【作者】 张晓光；

【导师】 惠延年；

【作者基本信息】 第四军医大学 ， 眼科学， 2010， 博士

【摘要】 【研究背景】视网膜色素上皮(retinal pigment epithelium,RPE)细胞层位于视网膜光感受器细胞与Bruch膜及脉络膜之间,支持着视网膜光感受器细胞,是维持视网膜结构和功能的重要组成部分。人RPE (human RPE,hRPE)细胞损伤性疾病,如年龄相关性黄斑病变(age-related macular degeneration,AMD)、增殖性玻璃体视网膜病变(proliferation vitreoretinopathy,PVR)以及视网膜色素变性等,是造成视力丧失的重要原因。RPE层一旦损伤,由病变区周围健康的RPE细胞向损伤区移行增生,以修复损伤区,这一过程对结构和功能的恢复十分重要。在细胞修复的过程中,有多种复杂的因素参与,其中伴有电场的作用。外加电场可以明显促进皮肤和角膜上皮损伤的修复。我们研究组前期的研究结果表明,在较短的时间内,电场能够引导hRPE细胞向负极移动,而且低于10 V/cm的电场强度对hRPE细胞的正常活性及分裂功能无明显不良影响。但是,单有外加电场的作用,似乎不足以促进hRPE细胞较快地移行修复缺损区。因此,我们试图寻找能够加快细胞在电场中移行的因子。有许多分子与细胞的移行有关。而人类甲酰肽受体(Formyl Peptide Receptor,FPR,1976年发现)是一种化学趋化受体,是介导细胞在一系列细胞因子的作用下移行的G蛋白偶联受体,与激动剂结合可以使PI3K,NF-κB和MAPK等激活,调节细胞的活化和移行。根据我们前期电场实验的结果,以及对FPR的文献回顾,推测可以将两者结合共同引入到促进RPE细胞移行修复的过程中,为RPE损伤性疾病的治疗提供一个新的治疗途径。【目的】1.确定FPR在hRPE细胞的表达及定位;观察激活FPR,电场引导hRPE细胞层的移行修复,以及在此过程中细胞间连接mRNA的表达和其超微结构的变化,细胞骨架的变化。2.研究此移行修复过程中PI3K/Akt信号通路的变化,Rho GTP ases家族蛋白的表达。3.研究此移行修复过程中细胞因子,如表皮生长因子(epidermal growth factor,EGF)、白介素(interleukin 8,IL-8)、单核细胞趋化蛋白-1(monocyte chemotactic protein-1)和转化生长因子-β1(transforming growth factor-β1,TGF-β1 )的表达和分泌。【方法】1.将hRPE细胞培养于含有fMLF(FPR经典激动剂)的培养液中,应用RT-PCR方法确定hRPE细胞表达FPR,免疫荧光染色方法着染FPR,从而进一步确定FPR在细胞上的表达与定位。根据本研究的前期实验结果,直接将细胞置于6 V/cm电场下,应用免疫荧光三标染色法着染FPR、F-actin以及胞核,通过激光共聚焦显微镜观察电场作用的hRPE细胞骨架F-actin和FPR的变化及定位关系。免疫荧光三标染色法着染细胞骨架F-actin、Vimentin及胞核,观察激活FPR促进电场引导hRPE细胞层的损伤修复以及电场引导下细胞骨架F-actin和Vimentin的变化。显微镜照相系统连续记录每1 h细胞移行的距离和细胞损伤范围的变化。并通过RT-PCR检测细胞间连接mRNA的表达,透射电子显微镜来观察细胞间连接的超微结构改变。2.初步研究激活FPR受体通过PI3K / Akt信号转导通路激活Rho GTP ases家族中Cdc42、Rac1和RhoA,促进电场引导下hRPE细胞层移行修复。应用RT-PCR检测Cdc42、Rac和RhoA mRNA的变化,应用Western blotting检测PI3K / Akt,以及RhoA GTPases家族中Cdc42、Rac1和RhoA蛋白表达的变化。3.观察激活FPR在电场作用下,采用RT-PCR和ELISA方法检测EGF、IL-8、MCP-1和TGF-β1的表达和分泌,以及加入FPR拮抗剂,PI3K/Akt阻断剂和Rho GTP ases阻断剂后因子的表达和分泌变化。【结果】1.首次确定了hRPE细胞表面存在FPR。应用FPR经典激动剂fMLF激活hRPE细胞的FPR,通过RT-PCR检测到hRPE细胞的FPR mRNA表达。同时揭示FPR与F-actin之间存在共定位关系。在电场作用下,hRPE细胞向阴极方向伸出伪足,朝向阴极的方向移行,FPR与F-actin在细胞的伪足上共定位,而在拮抗剂Boc和激动剂fMLF作用下,hRPE细胞的FPR表达减少。在电场和FPR的作用下,可以明显促进hRPE细胞层的移行和损伤修复。将hRPE细胞培养于无血清培养液组,20%血清培养液组及fMLF培养液组,电场作用3 h后,hRPE细胞层的移行距离分别为24.262±6.82μm,40.243±5.069μm(1.7倍),和56.926±7.821μm(2.4倍)。在无血清培养液组,20%血清培养液组和fMLF培养液组中可检测出CX-43和ZO-1 mRNA的表达,而E-cadherin mRNA的表达却分别在2 h,30 min和1 h时检测到。透射电镜可以观察到细胞间连接的的超微结构:包括紧密连接,缝隙连接,桥粒样结构和粘着斑。电场下细胞骨架的空间结构发生改变,细胞伪足朝向阴极方向,并表达F-actin,其荧光亮度在1 h时,达到峰值106.49±8.21,随后逐渐下降。2.在电场作用下,Western blotting结果显示pAkt在hRPE细胞中的表达,均在30 min时达到最高,无血清培养液组为0.6±0.012,20%血清培养液组1.36±0.093,含fMLF培养液组为2.44±0.089。加入FPR拮抗剂Boc后,发现pAkt蛋白的表达量明显降低。通过RT-PCR检测到Rho GTP ases家族中Cdc42、Rac1和RhoA mRNA的表达在激活FPR受体后在30 min时明显增加,Western blotting的结果提示其蛋白表达量在电场作用1 h时明显增加,在含fMLF培养液组增加明显,分别为3.5±0.2,5.5±0.21,4.78±0.22,加入PI3K/Akt通路阻断剂LY294002后,Rho GTP ases家族中Cdc42、Rac1和RhoA的表达明显降低。3.在电场作用下,RT-PCR结果显示EGF、IL-8、MCP-1和TGF-β1 mRNA表达在1 h时达到高峰,而ELISA结果提示培养液上清中四种因子的分泌量在2 h时,达到高峰,含fMLF培养液组四种因子的表达量和分泌量较其他组升高明显。加入FPR拮抗剂Boc,PI3K/Akt信号转导通路阻断剂LY294002以及Rho GTP ases阻断剂Y27632,发现EGF、IL-8、MCP-1和TGF-β1的表达量和分泌量下降。【结论】1.首次确定hRPE细胞表达FPR,并与细胞骨架F-actin共定位表达。激活FPR,在电场作用下,hRPE细胞发生形态和极性的改变,朝向阴极伸出伪足,向阴极移行,在细胞伪足上可见FPR与F-actin共定位,说明FPR的激活与hRPE细胞的移行修复有关。激活hRPE细胞FPR,显著促进了RPE细胞层在电场作用下的移行,并能引起细胞骨架蛋白F-actin的改变。细胞间连接mRNA的表达及其超微结构的观察,说明hRPE细胞层的损伤修复是成层移行的。2.激活hRPE细胞FPR,在电场作用下,通过PI3K/Akt信号转导通路的激活,活化Cdc42、Rac1和RhoA蛋白,说明FPR的作用至少是通过PI3K/Akt的信号转导通路实现的。3.激活hRPE细胞FPR,在电场作用下,诱导EGF、IL-8、MCP-1和TGF-β1的表达和分泌,而且与FPR激活、PI3K/Akt激活、Rho GTP ases活化相关。以上研究国内外未见相关报道。更多还原

