Basigin-2参与小胶质细胞调控氧诱导视网膜病变新生血管的生成

发布时间：2018-05-30 22:14

  本文选题：Basigin + 小胶质细胞　； 参考：《第四军医大学》2017年博士论文

【摘要】：【研究背景】早产儿视网膜病变(retinopathy of premature,ROP)是一种多发于早产儿和低体重婴儿的视网膜血管病变,是目前世界范围内的首要儿童致盲病因。高浓度氧疗阻断了早产儿尚未成熟的视网膜血管的正常发育进程,造成周边视网膜缺血缺氧,继而引发异常新生血管生成,导致视网膜出血、渗出及增殖。因此,缺氧诱发的异常血管增生是ROP病理发生的核心机制之一。视网膜新生血管的形成是机体应对缺氧环境的适应性反应,包括内皮细胞与多种细胞的交互作用,过程十分复杂,机制远未阐明。小胶质细胞是定居中枢神经系统(central nervous system,CNS)的组织巨噬细胞,参与免疫调节、组织发育、自身稳定及创伤修复过程。近来大量研究选用发育期的小鼠视网膜作为研究血管生成的模型,结果证实,视网膜小胶质细胞是血管生成的早期驱动力量。视网膜小胶质细胞与血管紧密相邻的解剖空间关系强烈提示,小胶质细胞在血管生成中的重要作用,但是其内在机制尚不清楚。此外,小胶质细胞具有很强的移动性,能感知生理及病理刺激,传递微环境的信号,迅速应对局部的趋化信号,通过分泌细胞因子及神经营养因子作用于周围神经及血管成分,完成细胞间的沟通。因此,小胶质细胞在ROP病变中极有可能传递局部的缺氧信号,产生适应性反应,促进视网膜新生血管生成。Basigin是一种高度糖基化的跨膜糖蛋白,因具有诱导基质金属蛋白酶(matrix metalloproteinase,MMP)生成的能力被命名为MMP诱导因子,简称EMMPRIN,也被称为CD147。Basigin具有多重生物功能,如免疫反应、基质降解及细胞移行。该分子尤其在肿瘤局部的缺氧环境下促进新生血管生成,从而加强肿瘤的侵袭和转移。它不仅具备蛋白酶诱导作用,促进MMP的生成,也能增加肿瘤及内皮细胞的可溶性血管内皮生长因子(vascular endothelial growth factor,VEGF)及血管内皮生长因子受体(vascular endothelial growth factor,VEGFR)2的分泌。在卵巢癌患者,Basigin可通过可溶性或者以外泌体形式分泌到微环境中,促进肿瘤血管生成。此外,有研究显示Basigin在单核-巨噬细胞的表达上调,增加其炎性活性。Basigin广泛参与缺氧条件下新生血管的生成,但在以缺氧为核心环节的ROP病变中,Basigin是否参与小胶质细胞与血管内皮细胞之间的交互作用还有待研究。【研究目的】探讨小胶质细胞通过Basigin-2调控缺氧相关的视网膜新生血管生成的作用及其机制,为拓展ROP等缺血缺氧性视网膜新生血管疾病的治疗提供实验依据。【研究方法】一、体内实验:Basigin参与小胶质细胞促进氧诱导视网膜病变(oxygen induced retinopathy,OIR)新生血管形成。(1)动物模型:建立小鼠OIR模型。(2)组织病理:制作冰冻切片和视网膜铺片,采用免疫组化及Basigin、IBA-1免疫双荧光染色,标记Basigin及小胶质细胞,观察两者在视网膜的表达及两者的共定位。(3)Western blot检测:测定OIR模型P12和P17,神经视网膜低氧诱导因子(hypoxia inducible factor-α,HIF-1α)和Basigin蛋白表达的变化。二、体外实验:小胶质细胞表达Basigin,增强视网膜脉络膜微血管内皮细胞血管生成能力。(1)细胞模型:应用小胶质细胞BV2细胞系,物理缺氧模式;(2)构建共培养体系:共培养BV2细胞及视网膜脉络膜血管内皮细胞(RF/6A),或应用BV2细胞条件培养基;(3)Western blot检测:测定不同处理条件下,小胶质细胞HIF-1α、Basigin、IGF-1、AKT、P-AKT、ERK及P-ERK等蛋白的表达变化以及RF/6A细胞VEGF和VEGFR-2的表达变化;(4)实时定量-PCR(quantitative reverse transcription PCR,qRT-PCR):检测BV2细胞在不同处理条件下表达的Basigin-1和Basigin-2及IGF-1变化,以及RF/6A细胞VEGF和VEGFR-2的表达变化;(5)细胞迁移实验:利用transwell小室测定RF/6A的迁移能力;(6)管腔形成实验:Matrigel铺胶,Image Pro Plus软件测量管腔形成的长度。(7)si RNA转染:采用脂质体包裹si RNA沉默小胶质细胞Basigin-2的基因表达;(8)细胞因子测定:应用细胞因子芯片,筛选出小胶质细胞缺氧后上调,且在Basigin-2敲减后出现下调的促进血管生成的可溶性功能分子。(9)药物干预:分别应用AKT抑制剂(LY294002)及MEK抑制剂(PD98549)抑制AKT或ERK激活,分析其对IGF-1生成的作用;外源性给予IGF-1蛋白或IGF-1抗体及IGF受体拮抗剂,阻断或激活IGF-1信号,观察对RF/6A管腔形成的影响。(10)统计学分析:应用统计学软件SPSS 17.0版进行分析。多个样本组间均数的比较采用单因素方差分析,任意组间两两比较采用Student-Newmann-Keuls(SNK)检验,P0.05为差异有统计学意义。【研究结果】一、体内实验:Basigin在OIR模型视网膜新生血管簇周围聚集的小胶质细胞内高表达。与正常对照组相比,OIR模型视网膜冰冻切片及视网膜铺片从不同角度均显示,视网膜新生血管簇有明显的IBA-1阳性的小胶质细胞聚集,且小胶质细胞标志物IBA-1与Basigin在新生血管区域有显著的共表达。OIR组P17神经视网膜Basigin的蛋白水平组较正常对照组显著增加(P=0.0012)。二、体外实验:缺氧环境下,小胶质细胞增加Basigin-2表达,促进血管内皮细胞血管生成能力及其分子机制。1.缺氧增加小胶质细胞HIF-1α及Basigin-2的表达小胶质细胞表达的HIF-1α及Basigin蛋白随着物理缺氧时间的延长而增加。Basigin的蛋白表达在缺氧12h(P=0.0097)及24h组(P=0.0003)均较对照组显著增加。qRT-PCR检测发现,缺氧后的小胶质细胞仅表达Basigin-2,且随缺氧时间延长而增加。2.缺氧处理后的小胶质细胞促进血管内皮细胞的管腔形成缺氧处理后的小胶质细胞与血管内皮细胞共培养或其条件培养基,均能促进血管内皮细胞的管腔形成。接种后6h,共培养组管腔形成的长度均值比对照组增加60.80%(P=0.0001);条件培养基组均值比对照组增加52.40%(P=0.0003)。但共培养组与条件培养基组相比无明显差别(P=0.4008),提示对管腔形成的促进作用不依赖于两种细胞的直接接触。3.脂质体包裹si RNA成功沉默Basigin-2使用qRT-PCR及Western blot测定使用脂质体包裹的si RNA敲减Basigin后的干涉效果。si RNA Basigin 317和458两种序列都能明显下调Basigin的核酸及蛋白表达,但使用两种序列联合敲减较单独敲减组的效果更佳。4.阻断小胶质细胞Basigin-2表达抑制血管内皮细胞血管生成能力干涉小胶质细胞Basigin-2表达后,共培养条件下血管内皮细胞的迁移及管腔形成功能被抑制。敲减后迁移细胞数减少65.93%(P=0.0098);接种后6h,管腔形成长度减少68.27%(P=0.0001)。5.