西地那非对血管瘤内皮细胞增殖和凋亡的影响及机制探讨

发布时间：2017-12-31 11:05

本文关键词：西地那非对血管瘤内皮细胞增殖和凋亡的影响及机制探讨　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景血管瘤(hemangiomas)是发生在婴幼儿的血管源性良性肿瘤,发病率高达10～12%,绝大多数在出生时或出生后不久发现,大部分可自行消退,但仍有20%的血管瘤不能消退。根据病变发展过程可分为增殖期、消退期和消退完成期。血管瘤虽然属于良性肿瘤,但发生在口腔颌面及颈部的病变,不仅严重影响面容美观,还可能由于病变阻塞呼吸道、消化道而影响发音、进食,甚至导致局部出血、窒息并危及生命。因此目前主张对该病应积极进行早期治疗。血管瘤常用的治疗方法有药物治疗、激光、手术切除、冷冻以及硬化治疗等。冷冻、激光及硬化治疗易导致增生或萎缩性瘢痕、色素沉着或色素减退等,手术切除仅适用于局限性的血管瘤的治疗。药物治疗则是一种治疗早期血管瘤安全、有效的方法。治疗血管瘤的药物有多种,目前国内外常用药物包括以下几类:1.β-肾上腺素受体阻滞剂,以普萘洛尔(propranolol)为主;2.皮质类固醇激素,以泼尼松(prednisolone)为主;3.其它:α-干扰素(IFN-α)、咪喹莫特(Imiquimod)、抗肿瘤药环磷酰胺(CTX)及长春新碱(VCR)等。2008年,在《新英格兰医学杂志》上,Leaute-Labreze C等首次报道口服普萘洛尔引起了鼻咽部血管瘤的消退。近年来,口服普萘洛尔逐渐成为治疗血管瘤的一线药物。2012年,Swetman等首次报道口服西地那非(sildenafil)治疗3名淋巴管畸形患儿后病变显著消退,发表于《新英格兰医学杂志》上,提示西地那非可作为治疗淋巴管畸形的一种新型药物。但该文章仅展示了患儿病变部位的照片和核磁共振平片(magnetic resonance imaging,MRI),病变均未经手术及病理证实为淋巴管畸形。根据MRI表现,我们认为此患儿更符合血管瘤伴发淋巴管畸形的诊断,且病变以血管瘤为主。西地那非是一种磷酸二酯酶-5(phosphodiesterase isoform-5,PDE-5)抑制剂,因而我们推测可能西地那非是通过抑制PDE-5的作用使该病变缩小的。在此启发下,我们通过免疫组织化学的方法研究了 PDE-5在静脉畸形、动静脉畸形、淋巴管畸形、血管瘤组织中的表达。结果表明PDE-5在血管瘤组织中呈阳性表达,在其它脉管畸形组织中无表达。因此,我们认为西地那非是对血管瘤而不是淋巴管畸形起作用。目前很多文献证实西地那非可增强某些类型的恶性肿瘤化疗效果并对血管平滑肌、肺动脉内皮细胞等有抗增殖及促进凋亡的作用。因此,我们通过体外实验进一步研究西地那非对血管瘤内皮细胞(HemECs)增殖及凋亡的影响,并对其相关机制进行探讨。目的1.检测PDE-5在血管瘤及各种脉管畸形组织中的表达。2.研究西地那非对人源性HemECs的增殖和凋亡的影响。3.研究西地那非对HemECs的增殖和凋亡作用与DNA分化抑制因子-1(Id-1)表达的关系,探讨该药的作用机制。方法1.收集山东大学齐鲁医院病理科2000—2013年血管瘤及脉管畸形组织块,通过免疫组织化学的方法分别检测石蜡包埋的10例淋巴管畸形、8例增殖期血管瘤、2例消退期血管瘤、10例静脉畸形以及10例动静脉畸形组织块中PDE-5的表达。血管瘤及脉管畸形的诊断是依据患者病史、体格检查、MRI表现以及最后的病理结果确定的。淋巴管内皮透明质酸受体-1(LYVE-1)、细胞膜表面分化抗原34(CD 34)和葡萄糖转运蛋白-1(GLUT-1)分别作为淋巴管内皮细胞、血管内皮细胞和血管瘤组织的特异性标记物。随后对PDE-5在血管瘤及各种脉管畸形组织的表达进行了检测。本研究通过了山东大学齐鲁医院医院伦理委员会授权,所有患者或监护人均知情同意。2.收集山东大学齐鲁医院手术切除并经病理证实为血管瘤组织标本,利用组织块贴壁培养法分离并培养HemECs。通过免疫细胞化学法检测第八因子相关抗原因子vWF、CD 34和PDE-5在HemECs中的表达。体外以梯度浓度西地那非(0μM,1 μM,3 μM,5 μM,10 μM 和 15 μM)处理 HemECs 24 h、48 h 和 72 h后,MTT法检测细胞的增殖能力。以梯度浓度的西地那非(0,1,5,10,15 μM)处理HemECs,20分钟后分别加入50ng/mlbFGF、50ng/mlVEGF。继续孵箱培育24 h。MTT法检测西地那非对bFGF及VEGF诱导的HemECs增殖的影响。HemECs分别经对照组(0 μM)、5 μM西地那非处理24 h后,采用流式细胞Annexin-V/PI staining assay 检测细胞凋亡率。3.组织块培养法获得的HemECs分别经0μM(对照组)、5μM(实验组)西地那非处理24 h后,用TRI法提取细胞总RNA并逆转录成Id-1 cDNA,再通过实时荧光定量RFQ-PCR扩增cDNA,利用比较Ct值法(2-△△Ct)计算Id-l基因和内参照基因阈值循环数差异,检测两组HemECs中Id-1基因的相对mRNA表达量。HemECs分别经0 μM(对照组)、5μ(实验组)西地那非处理24 h后,提取细胞总蛋白,通过免疫印迹法(western blot)检测两组HemECs中Id-1蛋白的表达水平,人舌鳞癌TCA 8113细胞系作为Id-1蛋白表达的阳性对照。结果1.免疫组织化学检测发现CD34、LYVE-I和GLUT-1分别作为血管内皮细胞、淋巴管畸形组织和血管瘤组织的特异性标记物表达均阳性。PDE-5表达在血管瘤组织HemECs胞浆内,但在淋巴管畸形、动静脉畸形、静脉畸形组织中均无表达。2.MTT法检测发现在西地那非浓度为5 μM时,24h后与对照组(0μM)相比开始出现显著差异(t =3.220,P0.01),出现明显的细胞抑制现象,且随时间延长抑制作用逐渐加强。西地那非作用于HemECs 72 h时细胞的增殖活性与48 h相比无明显差异(P0.05)。在检测西地那非对bFGF及VEGF诱导的HemECs增殖的抑制作用时,西地那非浓度为5 μ时,对bFGF及VEGF诱导的HemECs增殖也出现明显的细胞抑制现象(P0.05),且随时间延长抑制作用逐渐加强。然而,当西地那非浓度为15 μM时,与浓度为10 μM相比对HemECs的增殖抑制作用无明显差别(P0.05)。因此,浓度为5 μM和作用时间为24 h分别是西地那非抑制HemECs的最适有效浓度和最适有效作用时间。用5 μM西地那非处理HemECs 24 h后,在光学显微镜下可见到HemECs随着细胞间连接的破坏和胞浆内不断增加的颗粒,细胞形态发生了改变。流式细胞学分析结果表明5 μM西地那非处理HemECs 24 h后凋亡率为10.5%,高于对照组(2.53%)。因此,体外实验中浓度5 μM的西地那非处理24 h后显著抑制了 HemECs的增殖并促进了其凋亡。3.通过实时荧光定量RFQ-PCR方法,根据2-△△Ct公式,用5μM西地那非处理HemECs 24 h后,细胞内Id-1的mRNA相对表达量减少了 44.2%,(t = 9.749,df=4,P = 0.00060.01)。根据Western blot实验取得的免疫印迹条带的灰度值比较分析,用5 μM西地那非处理HemECs 24 h后,细胞内Id-1的蛋白表达也降低。结论1.血管瘤组织中存在磷酸二酯酶-5。2.西地那非在体外能抑制HemECs增殖和促进其凋亡。3.西地那非抑制HemECs增殖和促进其凋亡作用可能是通过下调Id-1基因来实现的。