【Abstract】 BackgroundRetinal pigment epithelial (RPE) cells form the monolayer between photoreceptors and the choroid, which play an important role in supporting the neural retina. RPE is essential for the integrity and function of neural retina. Damages to human RPE monolayer, such as happened in age-related mocular degeneration (AMD), proliferative vitreoretinopathy (PVR), and retinitis pigmentosa, may cause serious loss of vision. Once hRPE layer was wounded, normal hRPE cells surrounding the wound area will migrate and proliferate to cover the area. This process is crucial to recover the structure and function of the retina.During the wound healing process, many factors might play a role. And an endogenous electric field may yield. Moreover, the wound healing of the cornea and the skin can be accelerated by additional EFs. Our research group previously observed that EFs induced directed migration of hRPE cells and that limited period of exposure to EFs under 10 V/cm did not affect the normal viability of human RPE. But EFs exposure alone seems not sufficient to enhance migration of cells to restitute hRPE cell layer. We therefore tried to find some factors which can enhance the cell migration under exposure of EFs.Migration and wound healing process of RPE cells are a complex process. Many cytokines, growth factors take part in this process. Formyl Peptide Receptor (FPR) was first defined biochemically in 1976. Upon ligand binding, FPR undergoes a conformation change enabling interactions with G family proteins. Agonist-induced signaling also results in phosphorylation of protein kinases (PI3K,MAPK and NF-κB) and regulation of celluar activations and migration.Based on our previous study and understanding of FPR, we supposed that activation of FPR may enhance hRPE cell restitution under EFs.Aims1. To identify the expression of functional FPR in RPE cells and then observe that the activation of FPR can enhance migration of wounded RPE monolayer under EFs. And to demonstrate the cell sheet as a monolayer with CX-43, E-cadherin, ZO-1, and cell junctions.2. To evaluate the increase in cell migration to be associated with activation of PI3K/Akt, Cdc42, Rac1 and RhoA.3. To investigate the increase in cell migration to be associated with expressions and secretions of epidermal growth factor (EGF), interleukin 8 (IL-8),monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-β1 (TGF-β1 ) in the cells.Methods1. The expression of functional FPR in hRPE cells was analyzed with RT-PCR. Based on the previous study, the wound hRPE cells were exposed to EFs at 6 V/cm. FPR and F-actin were stained by immunofluoresence lableling assay and observed the location of FPR in hRPE cells and the change of F-actin of the cells. F-actin and Vimentin of hRPE cells were stained by immunity fluorescence labled assay and observed with a confocal microscope before and 1 h, 2 h and 3 h after exposure to EFs. Images of the monolayer migration were obtained at regular intervals under a Zeiss Axiovert microscope every 1 h. Cell junction, including CX-43, E-cadherin and ZO-1, were measured with RT-PCR. And the cell junctions in the hRPE monolayer were observed with transmission electron microscopy (TEM).2. In order to observe FPR activation enhancing the activation of Cdc42, Rac1 and RhoA through PI3K/Akt, the expression of Cdc42, Rac1 and RhoA was determined by RT-PCR and Western blotting. The expression of PI3K/Akt and pAkt was also determined by Western blotting.3. To observe FPR activation enhancing the expressions and secretions of EGF, IL-8, MCP-1 and TGF-β1 under EFs through PI3K/Akt or Rho GTP ases, the mRNA expressions of these cytokines were measured with RT-PCR. ELISA was also used to observe the