阻断小胶质细胞的Basigin-2表达抑制促血管生成因子的生成细胞因子芯片测定敲减小胶质细胞Basigin-2基因后血管生成因子的改变,筛选出IGFBP2,IGF-1,Pro-MMP9,VEGFR-1等4个因子在缺氧后上调;且在Basigin-2敲减后,出现下调。其中IGF-1表达改变最为显著,在缺氧后上调至基础值的3.11倍,敲减后下调为1.33倍。应用qRT-PCR及Western blot检测,BV2敲减Basigin-2后,IGF-1的转录水平下降(P=0.0001);蛋白表达下降(P=0.0082)。6.IGF-1促进内皮细胞的管腔形成培养基中分别加入IGF-1中和抗体及外源性IGF-1,结合对BV2的Basigin-2敲减,评价IGF-1对管腔形成的影响。接种后6h,模拟敲减联合IGF-1中和抗体组管腔形成减少28.83%(P=0.0098);Basigin-2敲减联合IGF-1可增加管腔形成60.32%(P=0.0002)。7.Basigin-2调控IGF-1表达的通路分析分别使用LY294002和PD98549阻断AKT或ERK通路,qRT-PCR检测BV2细胞不同处理组IGF-1的m RNA水平。Basigin-2敲减及AKT抑制剂能逆转缺氧后上调的IGF-1趋势。Western blot测定BV2细胞不同处理组HIF-1,Basigin,IGF-1,P-AKT,AKT,P-ERK及ERK水平。在蛋白水平,缺氧增加IGF-1表达,AKT抑制剂及Basigin-2敲减可以抑制IGF-1的产生,而ERK抑制剂不能抑制IGF-1的表达。8.IGF-1增强内皮细胞管腔形成能力的分子机制使用IGF-1受体中和抗体阻断内皮细胞的IGF-1信号,RF/6A的管腔形成长度减少101.33%(P=0.0001)。RF/6A表达的VEGF核酸和蛋白水平无明显改变;但VEGFR-2的转录下调(P=0.0091),蛋白表达减少(P=0.0006)。【结论】本研究首次报道了Basigin参与小胶质细胞对氧诱导的视网膜病变新生血管生成的调控作用,证实视网膜新生血管簇有明显的小胶质细胞聚集,且小胶质细胞Basigin表达水平显著增加。体外研究证实了缺氧后小胶质细胞表达Basigin-2对血管生成的正性调控作用,且该作用不依赖于细胞间的直接接触。继而以脂质体包裹si RNA为研究平台,重点观察沉默小胶质细胞Basigin-2表达后对于血管生成的影响,提出并初步揭示Basigin-2通过PI3K/AKT途径增加了促血管生成因子IGF-1的分泌,IGF-1上调内皮细胞VEGFR-2的生成,增强血管生成的内在分子机制。研究结果拓展了对小胶质细胞的认识,并且揭示了小胶质细胞表达Basigin-2作为递质,促进缺氧相关的视网膜新生血管生成的调控作用及机制,为拓展缺血缺氧性视网膜新生血管疾病的治疗提供了新的实验依据。
[Abstract]:[background] retinopathy of retinopathy of premature (ROP) is a kind of retinal vascular disease in premature and low weight infants. It is the leading cause of blindness in children worldwide. High concentration oxygen therapy blocks the normal development of retina vascular development in premature infants, resulting in peripheral optic network. Anoxia, which leads to abnormal neovascularization, causes retinal hemorrhage, exudation and proliferation. Therefore, abnormal vascular proliferation induced by hypoxia is one of the core mechanisms of ROP pathology. The formation of the retinal neovascularization is an adaptive response to the anoxic environment, including the interaction of endothelial cells with a variety of cells. The process is very complex and the mechanism is far from clarified. Microglia is the tissue macrophage that settles the central nervous system (CNS). It participates in the process of immunoregulation, tissue development, self stability and trauma repair. Recently, a large number of mouse optic membrane membranes have been used as a model to study angiogenesis. Membrane microglia is an early driving force for angiogenesis. The close spatial relationship between microglia and blood vessels strongly suggests the important role of microglia in angiogenesis, but the intrinsic mechanism of microglia is unclear. In addition, microglia has strong mobility and can perceive physiological and pathological stimuli. The signal of microenvironment is used to respond quickly to the localized chemotactic signal, and by secreting cytokines and neurotrophic factors to the peripheral nerve and blood vessel components to complete the communication between the cells. Therefore, the microglia may transmit the local anoxic signal in the ROP lesion, produce adaptive response, and promote the formation of.Bas in the retina neovascularization. Igin is a highly glycosylated transmembrane glycoprotein. The ability to induce matrix metalloproteinase (matrix metalloproteinase, MMP) generation is named MMP