CCR3在碱烧伤诱导角膜新生血管中的作用及机制

发布时间：2019-06-10 14:47

【摘要】：背景与目的血管的发生和形成是一基本的生物学过程,对健康和疾病均有至关重要的意义。新生血管形成(angiogenesis)在大约20种眼病中发挥着主要作用,其中角膜新生血管(corneal neovascularization, CRNV)在临床上的治疗仍是比较棘手的问题之一。目前关于角膜新生血管的发病机制尚未完全阐明,也无有效的治疗方法。碱烧伤诱导的角膜新生血管模型是一种敏感性强,重复性好,反应一致的炎症性模型,因其制作简单,便于观察等优点为研究炎症性角膜新生血管发生机制和治疗手段提供了理想的平台。 CCR3是CC类趋化因子的受体-3,是一种非特异性受体,可与特异性的或者非特异性的受体结合并趋化嗜酸性粒细胞、嗜碱性粒细胞、肥大细胞、TH2淋巴细胞等炎症细胞向炎症部位募集,从而发挥促进炎症作用,这些细胞和分子均与过敏因素引起的炎症有关。最近有研究发现CCR3及其配体与脉络膜新生血管的发生发展有关,但CCR3及其配体在角膜新生血管中的具体作用和内在机制仍不明确。因此本课题从建立小鼠碱烧伤CRNV模型着手,利用RT-PCR方法检测CCR3mRNA在受损角膜组织中各个时间点上的表达,并观察CCR3小分子拮抗剂SB328437对碱烧伤后CRNV形成的影响,利用RT-PCR方法、免疫组织化学法从分子水平和细胞水平来探讨CCR3在碱烧伤诱导的CRNV形成过程中的作用与机制。为临床治疗角膜新生血管性疾病提供一些线索和一定的理论依据。材料与方法 1、碱烧伤诱导建立CRNV模型的BALB/c小鼠30只,并于0d、2d、4d、7d、14d随机取角膜组织(每组6只),利用RT-PCR法检测CCR3mRNA在碱烧伤角膜组织中的表达情况。 2、CRNV小鼠模型80只,并随机平均分为八组(每组10只)：125μg/ml CCR3拮抗剂干预组、250μg/ml CCR3拮抗剂干预组、500μg/ml CCR3拮抗剂干预组、0.2%透明质酸钠(Sodium hyaluronate, HA)对照组分别进行早期和晚期干预。自碱烧伤后立即或者碱烧伤一周开始局部滴眼干预,每日3次,每次3u1,持续1w或2w。并于碱烧伤后14d和21d裂隙灯显微镜下观察各组角膜新生血管形成情况,并拍照记录。同时处死所有小鼠,获取眼球分离角膜,以角膜铺片CD31荧光免疫组织化学法检测各组角膜新生血管发生情况,统计分析新生血管区域占整个角膜的面积比例,并比较两种干预方法各组之间差异,分析CCR3拮抗剂在早期和晚期干预对碱烧伤CRNV形成的影响。 3、CRNV小鼠模型36只,随机分为500μg/ml CCR3拮抗剂干预组、0.2%HA对照组,每组18只小鼠。方法同早期干预,于碱烧伤后2d、4d、7d每组随机取6只小鼠处死,分离角膜组织,利用RT-PCR法检测并分析角膜组织中MCP-1、MCP-3mRNA表达的差异。 4、CRNV小鼠模型28只,随机分为500μg/ml CCR3拮抗剂干预组、0.2%HA对照组,每组14只小鼠。方法同早期干预,于碱烧伤后2d、4d每组随机取7只小鼠取眼球,行冰冻切片,免疫组织化学法检测并比较各组角膜组织切片中巨噬细胞的浸润数量。结果 1、CCR3mRNA在碱烧伤后0d、2d、4d、7d、14d的角膜组织呈动态表达,并且2d、4d、7d的表达明显高于0d,提示CCR3参与碱烧伤诱导角膜新生血管的发生发展。 2、重复性实验结果显示：在早期干预组中,碱烧伤后14d,500μg/ml CCR3拮抗剂干预组CRNV形成较对照组明显减少(P0.05),而125μg/ml和250μg/ml CCR3拮抗剂干预组与对照组之间比较,血管生成有下降的趋势,但差异没有统计学意义(P0.05)。而在晚期干预组中,各干预组与对照组相比较,CRNV形成情况没有明显的差异(P0.05),提示500μg/ml CCR3拮抗剂能在碱烧伤早期抑制角膜新生血管的形成。 3、碱烧伤后2d、4d500μg/ml CCR3拮抗剂干预组小鼠角膜组织中MCP-1、 MCP-3mRNA的表达水平与对照组相比,均明显降低(P0.05),而7d干预组MCP-1、MCP-3mRNA的表达水平与对照组相比,没有明显差异(P0.05),提示500μg/ml CCR3拮抗剂可能在碱烧伤早期通过下调角膜组织中MCP-1、MCP-3mRNA的表达而抑制CRNV的形成。 4、碱烧伤后2d、4d500μg/ml CCR3拮抗剂干预组小鼠角膜组织中巨噬细胞的浸润数量均较对照组低,其中4d两组间差异具有统计学意义(P0.05),提示500μg/ml CCR3拮抗剂在碱烧伤早期可能通过减少巨噬细胞向角膜组织中浸润,而达到抑制CRNV形成的作用。结论 1、CCR3参与碱烧伤诱导的角膜新生血管的发生发展过程,500μg/ml CCR3拮抗剂在碱烧伤早期干预能抑制CRNV形成。 2、CCR3拮抗剂抑制碱烧伤诱导的CRNV,可能与其下调碱烧伤早期角膜组织中MCP-1、MCP-3mRNA的表达,同时降低巨噬细胞向角膜组织的浸润有关。 3、局部应用CCR3拮抗剂干预,可能成为临床治疗CRNV一个新的途径。
[Abstract]:Background and Purpose The occurrence and formation of blood vessels is a basic biological process, which is of vital importance to both health and disease Angiogenesis plays a major role in about 20 kinds of eye diseases, and the clinical treatment of corneal neovascularization (CRNV) is still a thorny problem. I. At present, the pathogenesis of corneal neovascularization has not yet been fully clarified, and there is no effective treatment party The corneal neovascularization induced by alkali burn is a kind of inflammatory model with strong sensitivity, good repeatability and uniform response. Table. CCR3 is the receptor-3 of CC-type chemokines, is a non-specific receptor, and can be combined with specific or non-specific receptors and chemotactic eosinophils, basophils, mast cells, TH2 lymphocytes, and the like to the inflammation part. In order to play a role in promoting inflammation, these cells and molecules are associated with an allergic reaction. Recent studies have found that CCR3 and its ligands have been associated with the development of choroidal neovascularization, but the specific role and intrinsic mechanism of CCR3 and its ligands in corneal neovascularization remains The expression of CCR3 mRNA in the damaged corneal tissue was detected by RT-PCR and the effect of CCR3 on the formation of CRNV after alkali burn was observed by RT-PCR. RT-PCR was used to study the effect of CCR3 on the formation of CRNV after alkali burn. The expression of CCR3 in the formation of CRNV induced by alkali burn was discussed by the method of PCR and immunohistochemistry from the molecular level and the cell level. Function and mechanism. Provide some clues and must for clinical treatment of corneal neovascular disease. theoretical basis Materials and Methods 1,30 BALB/ c mice with the CRNV model were established by alkali burn induction, and the corneal tissue (6 rats in each group) was randomly taken at 0d, 2d, 4d, 7d, and 14d, and the CCR3 mRNA was detected by RT-PCR in the alkali burn corneal group. Expression in the weave. The CRNV mouse model was 