【作者】 李鹏程；

【导师】 胡燕华；

【作者基本信息】 华中科技大学 ， 眼科学， 2006， 博士

【摘要】 目的探讨大鼠角膜碱烧伤后基质细胞来源的因子(stromal cell-derived factor-1，SDF-1)及其受体CXCR4在鼠角膜的表达变化规律及其与角膜新生血管的关系；研究蛋白激酶C-ζ在SDF-1刺激的巨噬细胞的VEGF的mRNA转录中的调节机制。方法1)建立炎症性大鼠角新生血管动物模型；用逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹(Westenblot)分析SD大鼠角膜烧伤后0、2、5、7、10、14天时SDF-1／CXCR4的mRNA和蛋白在角膜中表达量的变化；免疫组织化学染色方法检测大鼠角膜烧伤后SDF-1／CXCR4蛋白在角膜各层中的分布。2)将碱烧伤大鼠分为治疗组和对照组，每组25眼。分别在伤后立即给予CXCR4特异性拮抗剂AMD310015ug或PBS缓冲液0.1ml球结膜下注射，每天一次，连续2周。观察伤后7天、10天、14天新生血管面积，并用蛋白质免疫印迹观察伤后各时间点角膜组织VEGF的含量。3)用Boyden小室法观察不同浓度的SDF-1对培养的大鼠巨噬细胞的趋化作用，以及不同浓度的蛋白激酶C抑制剂对其趋化作用的影响；用RT-PCR方法检测SDF-1对培养的U937细胞的VEGF的表达的影响以及蛋白激酶C抑制剂对其作用的影响。4)设计合成一对针对人PKC-ζ的mRNA的寡核苷酸序列，退火后将其连入载体。对重组质粒pSIRENp进行酶切鉴定。用脂质体介导的方法分别将干扰质粒pSIRENp转染U937细胞，用RT-PCR检测转染干扰质粒对PKC-ζ表达的影响；并检测转染前后的U937细胞对SDF-1刺激的VEGF的mRNA转录情况。结果1)正常大鼠角膜可检出微量SDF-1／CXCR4mRNA和蛋白；伤后2天，鼠角膜中可见SDF-1／CXCR4的mRNA和蛋白水平升高；伤后7—10天，角膜中SDF-1／CXCR4的mRNA和蛋白达高峰；伤后14天表达量开始下降。伤后SD大鼠角膜基质层，尤其是毗邻于烧灼区的浅基质层中有大量炎性细胞浸润，浸润的炎性细胞、角膜上皮细胞、基质细胞和新生血管内皮细胞均有SDF-1／CXCR4的蛋白表达。2)给予AMD3100的治疗组7天时新生血管面积是10.60±2.31mm~2，明显小于对照组的14.20±3.42 mm~2，在10天、14天时新生血管的面积也小于对照组，差异有显著性。VEGF的表达在各时间点稍小于对照组，差异无统计学意义。3)SDF-1对体外培养的大鼠巨噬细胞表现出剂量依赖的趋化作用，100ng／ml时作用最强，SDF-1作用12小时明显促进U937细胞VEGF表达；不同浓度的蛋白激酶C抑制剂对此二效应均有抑制作用，浓度为10uM时作用最强。4)酶切证实目的寡核苷酸片段已经被克隆到pSIRENp，转染pSIRENp质粒成功抑制了83％的PKC-ζ在U937细胞的表达；转染pSIRENp质粒的细胞VEGF基础表达量下降了79.7％，SDF-1刺激后细胞VEGF的mRNA的转录也明显低于对照。转染对照质粒对细胞PKC-ζ及SDF-1刺激的VEGF反应均无明显影响。结论角膜基质细胞、上皮细胞和炎性细胞分泌的SDF-1与其受体CXCR4的相互作用参与了角膜的创伤修复和炎症性角膜新生血管增殖的调控，对新生血管的生长有诱导和维持作用。AMD3100可以抑制炎症性新生血管的形成，其抑制作用不依赖VEGF，有潜在的应用价值。蛋白激酶C参与调控SDF-1对巨噬细胞的趋化作用和促进VEGF表达的作用，其中PKC-ζ可能通过影响NF-kappaB活性而参与调节U937细胞VEGF转录。更多还原

