 mRNA in the NP (- GC) group was 2.2175 (- 4974), NP (+ GC) group was 0.4617 (- 0.1367), and the control group was 0.4775 (- GC) and NP (- GC) respectively (P 0.05). Conclusion: 1. The expression of autophagy-related LC3-B gene and protein in nasal polyps is down-regulated and the expression of P62 is up-regulated compared with the normal control group, suggesting that the autophagy level of nasal polyps is down-regulated and autophagy may be involved in the occurrence of nasal polyps. 2. After methylprednisolone treatment, the expression of autophagy-related LC3-B gene and protein is up-regulated, while the expression of P62 is down-regulated. The elevated autophagy level indicates that methylprednisolone can treat nasal polyps by increasing the autophagy level of polyps, indicating that autophagy is the target of hormone intervention, and the treatment strategy for autophagy may provide a new treatment for nasal polyps.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R765.25
本文编号：2185555
[Abstract]:BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the nasal sinuses and nasal mucosa. It is characterized by a high degree of edema on the basis of chronic inflammation of the nasal mucosa. The incidence of CRSwNP in the population ranges from 1% to 4%, which has long been the focus of attention in rhinology. At present, CRSwNP is considered to be an inflammatory response caused by many factors, including anatomical abnormalities, infectious and non-infectious inflammation, immune, genetic factors, etc. But these are only hypotheses. The etiology and pathogenesis of CRSwNP are not completely clear, so there is controversy about the classification, treatment and the principle of reducing recurrence of nasal polyps. Autophagy is a ubiquitous biological phenomenon in eukaryotic cells. Autophagy is a metabolic and organelle renewal mechanism that occurs throughout cell growth, development and pathophysiology. It is important to maintain cell homeostasis. LC3-B and P62 are two autophagy-related genes.Autophagy is associated with tumors, infections, immune diseases, degenerative diseases, inflammatory diseases and so on.In recent years, the research on targeting autophagy has become a hot spot, expecting to achieve the purpose of treating diseases by regulating the level of autophagy.Currently, studies have proved that regulating the level of autophagy is the treatment of tumors, God. The treatment of nasal polyps mainly includes drug therapy and surgical treatment. Glucocorticoid therapy can be used before and after surgery. Local nasal spraying or oral administration of glucocorticoids can reduce the size of polyps and slow down the growth of polyps. It is known as "nasal polyps". Objective: To detect the expression of autophagy-related LC3-B, P62 gene and protein in the tissues of patients with chronic rhinosinusitis and nasal polyps before and after oral glucocorticoid (methylprednisolone) therapy. Methods: Thirty-eight patients (20 males and 18 females, aged 27-5 years) with CRSwNP diagnosed by pathology in Qilu Hospital of Shandong University from April 2015 to April 2016 were collected. Five years old, mean 43.3 years old. According to the Kunming Diagnostic and Therapeutic Guidelines of 2012 [4], the middle nasal meatus and nasal polyp tissues were forcefully taken under nasal endoscope before taking the medicine as NP (-GC) group. Then the patients were given praziquantel for 15 days (20 mg/qd before methylprednisolone, 8 mg/qd after the first 10 days, and NP (+GC) group again. Twenty patients (11 males and 9 females, aged 23-52, with an average age of 40.2 years) with nasal septum deviation were diagnosed at the first stage. The middle turbinate mucosa was clamped under the endoscope as the control group. There was no significant difference in age composition (P = 0.318) and sex composition (P = 0.460) between the two groups. Spraying antihistamines and antibiotics, excluding systemic diseases such as tumor and immunodeficiency, excluding simple cystic degeneration of sinuses, fungal sinusitis and so on. This study was examined and approved by the ethics committee of Qilu Hospital of Shandong University. The purpose and methods of the study have been explained to the patients and their families. All the participants signed the same information. In this study, HE staining was used to detect the general morphological characteristics of nasal polyps and the infiltration of inflammatory cells; immunohistochemical S-P method was used to detect the expression of autophagy-related LC3-B, P62 protein; real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the autophagy-related LC3-B, P62 gene mRNA table. Results: 1. HE staining results: Before taking methylprednisolone treatment, the surface of the polyp tissue was covered with pseudostratified ciliated columnar epithelium, the connective tissue was obviously loose, the cells were highly edematous, and the interstitial space was visible. The enlargement of internal glands was accompanied by hyperplasia and the enlargement of vascular lumen. In contrast, the nasal polyps of the patients treated with methylprednisolone showed a marked improvement. The epithelial edema was weakened and the infiltration of eosinophils and neutrophils was significantly reduced. Differences: In the control group, the high expression rates of NP (-GC) and NP (+GC) were 75.00%, 13.10% and 73.68%, respectively. LC3-B was significantly different between NP (-GC) and NP (+GC) (P 0.05), and between the control group and NP (-GC) (P 0.05). The difference of P62 protein expression among the control group was significant. In the control group, the high expression rates of NP (-GC) and NP (+GC) were 20.00%, 71.05% and 23.68%, respectively. P62 was significantly different between NP (-GC) and NP (+GC) (P 0.05), and between the control group and NP (-GC) (P 0.05). The levels and changes of autophagy-related gene mRNA were detected by RT-PCR. The expression of LC3-B mRNA was 1.009 (-GC) in NP (-GC) group, 2.685 (+GC) in NP (+GC) group and 3.291 (-GC) in NP (-GC) group, respectively. There was significant difference between P62 mRNA expression and NP (+ GC) in the control group and NP (- GC) (P 0.05). The expression of P62 mRNA in the NP (- GC) group was 2.2175 (- 4974), NP (+ GC) group was 0.4617 (- 0.1367), and the control group was 0.4775 (- GC) and NP (- GC) respectively (P 0.05). Conclusion: 1. The expression of autophagy-related LC3-B gene and protein in nasal polyps is down-regulated and the expression of P62 is up-regulated compared with the normal control group, suggesting that the autophagy level of nasal polyps is down-regulated and autophagy may be involved in the occurrence of nasal polyps. 2. After methylprednisolone treatment, the expression of autophagy-related LC3-B gene and protein is up-regulated, while the expression of P62 is down-regulated. The elevated autophagy level indicates that methylprednisolone can treat nasal polyps by increasing the autophagy level of polyps, indicating that autophagy is the target of hormone intervention, and the treatment strategy for autophagy may provide a new treatment for nasal polyps.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R765.25
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