人视网膜色素上皮细胞在电场中定向移行的实验研究

目的:观察外源性电场对培养人视网膜色素上皮humanretinalpigmentepithelial,hRPE)细胞移行方向及细胞内肌动蛋白表达的影响。方法:培养于DMEM+100g/LFBS的hRPE细胞暴露于强度为6V/cm的电场中,未暴露于电场的细胞作为对照。观察并记录2h中每隔15min细胞移行的变化图像,测量细胞移行距离、细胞移行方向及其与场线间夹角。2h后采用间接免疫荧光法检测实验组与对照组细胞内肌动蛋白表达情况。SPSS10.0及Image-ProPlus5.0软件处理结果。结果:未暴露于电场中的hRPE细胞在观察期间呈无序移行,2h后细胞分布未见特殊变化,细胞内肌动蛋白有弱阳性表达。细胞暴露于电场30min后开始出现阴极方向的移行趋势,大部分细胞长轴趋向垂直于场线方向排列。细胞的移行表现为移行速度和移行方向的一致性均随时间延长而增加(P<0.05)。暴露于电场2h后,大部分细胞内肌动蛋白表达阳性,且多集中分布于细胞质内朝向电场阴极一侧。结论:外加电场可诱导hRPE细胞向电场阴极方向定向移行,同时伴有细胞内肌动蛋白阳性表达及定向分布。电场对细胞的作用与暴露时间成正相关。视网膜脱离后的内源性电场的存在可能参与并诱导了RPE细胞的定向移行。     

电场对人视网膜色素上皮细胞生物学活性的影响

目的:观察电场作用对培养人视网膜色素上皮(human retinal pigment epithelial,hRPE)细胞活力及分裂过程的影响。方法:hRPE细胞置于强度为8V/cm的直流电场中暴露3h,停止电场作用后继续培养12h;未受电场作用的hRPE细胞作为对照组。显微摄像系统记录各时间点细胞图像,观察细胞形态变化;细胞行台盼蓝拒染活细胞计数及核仁嗜银蛋白(AgNORs)染色,图像分析染色结果;流式细胞仪检测细胞凋亡情况。结果:暴露于电场中的hRPE细胞伸长,垂直于场线方向排列,停止暴露后细胞恢复正常形态及分布;各时间点实验组与对照组细胞的台盼蓝拒染率差异无统计学意义(P>0.05);电场暴露前、暴露3h及停止暴露并继续培养12h后hRPE细胞核内AgNORs颗粒数分别为:6.2,6.5,7.3,与正常对照组细胞比较均无显著差异(P>0.05);流式细胞仪检测结果显示各组均未见明显细胞凋亡。结论:短时间电场作用对hRPE细胞的正常细胞活力及分裂无明显影响,提示该条件下的电场作用可能应用于促进RPE细胞修复的研究。     

外加直流电场对人晶状体上皮细胞移行和增殖活力的影响

目的:观察外加直流电场对人晶状体上皮细胞(lens epithelial cell,LEC)移行和增殖活力的影响.方法:将培养的人晶状体上皮细胞系HLE-B3细胞暴露于电场强度为100、200、400 mV/mm的直流电场中,未受电场暴露的细胞作为正常对照组.倒置显微镜观察并记录电场暴露前及暴露后的细胞图像,并计数细胞数目;流式细胞仪检测电场暴露前及暴露24 h后的细胞凋亡率和细胞周期.结果:强度为400 mV/mm的电场暴露3 h后,HLE-B3细胞呈现朝向电场阴极一侧的定向移行.电场持续暴露后,HLE-B3细胞数逐渐减少,在电场作用6h和12h时分别较正常对照组减少12.6%和18.6%,差异均具有统计学意义(P<0.05).100、200、400 mV/mm电场暴露24 h后,HLE-B3细胞凋亡率分别为(9.2±1.9)%、(23.9±2.6)%、(54±2.5)%,与正常对照组相比明显增加(P<0.05);细胞周期检测结果显示,HLE-B3细胞进入G2/M期的比例随电场强度增加逐渐上升,其中200 mV/mm和400mV/mm电场中G2/M期细胞比例分别为(13.8±2.2)%和(15.6±2.5)%,与正常对照组相比差异具有统计学意义(P<0.05).结论:外加直流电场可诱导HLE-B3细胞的定向移行,随电场作用时间的延长和电场强度增加,细胞生长受到抑制,同时伴随细胞凋亡增加和细胞周期G2/M期阻滞.     