【Abstract】 Objective To investigate the expression of stromal cell-derived factor-1 (SDF-1) and its receptor,CXCR4, in rat cornea after alkali burn and its significance in corneal neovascularization. And to clarify the role of PKC- ζ in the regulation of SDF-1 stimulated VEGF mRNA transcription.Methods 1) Inflammatory corneal neovascularization model was established in Sprayue-Dawley rats with alkali burn. SDF-1/CXCR4 was detected in corneal samples on 2,5,7,10,14th day after cornea cautery by RT-PCR and Westernblot and immunohistochemistry method was used to explore the distribution of SDF-1/CXCR4 at rat cornea after alkali burn. 2) The model eyes were divided into two groups and each group contains 25 eyes. PBS and 15ug AMD3100 was subconjunctivally injected respectively in eyes immediately after burn once a day. The areas of corneal neovascularization were measured on 7,10,14th day after treatment and the expression of VEGF of two groups was investigated by westenblot. 3) Boyden chamber method was used to observe the chemotaxis of macrophage to SDF-1 and the effect of PKC inhibitor on this chemotaxis. The VEGF mRNA was detected by RT-PCR in SDF-1 stimulated U937 cell and the PKC regulation on this procedure. 4) 64 bp reverse repeated motifs of PKC- C target sequence were synthesized and inserted into the plasmid to construct the plasmid expressing shRNA- PKC- ζ and the plasmid was transfected into U937 cell line. RT-PCR was used to detect the expression of PKC- ζ. The basic and induced VEGF mRNA by SDF-1 was also examined by RT-PCR.Results 1) There are trace SDF-1 and CXCR4 in normal cornea on both mRNA and protein level. The expression of SDF-1 and CXCR4 increased on 2nd day and peaked on 7th day and began to decrease at 14th day after corneal cautery. The rat corneas were infiltrated by massive inflammatory cells adjacent to the cautery lesion. Inflammatory cells, corneal epithelial cells and keratocytes showed positive expression of SDF-1 and its receptor, CXCR4. 2) The average area of corneal neovascularization in therapeutic group on 7th day is 10.60 ± 2.31mm~2, significantly lower than 14.20 ± 3.42 mm~2 of control group. And on 10,14th day, the areas of treatment group were also significantly lower than those in control group. There was no significant reduction of VEGF expression between two groups. 3) SDF-1 could induce chemotaxis of macrophage in a dose-dependent manner and peaked at the concentration of 100ng/ml and VEGF mRNA transcription is up-regulated in SDF-1 stimulated U937 cell. Both of the two above-mentioned effects were effectively inhibited by a broad-spectrum PKC inhibitor, chelerythrine chloride, at different concentration and 10uM is the most effective. 4) The recombinant plasmid of shRNA-PKC- ζ was successfully constructed and the recombinant plasmid nearly completely suppress the PKC- ζ expression in U937 cell line. After transfection the basic VEGF and the SDF-1-induced VEGF both significantly reduced compared to control.Conclusions The interaction of SDF-1 derived from inflammatory cells, epithelial cells and corneal keratocytes and its receptor, CXCR4, participate in corneal wound repair and inflammatory corneal neovascularization. SDF-1 and VEGF synergistically induce and support the neovascularization in rat cautery cornea. AMD3100 could effectively inhibit the proliferation of inflammation-induced corneal neovascularization independent of VEGF. PKC is involved in the regulation of chemotaxis of macrophage induced by SDF-1 and especially PKC- ζ , may be involved in the regulation of VEGF transcription through NF-kappa B signal passageway in U937 cell line.更多还原