[Abstract]:The research background of hemangioma (hemangiomas) occurred in vascular benign tumors in infants, the incidence rate of 10 ~ 12%, the vast majority at birth or shortly after birth, most can fade, but there are still not 20% hemangioma disappeared. According to the pathological development process can be divided into proliferating, involuting and complete regression period. Although hemangioma is benign tumor, but occurred in the oral maxillofacial and neck disease, not only affects the face appearance, also may be due to lesions of digestive tract and respiratory tract obstruction, influence of pronunciation, eating, and even lead to local hemorrhage, asphyxia and life-threatening. Therefore the claim of the disease should be performed early treatment treatment of hemangioma. Methods commonly used drugs, laser, surgery, refrigeration and hardening treatment. Freezing, laser hardening treatment and easily lead to hyperplasia or atrophic scars, pigmentation or pigment reduction Back, treatment of hemangioma resection is only applicable to limitations. Drug therapy is an early treatment of hemangioma is safe, effective method. There are many drugs for the treatment of hemangioma, the drugs commonly used at home and abroad include the following categories: 1. beta adrenergic receptor blockers, with propranolol (propranolol); 2. corticosteroids prednisone (prednisolone); the other 3.: interferon alpha (IFN- alpha), imiquimod (Imiquimod), antitumor drug cyclophosphamide (CTX) and vincristine (VCR),.2008, < > in the new England Journal of medicine, Leaute-Labreze C reported the first oral propranolol caused regression of nasopharyngeal hemangioma. In recent years, oral propranolol in the treatment of hemangioma has gradually become the first-line drugs.2012, Swetman reported the first oral sildenafil (sildenafil) treatment of 3 children with lymphatic malformation lesions were significantly. Back, published in the new England Journal of medicine < >, suggesting a new drug Sildenafil can be used as the treatment of lymphatic malformation. But the article only shows children lesion photos and MRI plain film (magnetic resonance imaging, MRI), the lesions were not confirmed by surgery and pathology of lymphatic malformation. According to the MRI, we think this is more in line with the children with hemangioma associated with lymphatic malformation diagnosis, and patients with hemangioma. Sildenafil is a phosphodiesterase -5 (phosphodiesterase isoform-5 PDE-5) inhibitor, so we may infer sildenafil by inhibition of PDE-5 activity of the lesions reduced. Under this inspiration, we study PDE-5 in venous malformation by immunohistochemical method, arteriovenous malformation, lymphatic malformations, expression of vascular tumor tissues. The results showed that PDE-5 was positive in hemangioma The expression of vascular malformations in other tissues without expression. Therefore, we believe that sildenafil is on hemangioma instead of lymphatic malformation effect. At present a lot of literatures