电场对人视网膜色素上皮细胞增生的影响

目的观察一定条件下电场对人视网膜色素上皮(human retinal pigment epitheli-um,hRPE)细胞增生的影响及相关机制。方法hRPE细胞置于电场强度为6V.cm-1的电场中连续作用12h。观察未受电场作用的正常对照组及电场作用12h的实验组细胞,显微摄像系统记录不同时间点细胞图像并计数细胞数,检测细胞密度和细胞的生长速度,观测指标包括:细胞密度,以每平方厘米细胞数表示;细胞生长速度,计算公式为:(Ni-N0)/N0×100%(Ni为12h细胞数;N0为0h细胞数);采用流式细胞仪测定细胞周期,计算细胞增生指数:(NS+NG2/M)/N(NS为S期细胞数;NG2/M为G2/M期细胞数;N为细胞总数);Western blotting检测细胞cyclinE蛋白的表达。结果对照组hRPE细胞在12h的观察期间内细胞密度缓慢上升,到12h时细胞密度由95×103cm-2增至130×103cm-2;而实验组在6V.cm-1电场作用下hRPE细胞数量逐渐上升,12h后细胞密度上升至150×103cm-2。实验组hRPE细胞生长速度为50.02%,较对照组(36.84%)明显升高(P<0.05);...     

缺氧及小干扰RNA转染对人RPE细胞增生及血小板源性生长因子-BB表达的影响

背景 增生性玻璃体视网膜病变（PVR）为视网膜表面发生无血管、纤维细胞性增生膜,其发生和发展的具体机制尚未完全阐明.人视网膜色素上皮（hRPE）及血小板源性生长因子（PDGF）在PVR发展中的作用是近几年研究的热点. 目的 探讨缺氧对体外培养hRPE细胞PDGF-BB表达及增生的影响. 方法 hRPE细胞在6孔板中培养,实验组用10、15、20、30、40 μmol/L CoCl2模拟体外培养hRPE细胞的缺氧环境,对照组用未加CoCl2的培养液培养hRPE细胞,采用逆转录PCR（RT-PCR）与ELISA法检测PDGF-BB mRNA和蛋白的表达,采用MTT法检测hRPE细胞增生率.依据转染siRNA的不同将细胞分为PDGF-BB siRNA1组、PDGF-BB siRNA2组、PDGF-BB siRNA3组（为针对PDGF-BB的3条不同的siRNA,其中有一条为有效siRNA）、β-actin siRNA组、无关siRNA组和只加Lip2000的空白对照组.转染4～6h后,除空白对照组外,其余各组加入对细胞PDGF-BB mRNA和蛋白及对hRPE细胞增生影响最明显的15 μmol/L CoCl2模拟细胞缺氧环境24 h,检测PDGF-BB mRNA和蛋白及hRPE细胞增生率. 结果 未加CoCl2的对照组未检测出PDGF-BBmRNA和蛋白的表达,不同浓度CoCl2培养hRPE细胞PDGF-BB mRNA和蛋白的表达量不同,差异均有统计学意义（F=43.737,P＜0.01;F=54.612,P＜0.05）,15μmol/L CoCl2组PDGF-BB的表达量最多,与其他各组比较差异均有统计学意义（P＜0.05）.MTT检测结果显示,不同浓度CoCl2处理后PDGF-BB蛋白表达及hRPE细胞增生率明显不同,差异均有统计学意义（F=95.379,P＜0.01;F=63.375,P＜0.05）,15 μmol/L CoCl2组hRPE细胞增生率明显高于其他浓度组,差异均有统计学意义（P＜0.05）,PDGF-BB蛋白表达量与hRPE细胞增生率呈线性正相关（r=0.994,P＜0.05）.各细胞转染组hRPE细胞中PDGF-BB mRNA及蛋白的表达整体比较,差异均有统计学意义（F=156.330、125.650,P＜0.01）,且PDGF-BB siRNA2组PDGF-BB mRNA较其他各组明显下降,差异均有统计学意义（P＜0.01）.MTT检测结果显示,各细胞转染组PDGF-BB蛋白的表达及hRPE细胞增生率明显不同,差异均有统计学意义（F=73.131、98.564,P＜0.01）,PDGF-BB siRNA2组PDGF-BB蛋白的表达及细胞增生率与其他各组比较明显下降,差异均有统计学意义（P＜0.05）,PDGF-BB蛋白表达与hRPE细胞增生率呈线性正相关（r=0.996,P＜0.05）.结论 缺氧能够促进PDGF-BB的表达,PDGF-BB的表达上调可显著促进hRPE细胞的增生.在转染靶向PDGF-BB siRNA后,PDGF-BB的表达受到抑制,可有效降低hRPE细胞的增生率.     