secretions of them in supernatants.Results1. The expression of functional FPR was observed and localized along actin filaments in the lamellipodia and filopodia of hRPE cells. After treated with Boc and fMLF, the fluorescence value of FPR was decreased. EFs and fMLF significantly increased the migration rate of the wound hRPE monolayer. The migrating distances of hRPE monolayers were measured as 24.262±6.82μm,40.243±5.069μm (1.7-fold) and 56.926±7.821μm (2.4-fold) in hRPE cells cultured with free serum, 20% serum,fMLF under EFs at 3 h, respectively (P<0.01). The mRNA expression of CX-43 and ZO-1 were detected in hRPE cells cultured with free serum, 20% serum and fMLF respectively; but the mRNA expression of E-cadherin was detectable until 2 h, 30 min and 1 h in hRPE cells cultured with free serum, 20% serum and fMLF under EFs respectively. TEM revealed that cell junctions formed. After exposed to EFs, the polyarity and spatial structure of RPE cells was changed, and the lamellipodial and filopodial of hRPE cells were getting along with the direction of the cathode. HRPE cells were migrated to the cathode. After exposure to an EFs for 1 h, the fluorescence value of cytoskeleton was 106.49±8.21, which was higher in the culture with fMLF than that of the control cultures (P<0.05).2. After exposed to EFs, western blotting reached a peak in pAkt of hRPE cells at 30 min. The level of pAkt in hRPE cells were measured as 0.6±0.012, 1.36±0.093 (1.8-fold), 2.44±0.089 (4-fold) in cells cultured with free serum, 20% serum and fMLF under EFs. The inhibitor of FPR, Boc abrogated the fMLF-induced increase in the level of pAkt in hRPE cells. The results of RT-PCR showed that a significant increase in Cdc42, Rac1 and RhoA mRNA at 30 min after exposure to EFs. Western blotting showed that a peak in Cdc42, Rac1 and RhoA protein expression of hRPE cells after exposure to EFs at 1 h. The Cdc42, Rac1 and RhoA of hRPE cells were measured as 3.5±0.2 (5 - fold, 1.6 - fold),5.5±0.21 (1.92 - fold, 1.34 - fold),4.78±0.22 (1.9 - fold, 1.9 - fold) in cell cultured with fMLF compared with the cells cultured with free serum and 20% serum, respectively. This up-regulation of Cdc42, Rac1 and RhoA was blocked by Boc and LY294002.3. After EFs exposure, the mRNA expression of EGF, MCP-1, IL-8 and TGF-β1 reached a peak at 1 h., the secretion of EGF, MCP-1, IL-8 and TGF-β1 reached a peak at 2 h. After activation of FPR, the level of expression and secretion of these cytokines were higher than control culture. This up-regulation of them was blocked by Boc and LY294002.Conclusion1. These results firstly showed that functional FPR express in hRPE cells. FPR localized along actin filaments in lamellipodial and dilopodial extrusions, which were getting along with the direction of the cathode. The FPR activation enhances hRPE cell migration and wound healing. Activation of FPR in hRPE cells and the distribution of the cytoskeleton in hRPE cells were changed after exposure to EFs. The expression of cell junction mRNA and cellular junctions shown by TEM further demonstrated the RPE sheet as a monolayer migrating under EFs.2. EFs induce directed hRPE cell migration and wound healing which can be enhanced by fMLF. The activation of FPR is associated with activation of PI3K/Akt and through PI3K/Akt-dependent activation of Rho GTP ases family members (Cdc42, Rac1, RhoA).3. Activation of FPR could induce expression and secretion of EGF, MCP-1, IL-8 and TGF-β1 in hRPE cells, which could be inhibited by Boc, LY294002 and Y 27632.更多还原