【作者】 韩静；

【导师】 惠延年；

【作者基本信息】 第四军医大学 ， 眼科学， 2008， 博士

【摘要】 【背景】组织的损伤修复对于维持组织正常结构和功能至关重要,在这一过程中,电场可能发挥着不容忽视的作用。当上皮细胞层受到破坏时,跨细胞层的离子流驱动电流流出,形成局部内源性电场。已证实多种细胞能够在与这种内源性电场大小相当的外加直流电场中呈现生物学特性的变化,尤其以移行能力的变化最为显著。研究显示,多种属、多组织来源的细胞在电场作用下可出现向阴极或阳极方向的定向移行,即具有趋电性;电场可明显促进皮肤、角膜上皮损伤的修复。因此,电场可能作为细胞移行的重要刺激因素参与损伤修复的调节。目前关于电场对细胞作用的机制仍处于探索中,一些研究认为细胞骨架蛋白和细胞膜表面生长因子受体的不对称分布、相关第二信使的激活均可能参与其中。在另一方面,细胞与细胞外基质(cell-extracellular matrix, ECM)间的粘附和相互作用是移行过程中的关键环节,而整合素家族在该过程中至关重要。整合素是细胞表面兼具粘附和信号传导功能的受体,通过胞外域与ECM、胞内域与信号传导分子结合,介导了细胞内外之间的双向信号传导,在调节细胞粘附、迁移、增生、分化等方面发挥重要作用。局部粘附激酶(focal adhesion kinase, FAK)是细胞内一种非受体型酪氨酸激酶,是整合素介导的胞外-胞内信号传导通路的基础性信号传递分子。它与整合素受体相连接并聚集其它分子于连接位点,形成信号复合体,将细胞外信号向细胞骨架传递,调节细胞的粘附、移行。PTEN (phosphatase and tensin homologue deleted on chromosome 10)为一种新发现的抑癌基因,能对FAK及其信号通路中多种信号分子进行脱磷酸调节,影响其正常细胞信号转导,导致细胞局部粘附和运动迁移功能受到抑制。了解整合素及其相关信号通路在电场中的变化情况有望为深入理解电场作用的机制开辟新领域。视网膜色素上皮(retinal pigment epithelium, RPE)位于视网膜光感受器细胞与Bruch膜之间,是维持视网膜结构和功能的重要组成部分,RPE损伤相关的病变可导致严重的视力丧失。然而RPE细胞所处位置的特殊性,使它易于同时受到神经视网膜和脉络膜的双重影响,同时由于各种外来干扰措施不易直接对其产生作用,因此造成相关疾病的治疗面临重重困难。在与RPE损伤相关的疾病中,年龄相关性黄斑变性(age-related macular degeneration, AMD)是患病率较高且危害较大的一种。AMD是50岁以上人群中视力障碍的主要原因,并随着人口老龄化趋势,该疾病的高危人群数量还在迅速扩大。AMD的发病中,在物质代谢异常、细胞功能变化、遗传和环境等因素的共同作用下,黄斑区脉络膜毛细血管、Bruch膜、RPE和光感受器细胞的结构发生慢性进行性改变。由于发病机制复杂,目前针对AMD的各种治疗措施效果均十分有限。分析AMD的组织病理学特点,认为其治疗关键在于促进病变区受损RPE细胞正常组织结构和功能的恢复。结合电场在其它组织损伤修复中的作用,推测可以将电场引入对RPE细胞诱导和AMD治疗的研究中来。【目的】建立电场对人RPE (hRPE)细胞的体外作用模型,观察电场对hRPE细胞生物学活性、移行、增生的影响,并检测此过程中整合素、FAK信号通路的变化以及PTEN基因对上述通路的调节作用。【方法】1)原代培养hRPE细胞,并根据文献描述建立电场对hRPE细胞的作用模型;细胞暴露于电场强度为0~10V/cm的电场中,显微照相系统记录电场作用前、作用3h及停止作用后12h的细胞图像;利用台盼蓝拒染活细胞计数法计算各时间点活细胞率;细胞核仁组成区嗜银蛋白(AgNORs)染色法计数细胞核内AgNORs颗粒数目;流式细胞仪检测各时间点细胞凋亡情况。2)观察电场对细胞移行的影响:hRPE细胞接受电场作用,选取电场强度分别为2、4、6、8、10V/cm,作用时间为3h,细胞分为无血清培养液组、无血清培养液+表皮生长因子(EGF)组、正常培养液组及正常培养液+EGF组。另选取部分正常培养液组细胞,于6V/cm电场强度中暴露3h后转换电极方向,继续暴露3h。显微照相系统连续记录每15min细胞图像,标记每个观察细胞核的中心,测量细胞移行距离、细胞移行方向与场线间的夹角,追踪细胞位移,用cosineФ描述细胞移行方向。观察参数包括:平均cosineФ、平均移行距离、平均移行速度、实际移行速度及移行指数。观察电场对细胞增生的影响:电场作用条件为6V/cm、12h,计数电场作用前后hRPE细胞的细胞密度、细胞生长速度;流式细胞仪测定细胞周期;采用Western blot检测细胞内cyclin E的表达情况。3)电场作用条件为6V/cm、3h,细胞松弛素B作为抑制剂,电场作用前与正常hRPE细胞共培养30min;免疫荧光染色方法检测正常hRPE细胞和细胞松弛素B处理细胞内F-actin、β1 integrin在电场作用前后的分布情况;RT-PCR、Western blot方法分别检测电场作用前后细胞内β1 integrin的mRNA和蛋白含量变化;Western blot方法检测细胞内FAK、pFAK(phosphorylated FAK)蛋白含量。4)利用脂质体转染法将PTEN的真核表达载体及其空载体转染hRPE细胞,经G418筛选(浓度为400mg/L)后获得稳定的克隆并进行鉴定;挑选细胞克隆扩大培养,接受电场作用,作用条件与第二部分实验相同;记录每15min细胞图像,标记每个观察细胞核的中心,测量细胞移行距离、细胞移行方向与场线间的夹角,追踪细胞位移,用cosineФ描述细胞移行方向;采用流式细胞仪测定电场作用前后的细胞周期;采用Western blot方法检测电场作用前后转染细胞中FAK、pFAK的表达。【结果】1)成功建立了电场对hRPE细胞的作用模型,各电场参数稳定,重复性好。电场强度小于2V/cm的电场暴露对hRPE细胞的形态无明显影响。暴露于2~10V/cm的电场后,hRPE细胞胞体伸长、细胞长轴垂直于场线方向排列;细胞暴露于电场3h后停止作用,继续培养12h,细胞恢复正常状态下的多角形或不规则形。台盼蓝和AgNORs染色显示各电场强度下各组细胞平均活细胞率和AgNORs颗粒数无显著差别(P>0.05)。流式细胞仪检测分析,电场作用前后hRPE细胞均未出现明显的凋亡现象。2)未受电场作用的正常对照组细胞随机分布,平均cosineФ值为0.02±0.10。电场作用后,hRPE细胞出现朝向电场阴极方向的定向移行,此现象随电场作用时间的延长和电场强度的升高更为明显,平均cosineФ值持续增大并趋近于1。血清能够影响hRPE细胞在电场中的定向移行。无血清培养液组中,电场强度低于6V/cm时细胞无明显的形态及移行变化,在6V/cm电场强度中方出现朝向垂直于场线方向的转位及微弱的阴极方向位移,平均cosineФ值为0.21±0.13。