枸杞多糖对蓝光损伤的视网膜色素上皮细胞的保护作用

目的:研究不同浓度的枸杞多糖对蓝光诱导损伤的体外培养人视网膜色素上皮(human retinal pigment epithelium,hRPE)细胞的保护作用。方法:通过蓝光诱导建立hRPE细胞光损伤模型,分别用不同浓度的枸杞多糖(分别为0.01,0.1,1mg/mL)对体外培养的hRPE细胞进行干预,通过流式细胞仪检测各实验组的细胞线粒体活性氧和凋亡率。实验组分为正常对照组、光照损伤组以及不同浓度枸杞多糖(0.01,0.1,1mg/mL)干预组。结果:线粒体活性氧检测:正常对照组荧光强度最小;蓝光损伤组荧光强度最大,不同浓度枸杞多糖处理组荧光度与蓝光损伤组强度相比,荧光强度差异有统计学意义(P<0.05)。Annexin V-FITC/PI凋亡检测显示不同浓度枸杞多糖干预组凋亡细胞数量与蓝光损伤组凋亡细胞相比差异均有统计学意义(P<0.05);1mg/mL枸杞多糖干预组凋亡细胞数量与对照组凋亡数量相比差异无统计学意义(P>0.05)。结论:枸杞多糖能抑制蓝光诱导损伤的hRPE细胞的凋亡,1mg/mL枸杞多糖抑制蓝光诱导的hRPE细胞凋亡的作用更强,其作用机制可能与抑制细胞线粒体产生活性氧有关。     

维生素E对大剂量蓝光诱导损伤视网膜色素上皮细胞的影响

目的:研究维生素E(Vit E)对大剂量蓝光诱导人视网膜色素上皮(hRPE)细胞损伤的影响,为减少蓝光损伤hRPE细胞的预后方案提供思路。方法:用3000±150Lx蓝光建立hRPE细胞损伤模型;利用流式细胞术检测照射时间分别为0、3、6、9、12、24h的6组hRPE细胞凋亡率及活性氧相对量;利用流式细胞术检测照射0h组、照射6h组、照射6h前或后加不同浓度Vit E组(Vit E的浓度分别为10、50、100μmol/L)的hRPE细胞凋亡情况和活性氧相对量,并采用Hoechst 33258试剂染色在荧光显微镜下观察hRPE细胞的荧光强度。结果:与照射0h组相比,照射3、6、9、12、24h组的hRPE细胞活性氧相对量明显增加(均P<0.01),照射6、9、12、24h组的hRPE细胞凋亡率也明显增加(均P<0.01),但照射3h组的hRPE细胞凋亡率无明显增加,差异无统计学意义(P=0.46)。与照射6h组相比,除照射6h前加入10μmol/L的hRPE细胞凋亡率(P=0.66)外,其他加Vit E的实验组hRPE细胞活性氧相对量和凋亡率明显减少、细胞中Hoechst 33258释放蓝色荧光逐渐减弱,且呈浓度依赖(均P<0.01)。与照射0h组相比,加Vit E的6组hRPE细胞活性氧相对量和凋亡率有差异(均P<0.01)。同一浓度的Vit E,除10μmol/L Vit E的hRPE细胞凋亡率在照射前或后加无差异(P=0.08)外,照射后加Vit E组比照射前加Vit E组的hRPE细胞活性氧相对量、凋亡率明显减少(均P<0.01)。结论:hRPE细胞经蓝光照射后出现胞内活性氧相对量增多的现象早于细胞凋亡,清除胞内活性氧是减少大剂量蓝光诱导hRPE细胞损伤的思路。Vit E可保护由大剂量蓝光诱导的RPE细胞损伤,这种效应随Vit E浓度增加而增强,且照射后加入比照射前加入的效果更好。但需要一定剂量的Vit E才能显效,而且无法完全修复损伤。     

