【作者】 安刚；

【导师】 洪晶；

【作者基本信息】 中国医科大学 ， 眼科学， 2007， 博士

【摘要】 目的视网膜色素上皮(Retinal Pigment Epithelium，RPE)具有复杂的结构和特殊的生理机能。RPE能吞噬脱落的视细胞外节膜盘(rod outer segment，ROS)，这对视细胞外节的更新及维持正常视觉功能至关重要，其功能障碍会导致严重视网膜疾病和一些严重的遗传性变性眼病，如Best病，Stargardt病和Leber先天性黑矇，年龄相关性黄斑变性(age-related macular degeneration，AMD)等。鉴于色素上皮的异常，应用RPE细胞移植治疗某些眼底疾病将是未来的一个研究热点，但目前的问题是如何得到可移植的RPE细胞。组织细胞工程尤其是干细胞工程的兴起为视网膜损伤的治疗提供了一种新的途径。干细胞(Stem Cell)、组织工程学以及神经细胞发育、分化调控与定向生长研究的进展，为我们最终有效治疗这些眼疾，恢复患者视功能提供了一个契机。骨髓间充质干细胞(mesenchymal stem cell，MSCs)是至今研究得最为广泛的组织干细胞，至少能分化形成10种组织的细胞，如成骨细胞、软骨细胞、脂肪细胞等。本研究采用体外培养的人RPE细胞，通过非接触共培养诱导的方式，诱导人MSCs向RPE细胞分化，并观察分化细胞的生物学特性，为治疗视网膜疾病提供种子细胞奠定基础。方法一、体外HRPE和人MSC共培养体系的建立1、HRPE细胞的培养及鉴定眼球取自中国医科大学附属医院眼科角膜移植术后的供体眼(年龄在24-36岁之间)，在无菌状态下，用胰酶消化法获取视网膜色素上皮细胞进行原代及传代培养。抗角蛋白抗体免疫组织化学染色鉴定。2、人MSC的培养及鉴定采用密度梯度离心法和贴壁筛选法从人穿刺骨髓血中分离和培养MSC，用免疫组化法鉴定MSC。3、HRPE和人MSC细胞共培养体系的建立取2代HRPE细胞接种在六孔Transwell上层培养板中，将人MSCs接种于Tanswell下层培养板中，共同培养2-4周，在显微镜下观察人MSC的形态学变化。二、对于分化的色素上皮样细胞进行鉴定1、对于分化的色素上皮样细胞进行免疫细胞化学鉴定将共培养后分化的色素上皮样细胞(pigment epithelial-like cells)接种于24孔板中，抗角蛋白抗体免疫组织化学染色。2、RT-PCR检测色素上皮样细胞的RPE65和Mertk基因(1)总RNA提取：应用Trizol总RNA抽提试剂盒(上海生工生物工程技术服务有限公司)分别提取MSCs、RPE、Transwell下层MSCs细胞总RNA，紫外分光光度计测定RNA浓度，-20℃保存备用。(2)引物设计：按照NCBI公布的RPE65和Mertk基因序列，应用互联网提供的软件Primer 3设计引物。(3) PCR：分别加入不同引物、GAPDH(内参照)引物，PCR终体系25μl。取PCR产物25μl经2％的琼脂糖凝胶电泳后用凝胶成像系统行扫描分析。三、检测分化的色素上皮样细胞的吞噬功能1、荧光标记法检测分化的色素上皮样细胞的吞噬功能参照Margaret等的双重荧光标记法。用含有终浓度为40μg／ml SR的生长液孵育90％融和的分化的色素上皮样细胞24-72小时后，弃SR染液并用新鲜的培养液轻轻冲洗细胞2次，加入FITC标记的ROS进行孵育，然后以非选择性滤光片，用荧光显微镜观察，SPOT INSIGH荧光数字图象采集系统记录分化的色素上皮样细胞结合及吞噬ROS的变化情况；将MSC细胞设为对照组。2、透射电镜检测分化的色素上皮样细胞的吞噬功能将分化的色素上皮样细胞与ROS孵育24h后，去除孵育液，用PBS轻轻冲洗一次，加入2.5％的戊二醛固定，备电镜观察。结果一、体外HRPE和人MSC共培养体系的建立1、HRPE细胞的培养及鉴定原代细胞镜下为圆形，大小不一，内含较多的色素颗粒，胞核无法辨认。原代细胞培养贴壁后3d增殖速度明显加快，至5—7d即可基本融合。荧光显微镜下观察RPE细胞提示角蛋白表达呈强阳性，荧光遍及胞质。2、人MSC的培养及鉴定原代MSCs培养72小时后，细胞贴壁，呈纺锤型、梭型、多角形，增殖迅速。荧光显微镜下观察MSC细胞CD44表达呈强阳性，荧光遍及胞质；P63表达呈强阳性，荧光遍及胞核；CD34表达呈阴性，未见明显荧光。3、HRPE和人MSC细胞共培养体系的观测人RPE细胞在Transwell聚碳酯膜上贴壁生长，呈多边形，细胞内充满了色素颗粒；与人RPE细胞共同培养的MSCs生长情况基本相似，MSCs 3小时贴壁，呈纺锤型、梭型、多角形，增殖迅速，7天左右，部分长梭形的MSCs逐渐变成多边形，细胞内出现典型的色素颗粒，10天后色素上皮样细胞越来越多，分化率为45±5.12纥。对照组MSCs始终为长梭形，未出现色素样细胞。二、对于分化的色素上皮样细胞进行鉴定1、对于分化的色素上皮样细胞进行免疫细胞化学鉴定通过光镜下观察，可见MSCs分化为色素上皮样细胞，对爬片细胞进行间接免疫荧光染色，发现色素上皮样细胞胞浆中有绿色荧光表达，说明细胞表达角蛋白；MSC则无荧光表达，说明细胞无角蛋白表达。2、RT-PCR检测色素上皮样细胞的RPE65和Mertk基因通过RT-PCR检测分化的色素上皮样细胞的RPE65和Mertk的mRNA的表达，结果HRPE细胞和分化的细胞两种基因有表达，MSCs无表达。三、检测分化的色素上皮样细胞的吞噬功能1、荧光标记法检测分化的色素上皮样细胞的吞噬功能孵育90 min时，观察到ROS结合到分化的色素上皮样细胞表面，48小时左右吞噬达到饱和；对照组MSC则无ROS结合与吞噬的现象。2、透射电镜检测分化的色素上皮样细胞的吞噬功能透射电镜显示了分化的色素上皮样细胞特异性吞噬的胞质内超微结构胞质内周围密度不均匀的溶酶体为吞噬了ROS的吞噬性溶酶体。结论1、MSC与RPE细胞在非接触共培养诱导方式下能分化成色素细胞。2、分化的细胞具有上皮细胞的特异性标志。3、分化的色素上皮样细胞具有RPE细胞特异的基因表达。4、分化的色素上皮样细胞具有吞噬ROS的功能。更多还原