正常培养液组中,6V/cm电场强度中细胞出现明显的阴极方向移行趋势,平均cosineФ值为0.63±0.04,并随电场强度升高而增强,该组细胞各电场强度下移行分布指标均高于无血清组(P<0.001)。EGF可增强hRPE细胞对电场作用的反应,在同时有血清存在时此作用更为明显。无血清组培养液中加入EGF后,在4V/cm电场中细胞平均cosineФ值为0.35±0.09,随电场强度的增高,此定向移行逐渐增强。细胞培养液中同时加入血清和EGF后,hRPE细胞在各电场强度中移行分布指标均较无血清组和正常培养液组升高。hRPE细胞移行方向随电极方向转换而改变,表现为始终朝向电场阴极一侧运动。对细胞增生情况的观察结果显示,电场作用12h后hRPE细胞密度和生长速度均较对照组上升,组间差异显著(P<0.05);流式细胞仪检测显示实验组G0/G1期细胞比例下降,S期和G2/M期细胞比例明显增多,与对照组间差别有统计学意义(P<0.05);Western blot结果表明电场作用下细胞内cyclin E的蛋白量较对照组升高。3)免疫荧光染色结果显示,正常hRPE细胞内F-actin在胞浆成网状分布;电场作用3h后actin纤维束向细胞周边聚集,尤其是朝向电场阴极一侧;β1 integrin在正常hRPE细胞内有弱表达,电场作用3h之后荧光强度明显升高并集中于细胞朝向电场阴极的一侧。细胞松弛素B预处理细胞内F-actin和β1 integrin均形成散在分布于胞浆内的点状沉积物,电场作用后未见有明显变化。RT-PCR检测结果显示,电场作用后β1 integrin的mRNA和蛋白表达均升高,该现象可被细胞松弛素B抑制。FAK蛋白在hRPE细胞中稳定表达,各组间未见显著性差异。pFAK在未受电场作用的细胞中有微弱表达,电场作用后表达迅速、持续上升,两组间差别显著(P<0.05)。4) PTEN基因被成功转染入hRPE细胞,转染有PTEN的hRPE细胞在电场作用前后细胞形态和移行均未发生明显变化,正常对照组细胞在10V/cm电场作用3h后平均cosineФ值为0.78±0.02,PTEN转染细胞降至0.19±0.02,组间差异显著(P<0.01)。流式细胞仪检测结果显示,PTEN转染后G1期细胞百分比上升,S期和G2/M期细胞数量下降,与对照组相比差异显著(P<0.01);6V/cm电场作用12h后,细胞周期的上述表现无明显变化(P>0.05)。Western blot检测结果显示,hRPE细胞中FAK蛋白的总水平在PTEN转染和电场作用前后均无显著变化;电场作用后细胞pFAK的蛋白量明显升高,PTEN转染则抑制了该现象,表现为仅有微弱的蛋白表达。【结论】1)短时间、低于10V/cm的电场作用对hRPE细胞的正常活力及分裂无明显不良影响,保证了进一步体外和在体实验的安全性。2)电场作用能够引导hRPE细胞的定向移行,该作用与电场强度、血清和生长因子有关;一定时间内电场作用能够通过上调cyclin E的表达促进hRPE细胞的增生。3)电场对hRPE细胞的作用可引起细胞骨架蛋白的聚合反应,进而触发整合素及整合素相关信号通路FAK的活化,说明电场作用至少部分的通过整合素及FAK信号通路进行调节。4) PTEN转染能够明显抑制hRPE细胞在电场作用后的移行、增生能力及FAK信号通路的活化,说明PTEN通过调节FAK信号传导通路参与调控hRPE细胞在电场中的移行、增生。以上研究在国内外尚未见报道。更多还原

【Abstract】 BackgroundWound healing is important for maintaining the normal structure and function of tissues. During this process, an endogenous electric field might play a role. When the epithelium is wounded, the transepithelial potential will drive current out of the disrupted area, and create the endogenous electric field in turn. It has been proven that many types of cells respond to direct current electric fields (EFs) by changing their biologic characters, especially the migration capacity. Studies have shown that cells from multiple species and tissues display directed migration to the EFs, known as galvanotaxis or electrotaxis. Further more, the wound healing of the skin and the cornea can be accelerated by EFs. With the accumulated experimental evidence, it can be suggested electric signals play an important role in the directed cell migration of wound healing.Up to now, the mechanisms that guide both cell migration to electric cues and EF-induced wound healing have not been illustrated thoroughly. However, some assumptions have been introduced, including the asymmetries of cytoskeletal molecules and growth factors receptors, and the activation of signalling pathways. In addition, cell attachment to the extracellular matrix (ECM) is critical for migration and is mediated by members of the integrin family. Integrins are heterodimeric transmembrane receptors and play a central role in regulating cell adhesion, migration, proliferation and differentiation by mediating interactions between the extracellular