缺氧对视网膜Muller细胞上水通道蛋白-4表达的影响

目的利用体外培养的视网膜Muller细胞，研究在缺氧条件下视网膜Muller细胞上水通道蛋白-4（AQP-4）表达的变化。方法采用组织块培养法从新西兰大白兔视网膜中获取Mullerr细胞，在含20％胎牛血清的DMEM培养液中原代培养，培养的细胞通过胶质纤维酸性蛋白（GFAP）免疫细胞化学染色及透射电子显微镜进行鉴定。取第2代细胞进行实验，将化学缺氧诱导剂CoCl，联合DMEM培养液培养24h的细胞作为缺氧组；将DMEM培养液单独培养24h的细胞作为对照组。采用免疫细胞化学法及半定量RT．PCR法分别检测2组Muller细胞上AQP-4蛋白及AQP-4mRNA的表达。结果Muller细胞（第2代）上GFAP的阳性率为90％以上，细胞质染色呈棕色。细胞内含特征性的中间丝，细胞表面有微绒毛，细胞质内含丰富的细胞器。CoCl，联合DMEM培养液培养24h后Muller细胞上AQP-4蛋白的表达较对照组明显增加（t=6．74，P〈0．05）；AQP-4mRNA的表达亦明显增加（t=21．79，P〈0．05）。结论缺氧能增强Muller细胞上AQP-4的表达，进而使视网膜内液体的积聚增加。提示Muller细胞在糖尿病视网膜病变（DR）或增生性视网膜病变的视网膜水肿形成过程中起重要作用。     

siRNA抑制缺氧人视网膜色素上皮细胞ILK的表达及对HIF-1α的影响

目的:探讨干扰RNA(siRNA)沉默缺氧培养下人的视网膜色素上皮细胞(hRPE)中整合素连接激酶(ILK)的表达及其对缺氧诱导因子1α(HIF-1α)表达的影响。方法:CoCl2建立hRPE细胞的化学缺氧模型,Western blot和RT-PCR方法半定量检测不同缺氧时间(0,6,12,24,48h)hRPE细胞中ILK、HIF-1α蛋白及其mRNA的表达;阳离子脂质体转染ILK的干扰片段抑制缺氧24h hRPE细胞中ILK的表达,同上方法检测转染后缺氧24h hRPE细胞中ILK的表达及其对HIF-1α表达的影响。结果:ILK表达于正常及缺氧培养的hRPE细胞中。随着hRPE细胞缺氧时间的延长,在蛋白和mRNA水平ILK、HIF-1α都呈现逐渐增加的表达趋势。siRNA抑制ILK在缺氧24h hRPE细胞中的表达,同时显著抑制了HIF-1α的表达,较阴性对照组、单纯脂质体组有显著差异(P<0.01)。结论:ILK可以通过HIF途径应答RPE细胞的缺氧反应。     

整合素连接激酶对人视网膜色素上皮细胞增殖的影响

目的研究整合素连接激酶(integrin linked kinase,ILK)小分子干扰RNA(siRNA)对人视网膜色素上皮(human retinal pigment epithelium,hRPE)细胞增殖的影响。方法培养hRPE细胞,角蛋白鉴定。以ILK为靶基因设计合成特异性的siRNA干扰片段转染hRPE细胞。荧光显微镜下观察转染效率,选择转染效率最高的干扰片段进行细胞转染。RT-PCR检测siRNA对ILK基因表达的抑制作用,MTT法检测转染前后hRPE细胞增殖活性。结果 ILK-siRNA可以成功转染至hRPE细胞,转染24h后hRPE细胞中ILK mRNA的相对表达水平在正常对照组、单纯脂质体组、阴性对照组及转染组中分别为0.44±0.48、0.43±0.38、0.42±0.47、0.32±0.71,正常对照组、单纯脂质体组和阴性对照组中ILKmRNA表达差异无统计学意义(P>0.05),转染组ILKmRNA的表达较正常对照组明显降低,差异有显著统计学意义(F=21.78,P<0.01)。不同浓度ILK-siRNA转染组在不同时间点细胞增殖较正常培养组明显降低(P<0.01),50nmol·L-1浓度转染组对hRPE细胞的生长抑制最明显。结论 hRPE细胞表达ILK,siRNA抑制ILKmRNA的表达,在一定范围呈时间浓度依赖性抑制hRPE细胞增殖。     