inducer, called EMMPRIN, also known as CD147.Basigin has multiple biological functions, such as immune response, basic degradation and cell migration. This molecule is especially localized in the tumor. In anoxic environment, it promotes neovascularization and strengthens the invasion and metastasis of tumor. It not only has protease induction, promotes the formation of MMP, but also increases the soluble vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and vascular endothelial growth factor receptor (vascular endothelial g) in tumor and endothelial cells (vascular endothelial g). Rowth factor, VEGFR) 2 secretion. In patients with ovarian cancer, Basigin can be secreted into the microenvironment through soluble or exudate forms to promote tumor angiogenesis. In addition, studies have shown that the expression of Basigin in mononuclear macrophages is up-regulated and its inflammatory activity.Basigin is widely involved in the formation of neovascularization under hypoxia. Whether Basigin participates in the interaction between microglia and vascular endothelial cells in ROP lesions with hypoxia as the core link remains to be studied. [Objective] to explore the role and mechanism of microglia cells through Basigin-2 to regulate the formation of retinal neovascularization related to hypoxia, in order to develop ROP and other hypoxic retinal neovascularization. The treatment of vascular disease provides experimental basis. [method] one, in vivo experiment: Basigin participates in microglia to promote the formation of oxygen induced retinopathy of oxygen induced retinopathy (OIR). (1) animal model: establish a mouse OIR model. (2) tissue disease: making frozen section and retina sheet, immunohistochemistry and Bas Igin, IBA-1 immunofluorescence staining, labeling Basigin and microglia, and observing their expression in the retina and their co localization. (3) Western blot detection: the determination of OIR model P12 and P17, the changes of the nerve retinal hypoxia inducible factor (hypoxia inducible factor- alpha, HIF-1 a) and the expression of protein. Two, in vitro experiment: microglia Cell expression Basigin, enhancing the angiogenesis of retinal choroidal microvascular endothelial cells. (1) cell model: the use of microglia BV2 cell line, physical hypoxia model; (2) co culture system: co culture BV2 cells and retinal choroidal vascular endothelial cells (RF /6A), or the application of BV2 cell conditioned medium; (3) Western blot detection: Test: Test The changes in the expression of HIF-1 alpha, Basigin, IGF-1, AKT, P-AKT, ERK and P-ERK in microglia and the changes in the expression of VEGF and VEGFR-2 in RF/6A cells were determined under different treatment conditions. (4) real time quantitative -PCR (Quantitative) Changes, and changes in the expression of VEGF and VEGFR-2 in RF/6A cells; (5) cell migration experiments: using Transwell chamber to determine the migration ability of RF/6A; (6) lumen formation experiment: Matrigel paving, Image Pro Plus software to measure the length of the cavity formation. (7) Si RNA transfection: liposomes encapsulated the gene expression of Si silenced microglia; 8) cytokine assay: using cell factor chip to screen out the up regulation