matrix and the cell. Integrins exist in several activation states on the cell surface and activation induces integrin clustering. This leads to recruitment of multiple signaling molecules and the regulation of different signaling pathways. Furthermore, cell-to-substratum linkage sites mediated by integrin binding to matrix proteins must be regulated in order for cells to generate traction forces and to initiate directional movement. Focal adhesion kinase (FAK) plays an important role during this process. FAK is a nonreceptor protein-tyrosine kinase that localizes to focal contact sites. It associates with integrin receptors and recruits other molecules to the site of this interaction, thus forming a signaling complex that transmits signals from the extracellular matrix to the cell cytoskeleton. The tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylates FAK and is a key negative regulator of FAK signalling. Taken together, to investigate the effects of EFs on integrins and integrin-mediated signaling might be helpful to further understand the mechanisms of EFs on cells.Retinal pigment epithelium (RPE) is essential for the integrity and function of neural retina. Dysfunction or injury to RPE has been linked to many devastating eye disorders. However, RPE cells are under the influence from both neural retina and choroid, and treatment targeting the RPE cells usually has poor effect.Among the diseases which relate to the RPE disfunction, age-related macular degeneration (AMD) is a common cause for legal blindness. AMD is a complex and multifactorial disease and prevalence will further increase given demographic developments in ageing populations. A complex interaction of metabolic, functional, genetic and environmental factors seems to create a stage for chronically developing changes in ocular structures of the macular region (choriocapillaries, Bruch’s membrane, retinal pigment epithelium, photoreceptors) which may contribute to varying degrees to the onset and final picture of AMD. Current therapeutic options are limited for this disease so far. According to the histopathology of AMD, recovery of the normal structure and function of RPE cells seems to be the key point for proper treatment. Based on the effects of EFs on wound healing, we suppose that applying EFs on the RPE cells may be helpful in controlling RPE cell movements and more importantly, for AMD treatment.AimsTo establish the experiment model of RPE cells exposed to EFs, and investigate the effects of EFs on the viability, migration and proliferation of human RPE (hRPE) cells and the possible mechanisms.Methods1) A RPE cell exposed to EF model was established. Cultured hRPE cells were exposed to EFs within the range of 0~10 V/cm and images of the cells were obtained. The viability of the cells was determined by means of staining with trypan blue and AgNORs during and after exposure to EFs. Flow cytometry was applied to assess the apoptosis of the cells.2) To