Movements of cultured corneal epithelial cells and stromal fibroblasts in electric fields

The effects of an externally applied direct-current electric field on the movement of cultured rabbit corneal epithelial cells and stromal fibroblasts were studied. After a latency of approximately 20 minutes in an electric field, both epithelial cells and stromal fibroblasts became spindle shaped and underwent galvanotropism by aligning their long axes perpendicular to the applied electric field. The electric field stimulus thresholds for galvanotropic movements in epithelial cells and stromal fibroblasts were 4V/cm and 6 V/cm, respectively. After an additional latency of 30 minutes, both cell types manifested galvanotaxic movements: epithelial cells commenced migration in the cathodal (downfield) direction and stromal fibroblasts in the anodal (upfield) direction. For both types of cells, ruffled membranes and lamellipodia were abundant at the leading edges of migrating cells and cell processes underwent retraction at the trailing edges. At field strengths of above 10 V/cm, evidence of cellular damage (manifested by cellular rounding and detachment), attributable to the electric field treatment, was observed after 4 hours. These preliminary results suggest that galvanotaxic responses could be exploited clinically in the enhancement of corneal wound healing.     

Effects of steady electric fields on human retinal pigment epithelial cell orientation and migration in culture.

Low-level, steady electric fields of 6-10 volts/cm stimulated directional orientation and translocation of cultured human retinal pigment epithelial cells. The orientative movements (galvanotropism) consisted of somatic elongation of the cells into spindle shapes, followed by pivotal alignment orthogonal to the field. The anodal edges of the cells underwent retraction of their plasmalemmal extensions, while the cathode edges and the longitudinal ends developed lamellipodia and ruffled membranes. These tropic movements were followed by a translocational movement (galvanotaxis) of the cells towards the cathode. Staining of these migrating cells for actin showed the accumulation of stress fibers at the leading (cathodal) edge, as well as at the longitudinal ends of the elongated somata. These results suggest that endogenous, biologically-generated electric fields (eg., injury currents) may play a role in the guidance and migration of retinal pigment epithelial cells after retinal injury.     

Epidermal growth factor receptor in cultured human retinal pigment epithelial cells

BACKGROUND: Migration and proliferation of retinal pigment epithelial (RPE) cells play an important role in proliferative vitreoretinopathy. Epidermal growth factor receptor (EGFR) is a cell surface receptor with intrinsic tyrosine kinase activity. The engagement of the receptor by its ligand can induce intracellular mitogenic signal transduction pathways and stimulate proliferation, migration and differentiation of cells. This experiment aimed to investigate the activation and role of EGFR signal transduction pathway in proliferation of human RPE cells. METHODS: Cultured human RPE cells of the 3rd to 6th passages were studied by colorimetric assay for cellular growth and survival (MTT assay) to test the effects of EGF (0.1, 1, 10, 50, and 100 ng/ml) and fetal bovine serum (FBS) on proliferation of human RPE cells. An in vitro wound healing model was also set up, and the number of cells that had entered the denuded area was counted. The human RPE cells were cultured for 3 days with 0.1% FBS, 10% FBS, 10 ng/ml EGF + 0.1% FBS and a combination of EGF and 10% FBS, respectively. Immunohistochemical staining and in situ hybridization were used to observe the expressions of EGFR protein and mRNA, respectively. Activation of mitogen-activated protein kinase (MAPK) was detected by immunohistochemical method with specific antiphosphorylated extracellular signal-regulated kinase (ERK)1/2 antibody. RESULTS: EGF stimulated proliferation and migration of cultured human RPE cells in a concentration-dependent manner. The maximum of the proliferation rate of RPE cells was 81.8% with EGF at a concentration of 10-100 ng/ml of EGF in serum-free Dulbecco's modified essential medium (DMEM) and 122.7% at a concentration of 1-10 ng/ml of EGF in 5% FBS DMEM (p < 0.001); there was a significant difference between serum-free DMEM groups and 5% FBS DMEM groups. The maximum of the migration rate of the cells was 438.9% at a concentration of 10-100 ng/ml of EGF in 10% FBS DMEM, 147% with 10% FBS, and only 36% with EGF in 0.1% FBS at the concentration of 10 ng/ml (p < 0.001). EGF promoted the expression of EGFR protein and mRNA in RPE cells. FBS cooperated with EGF in the stimulation of EGFR expression, and it had a stronger effect in the process than EGF alone. After 3 days of incubation with EGF, phosphorylated ERK1/2 was detectable in the nucleus of RPE cells, whereas cells presented immunostaining positive for phosphorylated ERK1/2 in the cytoplasm before stimulation, indicating that EGF could induce MAPK nuclear translocation. CONCLUSION: EGF could induce EGF-EGFR-MAPK signal transduction pathway in human RPE cells in a concentration-dependent manner in vitro, which may play a key role in the activation of human RPE cell proliferation and migration.     