80, and was randomly divided into eight groups (10 in each group):125. mu.g/ ml of CCR3 antagonist intervention group,250. mu.g/ ml of CCR3 antagonist intervention group,500. mu.g/ ml of CCR3 antagonist intervention group, 0.2% sodium hyaluronate (HA) control group, Early and late intervention. Local drop-eye intervention was initiated once a week after an alkali burn or a week after the base burn,3 times a day,3 u each time 1. Continuous 1w or 2w. The corneal neovascularization in each group were observed under a slit lamp microscope of 14 d and 21 d after alkali burn. In the same time, all the mice were sacrificed, the eyes were isolated, and the corneal neovascularization in each group was detected by using the CD31 fluorescence immunoassay method of the cornea. The area ratio of the new blood vessel area to the whole cornea was statistically analyzed and the two types of dry blood vessels were compared. The effect of CCR3 antagonist on alkali burn in early and late intervention was analyzed by pre-method. The effects of the formation of CRNV.3, the CRNV mouse model 36 was randomly divided into 500. m u.g/ ml of the CCR3 antagonist intervention group, 0.2% HA In the control group,18 mice in each group were randomly divided into 6 mice at 2 days,4 days and 7 days after the alkali burn, and the corneal tissue was isolated, and the MCP-1 and MCP in the corneal tissue were detected and analyzed by RT-PCR. -3 mRNA expression differences.4, CRNV mouse model 28, randomly divided into 500. mu.g/ ml of CCR3 antagonist intervention group, 0.2% In the control group,14 mice in each group were randomly divided into two groups: the first intervention, the second day after the alkali burn,4 days,7 mice were randomly divided into the eyeball, the frozen section and the immunohistochemical method were used to detect and compare the groups of the groups. weaving The results showed that the expression of CCR3 mRNA in the corneal tissue of 0 d,2 d,4 d,7 d, and 14 d after alkali burn was significantly higher than that of 0 d, and that CCR3 was involved in the base. The occurrence and development of corneal neovascularization induced by burn.2. The results of repeated experiments showed that in the early intervention group, there was a significant decrease of CRNV in the treatment group (P0.05), and the intervention group of 125. m u.g/ ml and 250. m u.g/ ml of CCR3 antagonist was significantly reduced (P0.05). As a result of the comparison between the groups, there is a downward trend in the generation of blood vessels, However, there was no significant difference between the intervention group and the control group (P0.05). There was no significant difference in the formation of CRNV (P0.05). The expression of MCP-1 and MCP-3 mRNA in the corneal tissue was significantly lower than that in the control group (P0.05), while the expression level of MCP-1 and MCP-3 mRNA in the 7-day intervention group was significantly lower than that of the control group (P0.05). Compared with the control group, there was no significant difference (P0.05). It was suggested that 500. m u.g/ ml of CCR3 antagonist could decrease the MCP-1 and MCP in the corneal tissue in the early stage of alkali burn. the expression of -3 mrna inhibited the formation of crnv.4, the number of macrophages in the corneal tissue of the mice was lower than that of the control group after 2 d,4 d500. m u.g/ ml of the ccr3 antagonist after the alkali burn, There was a significant difference between the two groups (P0.05). It was suggested that 500. m u.g/ ml of CCR3 antagonist could decrease the angle of the macrophage to the angle early in the early stage of alkali burn. membrane group Conclusion 1, CCR3 is involved in the development of corneal neovascularization induced by alkali burn and 500. m u.g/ ml C CR3 antagonist can inhibit the formation of CRNV in the early intervention of alkali burn. The CCR3 antagonist can inhibit the CRNV induced by alkali burn and may lower the MCP-1, MCP-3 m in the early corneal tissue of the alkali burn. the expression of RNA, at the same time, decreased the infiltration of macrophages into the corneal tissue.3. Local application C
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.1

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