observe the effect of EFs on hRPE cell migration, the hRPE cultured in the medium with and without serum and EGF (epidermal growth factor) were exposed to EFs at 2, 4, 6, 8, 10 V/cm for 3h. Images of the cells were obtained every 15 min and directedness of movement were measured by tracing the position of cell nuclei before and after EF application, the directionality of migration was described by cosineФ, whereФwas the angle between the field axis and the vector drawn by the net cell translocation path. The parameters used to quantify cell migration in this study include the average cosineФ, the average distance, the average velocity and the migration index. The cell number and cell density at different time point were calculated, and the cell circle was analyzed by flow cytometry. The expression cyclin E was analyzed by Western blot.3) In the hRPE cells pretreated with or without cytochalasin B and EFs, the distribution of F-actin andβ1 integrin was measured by immunohistochemistry. The expression ofβ1 integrin was determined by PCR and Western blotting and the expression of FAK and pFAK was determined by Western blotting.4) Using liposome mediated method, PTEN were transfected into hRPE cells. The transfected cells were selected with 400 mg/L G418 and clones were picked and expanded. The transfected cells were exposed to the EFs as indicated above, and the directedness of the cell movement was measured. Flow cytometry was used to assess the cell circle, and Western blot was used to detect the expression of FAK and pFAK in the cells.Results1) The EF-exposure model for the experiment was stable and reliable. EFs below 2 V/cm did not affect the hRPE cell shape. After exposed to the EFs of 2~10 V/cm for 3 h, hRPE cells were elongated and oriented with their long axes perpendicular to the vector of the field. After stopped the EF exposure and cultured for an additional 12 h, the cells regained their normal shape and randomly distributed in the culture medium. Trypan blue and AgNORs staining showed no effect of EFs on the viability of RPE cells during 3 h exposure (P>0.05). The result of flow cytometry showed no obvious apoptosis in hRPE cells before and after EF exposure.2) Without exposure to EFs, RPE cells in the culture were randomly distributed with no obvious changes to their motility during the course of the experiment, and the cosineФwas 0.02±0.10. After exposed to EFs, hRPE cells were migrated to the cathode, and this directed translocation was more conspicuous with the value of cosineФclose to 1 when the field strength increased. Cells cultured in serum free medium showed slight polarization and the cosineФwas 0.30±0.12. Cultured in the medium with serum or serum plus added EGF, cells showed obvious cathodal migration in EFs. Increased electric field strength could enhance this cathodal directedness. When the polarity of the electric field was reversed, the cells reversed their direction of migration accordingly. In