of microglia after hypoxia and down regulation of soluble functional molecules to promote angiogenesis after Basigin-2 knockout. (9) drug intervention: AKT inhibitor (LY294002) and MEK inhibitor (PD98549) were used to inhibit AKT or ERK activation, and the effect on IGF-1 formation was analyzed. Exogenous administration of IGF-1 protein or IGF-1 antibody and IGF receptor antagonist, blocking or activating IGF-1 signal, and observing the effect of RF/6A on the formation of RF/6A. (10) statistical analysis: the application of statistical software SPSS 17 edition for analysis. The comparison of the average number of multiple sample groups using single factor analysis of variance, 22 of any groups using Student-Newmann-K Euls (SNK) test and P0.05 were statistically significant. [results] 1. In vivo experiments: Basigin was highly expressed in the microglia gathered around the neovascular cluster of the retina of the OIR model. Compared with the normal control group, the OIR model retina frozen section and the retina spread were shown from different angles, and the neovascular clusters of the retina were clustered in different angles. IBA-1 positive microglia aggregated, and microglia marker IBA-1 and Basigin had significant co expression in the neovascular region. The protein level group of P17 nerve retina Basigin in group.OIR increased significantly (P=0.0012). Two. In vitro experiment: microglia increased the expression of Basigin-2 and promoted blood vessels in the hypoxic environment. Endothelial cell angiogenesis and its molecular mechanism.1. hypoxia increases HIF-1 A and Basigin-2 in microglia and the expression of HIF-1 A and Basigin protein in microglia and the increase of.Basigin protein expression in the hypoxia 12h (P=0.0097) and 24h group (P=0.0003) increased significantly compared with the control group. It was found that the microglia after anoxia only expressed Basigin-2, and the microglia after hypoxia treatment increased with the prolonged anoxic time to promote the formation of endothelium cells by co culture of microglia and vascular endothelial cells after anoxic treatment of the endothelium of vascular endothelial cells. After 6h, the mean length of the lumen formation in the co culture group was 60.80% higher than that of the control group (P=0.0001), and the mean value of the conditioned medium was 52.40% (P=0.0003) higher than that of the control group, but the co culture group had no significant difference compared with the conditioned medium group (P=0.4008), suggesting that the promoting effect on the formation of the lumen was not dependent on the direct contact with the.3. liposomes of two cells. Parcel Si RNA successfully silence Basigin-2 using qRT-PCR and Western blot to determine the interference effect after Si RNA encapsulated by liposomes.Si RNA Basigin 317 and 458 sequences can obviously reduce the nucleic acid and protein expression, but the effect of the combination of two sequence knockout is better than that of the separate knockout group. The cell Basigin-2 expression inhibited the angiogenesis of vascular endothelial cells and interfered with the expression of Basigin-2 in microglia. The migration of vascular endothelial cells and the function of the lumen formation were inhibited under co culture conditions. The number of migratory cells decreased by 65.93% (P=0.0098) after subtraction. After 6h, the length of the lumen decreased by 68.27% (P=0.0001).5. to block the microglia The expression of Basigin-2 expression inhibited angiogenesis factor by cell factor chip to determine the changes in angiogenesis factor after knockout of Basigin-2 gene in glial cells. 