demonstrate that sildenafil can enhance chemotherapy effect of certain types of vascular smooth muscle, anti proliferation and promote the apoptosis of pulmonary artery endothelial cells. So, we study the effect of sildenafil on vascular endothelial cells and further in vitro experiments (HemECs) proliferation and apoptosis, and the related mechanisms were discussed. The expression of.2. on proliferation and apoptosis of sildenafil in human HemECs.3. effect of sildenafil on HemECs proliferation and apoptosis and DNA differentiation inhibitory factor -1 1. detection PDE-5 in the form of abnormal tissue vascular hemangioma (Id-1) and the expression of the relationship, to explore the mechanism of action of the drug. Methods 1. collection of Shandong University Qilu medicine Institute of disease science from 2000 to 2013 in hemangioma and vascular malformation tissue were detected in 10 cases of paraffin embedded lymph tube defects by immunohistochemical method, 8 cases of hemangioma, 2 cases of hemangioma, 10 cases of venous malformation and 10 cases of arteriovenous malformation tissue expression of PDE-5 in vascular. Hemangioma and vascular malformation diagnosis is based on patient history, physical examination, MRI and final pathological results determined. Lymphatic endothelial hyaluronan receptor -1 (LYVE-1), cell surface differentiation antigen 34 (CD 34) and glucose transporter -1 (GLUT-1) respectively as lymphatic endothelial cell specific markers. From vascular endothelial cells and tumor tissues. The expression of PDE-5 in hemangioma and vascular malformations tissue were detected. This study through the authorization of Qilu Hospital of Shandong University hospital ethics committee, and all patients or guardians Informed consent was obtained from Qilu Hospital of Shandong University.2. surgery and pathologically confirmed hemangioma tissues by tissue adherent method were isolated and cultured by HemECs. factor related antigen eighth factor immunocytochemical method for detection of vWF expression of CD 34 and PDE-5 in HemECs. In vitro with gradient concentration of sildenafil (0 M, 1 M, 3 M, 5 M, 10 M and 15 M) HemECs 24 h, 48 h and 72 h, the cell proliferation was determined by MTT. The concentrations of sildenafil (0,1,5,10,15 M) HemECs, 20 minutes after 50ng/ mlbFGF were added to 50ng/mlVEGF., continue to affect the hatch box cultivate 24 h.MTT method to detect the effect of sildenafil on the proliferation of HemECs induced by bFGF and VEGF.HemECs respectively by the control group (0 M), 5 M sildenafil treatment after 24 h Annexin-V/PI staining assay by flow cytometry to detect the apoptosis rate of.3. tissue culture Method to obtain HemECs respectively by 0 M (control group), 5 M (experimental group) and sildenafil treatment after 24 h extracted with TRI cell total RNA and reverse transcription into Id-1 cDNA by real-time fluorescent quantitative RFQ-PCR amplification of cDNA, using the Ct value comparison method (2- Delta Ct) gene and Id-l calculation the reference gene threshold cycle number difference, relative to the mRNA detection of Id-1 two in the HemECs group the