cultures with EFs, hRPE cell density and cell growth rate were increased higher than that of the control cultures (P<0.05). Flow cytometry showed the percent of the cell population in the G0/G1 phase in EF-exposed cells decreased, whereas the percentage of the cell population in the S and G2/M phases increased significantly. Western blot analysis of hRPE cells showed that EF exposure induced a significant increase in the expression of cyclin E.3) Immunofluorescence staining revealed that F-actin formed a stress fiber network across the cytoplasm in the normal RPE cells. After exposure to an EF for 3 h, the actin bundles accumulated at the lateral borders of RPE cells, especially towards the cathode side.β1 integrin was weakly expressed in normal RPE cells. Three hours after exposure to EFs, the staining ofβ1 integrin had increased in hRPE cells, with the stain density accumulating at the side facing cathode. After treated with cytochalasin B, a disruption of F-actin stress fibers in the cells was observed and random distributed deposits ofβ1 integrin staining were also detected. Exposure to EFs did not reverse these phenomena. The results of RT-PCR and Western blot showed an increase inβ1 integrin mRNA and protein expression of hRPE cells after exposure to EFs. This up-regulation ofβ1 integrin was blocked by cytochalasin B. FAK was steadily detected in normal RPE cells or in the cells exposed to EFs. The level of pFAK was low in RPE cells without exposure to EFs. After the cells were exposed to EFs, the relative intensity between the band density of pFAK and that ofβ-actin significantly increased compared with the controls (P<0.05).4) PTEN expressing vector was successfully transfected into hRPE cells and dramatically inhibited the polarization and directed migration of hRPE cells in EFs. Flow cytometry showed the percentage of the cell population in the S and G2/M phases in the transfected cells decreased whereas the percent of the cell population in the G1 phase increased significantly (P<0.01). EF exposure did not reverse this cell circle inhibition. Western blot analysis revealed that the expression of FAK in hRPE cells was not affected by PTEN transfection or EF exposure. However, the increased expression of pFAK in hRPE cells after EF exposure was blocked by PTEN transfection.Conclusions1) Limited period of exposure to EFs under 10 V/cm does not affect the normal viability of hRPE cells.2) EFs induce directed migration of hRPE cells, which can be enhanced by serum or EGF. EFs promote the proliferation of hRPE cells in certain exposure period by up-regulating the expression of cyclin E.3) Exposure to EFs induces the polymerization of cytoskeleton and up-regulate the expression of integrin. Activation of FAK signaling pathway may also involved in this interation. 4) FAK and PTEN mediate directional sensing of cell migration in response to electric signals.Similar research has not been reported at home and abroad.更多还原