4 factors, such as IGFBP2, IGF-1, Pro-MMP9, VEGFR-1 and so on, were screened out after hypoxia and decreased after Basigin-2 knockout. The most significant change in IGF-1 expression was in the absence of IGF-1 expression. After qRT-PCR and Western blot detection, the transcription level of IGF-1 decreased (P=0.0001) after BV2 knockout Basigin-2, and protein expression decreased (P=0.0082).6.IGF-1 promoted the endothelium cell culture medium with IGF-1 neutralization antibody and exogenous IGF-1, combined with BV2 Asigin-2 knockout to evaluate the effect of IGF-1 on the formation of the lumen. After 6h, the formation of the lumen in the combined IGF-1 neutralization antibody group was reduced by 28.83% (P=0.0098); Basigin-2 knockout combined with IGF-1 could increase the pathway of the cavity formation of 60.32% (P=0.0002).7.Basigin-2 regulated IGF-1. PCR detection of M RNA level.Basigin-2 knockout in different treatment groups of BV2 cells and AKT inhibitor can reverse the increase of IGF-1 trend after hypoxia. ERK inhibitors do not inhibit the molecular mechanism of IGF-1 expression.8.IGF-1 to enhance endothelium cell formation ability by using IGF-1 receptor neutralizing antibodies to block the IGF-1 signal of endothelial cells, and there is no significant change in VEGF nucleic acid and protein levels of 101.33% (P=0.0001).RF/6A expressed by RF/6A in the length of RF/6A, but the transcription of VEGFR-2 (P=0.0091) and protein expression reduction (P=0.0006). [Conclusion] this study was the first to report the role of Basigin in the regulation of microglia induced angiogenesis in oxygen induced retinopathy. It was confirmed that the retinal neovascularization clusters have obvious microglia aggregation, and the expression level of microglia Basigin was significantly increased. The positive regulation of Basigin-2 on angiogenesis after hypoxia is confirmed, and the effect is not dependent on the direct contact between cells. Then the liposomes are wrapped with Si RNA as the research platform, and the effects of Basigin-2 expression on the angiogenesis of silenced microglia are observed, and Basigin-2 is preliminarily revealed through PI3K. The /AKT pathway increases the secretion of angiogenic factor IGF-1, IGF-1 up-regulates the formation of VEGFR-2 in endothelial cells and enhances the intrinsic molecular mechanism of angiogenesis. The results extend the understanding of microglia and reveal the regulation of the expression of Basigin-2 as a transmitter in the hypoxia related retinal neovascularization. The action and mechanism provide a new experimental basis for expanding the treatment of ischemic and hypoxic retinal neovascularization.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R774.1
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