整合素连接激酶对人视网膜色素上皮细胞增殖和迁移的影响

目的:探讨RNA干扰抑制整合素连接激酶（integrin-linked kinase, ILK）对人视网膜色素上皮（human retinal pigment epithelium,hRPE）细胞增殖和迁移的影响。方法:体外培养hRPE细胞,设计并合成特异性人ILK的RNA干扰片段,阳离子脂质体转染入人视网膜色素上皮细胞,利用RT-PCR及Western blot半定量检测siRNA对ILK基因及蛋白表达的抑制作用,MTT方法检测转染前后siRNA对细胞增殖的抑制作用。损伤愈合实验和Transwell实验观察人视网膜色素上皮细胞迁移能力的改变。结果:培养的hRPE细胞存在ILK的基因转录表达,siRNA显著抑制hRPE细胞ILK的mRNA和蛋白表达（P<0.01）。MTT、损伤愈合实验和Transwell实验提示转染ILK-siRNA后人视网膜色素上皮细胞的增殖、迁移能力有明显下降,与正常对照组相比有统计学差异（P<0.05）。结论:特异性ILK-siRNA能有效抑制ILK在hRPE的mRNA和蛋白的表达并显著降低hRPE的增殖和迁移能力。     

Electrical estimulation of retinal pigment epithelial cells

We investigated and characterized the effect of externally applied electric fields (EF) on retinal pigment epithelial (RPE) cells by exposing primary cultures of human RPE cells (hRPE) and those from the ARPE19 immortalized cell line to various strengths of EF (EF-treated cells) or to no EF (control cells) under different conditions including presence or absence of serum and gelatin and following wounding. We evaluated changes in RPE cell behavior in response to EF by using a computer based image capture and analysis system (Metamorph). We found that RPE cells responded to externally applied EFs by preferential orientation perpendicular to the EF vector, directed migration towards the anode, and faster translocation rate than control, untreated cells. These responses were voltage-dependent. Responses were observed even at low voltages, of 50-300 mV. Furthermore, the migration of hRPE cell sheets generated by wounding of confluent monolayers of cells at early and late confluence could be manipulated by the application of EF, with directed migration towards the anode observed at both sides of the wounded hRPE. In conclusion, RPE cell behaviour can be controlled by an externally applied EF. The potential for externally applied EF to be used as a therapeutic strategy in the management of selected retinal diseases warrants further investigation.     

Effects of electric fields on cytoskeleton of corneal stromal fibroblasts

Low-level, steady electric fields (6-10 volts/cm) stimulated cultured corneal stromal fibroblasts to undergo directional orientation and translocation. The orientative movements (galvanotropism) consisted of somatic elongation of the cells into spindle shapes along an imaginary axis perpendicular to the field; the cathodal edge of the cell underwent retraction, while the anodal edge and the longitudinal ends developed ruffled membranes and lamellipodia. The translocational movements (galvanotaxis) consisted of directed migration of the cells towards the anode. While most actin-containing stress fibers became aligned along the long axes of the elongated fibroblasts (with distal ends of the stress fibers terminating at the longitudinal extremes of the cells), some were aligned towards the anodal direction (with distal terminations inside ruffled membranes and lamellipodia on the leading anodal edge of cells). The distal ends of stress fibers were associated with discrete foci of vinculin, ie, focal indicators of cell-to-substrate adhesion; these foci were abundant at the longitudinal ends and at the anodal edge of the elongated cells. The observed cytoskeletal changes are consistent with an active, rather than passive, directed migration of stromal fibroblasts in response to constant electric fields.     

