甲潑尼龍對鼻息肉中自噬相關基因LC3-B和P62表達影響的研究
發(fā)布時間：2018-08-16 09:19
【摘要】：背景:慢性鼻-鼻竇炎伴鼻息肉(Chronic Rhinosinusitis with Nasal Polyps,CRSwNP)是鼻竇及鼻腔粘膜的慢性炎癥性疾病,其特點是在鼻粘膜慢性炎癥的基礎上伴發(fā)高度水腫的炎性組織。CRSwNP在人群中的發(fā)病率為1%～4%,是鼻科學長期關注的焦點問題之一。目前CRSwNP被認為是多種因素導致的炎癥反應,包括解剖異常、感染和非感染性炎癥、免疫、遺傳因素等。但這些都只是假說,其病因及發(fā)病機制并不完全清楚,以至于對于鼻息肉的分型、治療及減少復發(fā)原則方面存在爭議,目前有研究表明鼻息肉中自噬水平降低[1,2],自噬參與了鼻息肉的發(fā)生。細胞自噬(autophagy)是真核生物細胞中廣泛存在的生物現(xiàn)象,其通過多種酶實現(xiàn)自身代謝及細胞器的更新,貫穿于細胞生長發(fā)育及病理生理過程,對維持細胞穩(wěn)態(tài)有重要意義,其中LC3-B及P62是兩種自噬相關基因。自噬與腫瘤、感染、免疫疾病、退行性病變、炎癥性疾病等有關,而且近年來對于靶向自噬的研究也成為熱點,期待通過調節(jié)細胞自噬水平達到治療疾病的目的。目前有研究證明調節(jié)自噬水平是治療腫瘤、神經(jīng)退行性病變、免疫疾病以及炎癥性疾病是一種有效途徑。鼻息肉的治療主要包括藥物治療和手術治療,其中糖皮質激素的治療可貫穿于手術治療的前后,局部鼻噴或口服激素可縮小息肉的體積并延緩息肉的生長速度,被形象的稱為"藥物性鼻息肉切除"[3],但是口服激素對鼻息肉患者自噬水平是否有影響還有待進一步研究。目的:本課題從組織學入手,檢測口服糖皮質激素(甲潑尼龍)治療前后慢性鼻-鼻竇炎伴鼻息肉患者病變組織中自噬相關LC3-B、P62基因及蛋白表達水平的變化情況,分析激素對鼻息肉中自噬水平表達的影響,探討自噬及其在鼻息肉中的治療潛力,以探討甲潑尼龍治療鼻息肉的藥理機制。方法:收集2015.04-2016.04于山東大學齊魯醫(yī)院經(jīng)病理確診的CRSwNP患者38例(男20例,女18例,年齡27～55歲,平均43.3歲),診斷標準參照2012年昆明診療指南[4],服藥前于鼻內鏡下鉗取中鼻道鼻息肉組織為NP(-GC)組,然后給予患者口美卓樂激素治療15天(甲潑尼龍,前10天20mg/qd,后5天8mg/qd)后,再次鉗取同一側的息肉組織為NP(+GC)組。選取我科同時期確診的鼻中隔偏曲患者20例(男11例,女9例,年齡23～52歲,平均40.2歲),行鼻內鏡手術時,于鏡下鉗取的中鼻甲黏膜作對照組。各組患者之間的年齡構成差異(P=0.318)及性別構成差異(P=0.460)之間無統(tǒng)計學意義。所有患者術前1月均未口服或鼻噴抗組胺類及抗生素類等藥物,排除腫瘤、免疫缺陷等全身性疾病,排除鼻竇單純囊性變、真菌性鼻竇炎等。本研究經(jīng)山東大學齊魯醫(yī)院醫(yī)院倫理委員會審核并批準,研究的目的及方法均已向患者及家屬說明,均簽署參與本研究的知情同意書。本實驗采用HE染色檢測鼻息肉組織的一般形態(tài)特征和炎性細胞的浸潤情況;應用免疫組化S-P法檢測自噬相關LC3-B、P62蛋白表達情況;采用實時定量反轉錄聚合酶鏈反應(quantitativereal-timePCR,RT-PCR)技術檢測自噬相關LC3-B、P62基因mRNA表達的情況。采用SPSS21.0軟件進行統(tǒng)計學分析,P0.05為差異具有統(tǒng)計學意義。結果:1、HE染色結果:服用甲潑尼龍治療前的CRSwNP患者息肉組織粘膜表面被覆假復層纖毛柱狀上皮,粘膜下見結締組織呈明顯疏松狀態(tài),細胞高度水腫,可見組織間隙內腺體增大明顯伴增生,血管管腔增寬。與之形成明顯對比,經(jīng)甲潑尼龍治療后患者鼻息肉組織可見明顯好轉,主要表現(xiàn)為上皮水腫減弱,嗜酸性粒細胞及中性粒細胞浸潤均可見大幅度減少。2、免疫組化染色結果①各組間LC3-B蛋白的表達差異:在對照組、NP(-GC)及NP(+GC)高表達率為分別為 75.00%、13.10%和73.68%。LC3-B在NP(-GC)與NP(+GC)之間(P0.05)、在對照組與NP(-GC)之間(P0.05),差異有統(tǒng)計學意義;②各組間P62蛋白的表達差異:在對照組、NP(-GC)及NP(+GC)高表達率為分別為 20.00%、71.05%和 23.68%。P62 在 NP(-GC)與 NP(+GC)之間(P0.05),在對照組與NP(-GC)之間(P0.05),差異有統(tǒng)計學意義。3、RT-PCR檢測自噬相關基因mRNA水平及變化:①LC3-B mRNA 的表達情況:NP(-GC)組為 1.009±0.4583,NP(+GC)組為 2.685±0.5284,對照組為 3.291±0.8650。兩兩比較:LC3-B 在 NP(-GC)與NP(+GC)之間(P0.05)、在對照組與NP(-GC)之間差異有統(tǒng)計學意義(P0.05)。②P62mRNA 的表達情況:P62mRNA:NP(-GC)組為 2.2175±0.4974,NP(+GC)組為 0.4617±0.1367,對照組為 0.4775±0.3270。兩兩比較:P62在NP(-GC)與NP(+GC)之間(P0.05),在對照組與NP(-GC)之間差異有統(tǒng)計學意義(P0.05)。結論:1、鼻息肉組織自噬相關LC3-B基因及蛋白較正常對照組表達下調,P62表達上調,說明鼻息肉組織自噬水平降低,自噬可能參與了鼻息的發(fā)生;2、甲潑尼龍作用后,息肉組織中自噬相關LC3-B基因及蛋白表達上調,P62表達下調,自噬水平升高,說明甲潑尼龍能夠通過上調息肉組織的自噬水平來治療鼻息肉。表明自噬是激素干預的靶點,并且針對自噬的治療策略可能為治療鼻息肉提供新的治療方法。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R765.25
本文編號：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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R765.25
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