【摘要】：背景與目的 血管的發(fā)生和形成是一基本的生物學(xué)過程,對健康和疾病均有至關(guān)重要的意義。新生血管形成(angiogenesis)在大約20種眼病中發(fā)揮著主要作用,其中角膜新生血管(corneal neovascularization, CRNV)在臨床上的治療仍是比較棘手的問題之一。目前關(guān)于角膜新生血管的發(fā)病機(jī)制尚未完全闡明,也無有效的治療方法。堿燒傷誘導(dǎo)的角膜新生血管模型是一種敏感性強(qiáng),重復(fù)性好,反應(yīng)一致的炎癥性模型,因其制作簡單,便于觀察等優(yōu)點(diǎn)為研究炎癥性角膜新生血管發(fā)生機(jī)制和治療手段提供了理想的平臺(tái)。 CCR3是CC類趨化因子的受體-3,是一種非特異性受體,可與特異性的或者非特異性的受體結(jié)合并趨化嗜酸性粒細(xì)胞、嗜堿性粒細(xì)胞、肥大細(xì)胞、TH2淋巴細(xì)胞等炎癥細(xì)胞向炎癥部位募集,從而發(fā)揮促進(jìn)炎癥作用,這些細(xì)胞和分子均與過敏因素引起的炎癥有關(guān)。最近有研究發(fā)現(xiàn)CCR3及其配體與脈絡(luò)膜新生血管的發(fā)生發(fā)展有關(guān),但CCR3及其配體在角膜新生血管中的具體作用和內(nèi)在機(jī)制仍不明確。 因此本課題從建立小鼠堿燒傷CRNV模型著手,利用RT-PCR方法檢測CCR3mRNA在受損角膜組織中各個(gè)時(shí)間點(diǎn)上的表達(dá),并觀察CCR3小分子拮抗劑SB328437對堿燒傷后CRNV形成的影響,利用RT-PCR方法、免疫組織化學(xué)法從分子水平和細(xì)胞水平來探討CCR3在堿燒傷誘導(dǎo)的CRNV形成過程中的作用與機(jī)制。為臨床治療角膜新生血管性疾病提供一些線索和一定的理論依據(jù)。材料與方法 1、堿燒傷誘導(dǎo)建立CRNV模型的BALB/c小鼠30只,并于0d、2d、4d、7d、14d隨機(jī)取角膜組織(每組6只),利用RT-PCR法檢測CCR3mRNA在堿燒傷角膜組織中的表達(dá)情況。 2、CRNV小鼠模型80只,并隨機(jī)平均分為八組(每組10只)：125μg/ml CCR3拮抗劑干預(yù)組、250μg/ml CCR3拮抗劑干預(yù)組、500μg/ml CCR3拮抗劑干預(yù)組、0.2%透明質(zhì)酸鈉(Sodium hyaluronate, HA)對照組分別進(jìn)行早期和晚期干預(yù)。自堿燒傷后立即或者堿燒傷一周開始局部滴眼干預(yù),每日3次,每次3u1,持續(xù)1w或2w。并于堿燒傷后14d和21d裂隙燈顯微鏡下觀察各組角膜新生血管形成情況,并拍照記錄。同時(shí)處死所有小鼠,獲取眼球分離角膜,以角膜鋪片CD31熒光免疫組織化學(xué)法檢測各組角膜新生血管發(fā)生情況,統(tǒng)計(jì)分析新生血管區(qū)域占整個(gè)角膜的面積比例,并比較兩種干預(yù)方法各組之間差異,分析CCR3拮抗劑在早期和晚期干預(yù)對堿燒傷CRNV形成的影響。 3、CRNV小鼠模型36只,隨機(jī)分為500μg/ml CCR3拮抗劑干預(yù)組、0.2%HA對照組,每組18只小鼠。方法同早期干預(yù),于堿燒傷后2d、4d、7d每組隨機(jī)取6只小鼠處死,分離角膜組織,利用RT-PCR法檢測并分析角膜組織中MCP-1、MCP-3mRNA表達(dá)的差異。 4、CRNV小鼠模型28只,隨機(jī)分為500μg/ml CCR3拮抗劑干預(yù)組、0.2%HA對照組,每組14只小鼠。方法同早期干預(yù),于堿燒傷后2d、4d每組隨機(jī)取7只小鼠取眼球,行冰凍切片,免疫組織化學(xué)法檢測并比較各組角膜組織切片中巨噬細(xì)胞的浸潤數(shù)量。結(jié)果 1、CCR3mRNA在堿燒傷后0d、2d、4d、7d、14d的角膜組織呈動(dòng)態(tài)表達(dá),并且2d、4d、7d的表達(dá)明顯高于0d,提示CCR3參與堿燒傷誘導(dǎo)角膜新生血管的發(fā)生發(fā)展。 2、重復(fù)性實(shí)驗(yàn)結(jié)果顯示：在早期干預(yù)組中,堿燒傷后14d,500μg/ml CCR3拮抗劑干預(yù)組CRNV形成較對照組明顯減少(P0.05),而125μg/ml和250μg/ml CCR3拮抗劑干預(yù)組與對照組之間比較,血管生成有下降的趨勢,但差異沒有統(tǒng)計(jì)學(xué)意義(P0.05)。而在晚期干預(yù)組中,各干預(yù)組與對照組相比較,CRNV形成情況沒有明顯的差異(P0.05),提示500μg/ml CCR3拮抗劑能在堿燒傷早期抑制角膜新生血管的形成。 3、堿燒傷后2d、4d500μg/ml CCR3拮抗劑干預(yù)組小鼠角膜組織中MCP-1、 MCP-3mRNA的表達(dá)水平與對照組相比,均明顯降低(P0.05),而7d干預(yù)組MCP-1、MCP-3mRNA的表達(dá)水平與對照組相比,沒有明顯差異(P0.05),提示500μg/ml CCR3拮抗劑可能在堿燒傷早期通過下調(diào)角膜組織中MCP-1、MCP-3mRNA的表達(dá)而抑制CRNV的形成。 4、堿燒傷后2d、4d500μg/ml CCR3拮抗劑干預(yù)組小鼠角膜組織中巨噬細(xì)胞的浸潤數(shù)量均較對照組低,其中4d兩組間差異具有統(tǒng)計(jì)學(xué)意義(P0.05),提示500μg/ml CCR3拮抗劑在堿燒傷早期可能通過減少巨噬細(xì)胞向角膜組織中浸潤,而達(dá)到抑制CRNV形成的作用。結(jié)論 1、CCR3參與堿燒傷誘導(dǎo)的角膜新生血管的發(fā)生發(fā)展過程,500μg/ml CCR3拮抗劑在堿燒傷早期干預(yù)能抑制CRNV形成。 2、CCR3拮抗劑抑制堿燒傷誘導(dǎo)的CRNV,可能與其下調(diào)堿燒傷早期角膜組織中MCP-1、MCP-3mRNA的表達(dá),同時(shí)降低巨噬細(xì)胞向角膜組織的浸潤有關(guān)。 3、局部應(yīng)用CCR3拮抗劑干預(yù),可能成為臨床治療CRNV一個(gè)新的途徑。
[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
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R779.1

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7 李之U，

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