培养人视网膜色素上皮细胞牵拉模型中细胞骨架的改变

目的研究培养人视网膜色素上皮细胞（RPE）受机械牵拉力后细胞骨架的变化。方法将胶原包被的磁珠悬液加入培养的人RPE细胞后孵育,洗去未结合的磁珠。在磁场垂直方向力作用下0、4、8、12和24h,以及加入细胞松弛素D（0．05mmol／L,25min）预处理后,用免疫荧光双标染色法着染细胞中的肌动蛋白和波形蛋白,并用激光共聚焦显微镜观察。结果机械牵拉作用4h后,人RPE细胞的形态和微丝的极性发生改变。肌动蛋白微丝的排列与受力方向一致。细胞骨架微丝表达主要在细胞核周及磁珠聚集处周围。而加入细胞松弛素D预处理的细胞,在8h时发生以上变化。结论机械牵拉可引起人RPE细胞骨架分布的改变,使其表现出肌细胞的某些生物学特征,这对细胞的移行和增生可能有促进作用。     

载脂蛋白A-Ⅰ对高糖环境下人视网膜血管内皮细胞生物行为及VEGF表达的抑制作用

背景 视网膜血管内皮细胞(RVECs)是视网膜微血管的主要成分,通过增生、迁移以及血管发生等生物行为在糖尿病视网膜病变(DR)的发生和发展中发挥关键作用.载脂蛋白A-Ⅰ (ApoA-Ⅰ)是高密度脂蛋白中主要的载脂蛋白,既往研究表明ApoA-Ⅰ在糖尿病患者视网膜组织内呈高表达,且在不同微环境中对RVECs发挥不同作用,但其在高糖环境下与RVECs生物学行为的关系仍不清楚. 目的 研究ApoA-Ⅰ对高糖环境下培养的人RVECs(hRVECs)生物行为及血管内皮生长因子(VEGF)表达的抑制作用. 方法 用含体积分数10％胎牛血清的DMEM体外培养hRVECs并传代,取3～6代细胞用于实验.将培养的细胞分为低糖组、低糖+ApoA-Ⅰ组、高糖组和高糖+ApoA-Ⅰ组,按照分组分别在DMEM中添加葡萄糖和ApoA-Ⅰ,低糖浓度为5 mmol/L,高糖浓度为25 mmol/L,ApoA-Ⅰ终质量浓度为30 μg/ml.采用细胞计数试剂盒-8、CCK-8法检测细胞增生能力(吸光度,A值);采用细胞划痕法检测细胞的迁移率;采用管腔形成实验检测培养细胞的体外成管能力;分别采用实时荧光定量PCR法和Western blot法检测各组细胞中VEGF mRNA及其蛋白的相对表达量.结果 高糖组细胞增生值(A值)和细胞迁移率明显高于低糖组,高糖+ApoA-Ⅰ组细胞增生值和细胞迁移率明显低于高糖组,差异均有统计学意义(A值:P=0.001、0.033;迁移率:P=0.001、0.010).低糖组、低糖+ApoA-Ⅰ组、高糖组和高糖+ApoA-Ⅰ组管腔数分别为(7.250±2.217)、(9.250±2.630)、(19.000±3.916)和(11.500±3.697)个,组间总体比较差异有统计学意义(F=10.335,P=0.001).高糖组管腔数较低糖组显著增加,高糖+ApoA-Ⅰ组较高糖组管腔数显著减少,差异均有统计学意义(P=0.001、0.037).低糖组、低糖+ApoA-Ⅰ组、高糖组和高糖+ApoA-Ⅰ组细胞中VEGF mRNA相对表达量分别为0.944±0.083、1.117±0.204、1.768±0.164和1.301±0.077,VEGF蛋白相对表达量分别为1.000±0.130、1.217±0.152、1.871+0.101和1.609±0.087,组间总体比较差异均有统计学意义(mRNA:F=18.640,P=0.001;蛋白:F=10.335,P=0.001),其中高糖组细胞中VEGF mRNA及其蛋白的相对表达量均明显高于低糖组和高糖+ApoA-Ⅰ组,差异均有统计学意义(mRNA:P=0.000、0.004;蛋白:P=0.000、0.029). 结论 ApoA-Ⅰ对高糖环境下hRVECs的增生、迁移及管腔形成具有抑制作用,可能与其下调细胞VEGF的表达有关.