expression of.HemECs gene respectively by 0 M (control group), 5 (experimental group) and sildenafil treatment after 24 h, the total proteins were extracted, by immunoblotting (Western blot) to detect the expression of Id-1 two in the HemECs group protein, positive control of human tongue squamous cell carcinoma cell line TCA 8113 as the expression of Id-1 protein. Results 1. immunohistochemical detection of chemical found in CD34, LYVE-I and GLUT-1 respectively as vascular endothelial cells, lymphatic malformations and vascular tumor specific marker expression were positive expression of.PDE-5 in blood The tube of HemECs in tumor tissue in the cytoplasm, but in lymphatic malformations, arteriovenous malformation, venous malformation tissues were not found in the.2.MTT method to detect the expression of sildenafil concentration is 5 M, 24h and control group (0 M) appeared significant difference compared (t =3.220, P0.01), apparent cell inhibition, and the longer the time the inhibition effect gradually strengthened. The proliferation activity of sildenafil in HemECs 72 h cells and 48 h showed no significant difference (P0.05). The inhibition induced by bFGF and VEGF in the detection of sildenafil HemECs proliferation, sildenafil concentration is 5, the induction of bFGF and VEGF the proliferation of HemECs cell inhibition phenomenon is obvious (P0.05), and with the prolonging of the inhibition effect gradually strengthened. However, when the concentration of sildenafil is 15 M, and the concentration of 10 M compared to the inhibitory effect of HemECs was no significant difference (P0.05). Therefore, concentrated Of 5 M and reaction time was 24 h sildenafil inhibited HemECs optimal effective concentration and the optimal effective time. With 5 M HemECs 24 h after sildenafil treatment, under the microscope can be seen with the HemECs connection between the cell damage in the cytoplasm and increasing particles changed cell morphology. Flow cytometry analysis showed that 5 M sildenafil treatment HemECs after 24 h apoptosis rate was 10.5%, higher than the control group (2.53%). Therefore, the concentration of the in vitro experiments of 5 M sildenafil treatment after 24 h significantly inhibited the proliferation of HemECs and promote the apoptosis of.3. by real-time fluorescence quantitative RFQ-PCR method. According to the 2- Delta Ct formula, with 5 M HemECs 24 h after sildenafil treatment, intracellular Id-1 mRNA relative expression decreased by 44.2% (t = 9.749, P = df=4, 0.00060.01). According to the immune blot achieved Western blot Experimental Zone Comparative analysis of gray value, with 5 M HemECs 24 h after sildenafil treatment, the expression of Id-1 protein also decreased. Conclusion there are 1. hernangioma phosphodiesterase -5.2. Sildenafil can promote the apoptosis of.3. and inhibit the proliferation of HemECs and sildenafil in inhibiting the proliferation of HemECs cells and promote their apoptosis through down-regulation of Id-1 gene may be realized in in vitro.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R732.2

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