【Abstract】 ObjectiveThe retinal pigment epithelium (RPE) is a vital tissue for the maintenance of photoreceptor function. Amongst the many important functions of the RPE, it has the ability to phagocytosis and digest the rod outer segments (ROS) which is shed on a daily basis. The malfunction of RPE leads to retinal diseases, such as retinitis pigmentosa(RP), Best’s disease, Stargardt’s disease,Leber’s congenital amaurosis and age-related macular degeneration. For the abnormal lity of RPE,human RPE transplantation is the highlight nowadays. However, the ability to obtain an adequate source of autologous RPE and that homologo us cells still is an unsettled problem.The development of tissue engineering, especially stem cell engineering provide a new approach.The research in the fields of stem cell, tissue engineer ing, neural development,inducement and differentiation modulation provide a opportunity to rehabilitate and treat these diseases. mesenchymal stem cell (MSCs) is widely studied. Previous studies have shown that these progenitor cells express pluripotential. They were shown to differentiate into osteoblasts, cartilages, skeletal muscle cells, endothelium, cardiac muscle cells, hepatocyt, and neuroectoderm both in vitro and in vivo. In this study we cultured RPE in vitro and non-contact induce the MSCs to be pigment epithelial-like cells and observe their physiological properties. It can establish found to treat retina 1 disease by providing seed-cells. Methods1. Establishing indirect co-culture system of HRPE and MSC in vitro(1) HRPE culture and lablingHRPE cells were cultured from human donor eyes for keratoplasty after death (Peking University Eye Center,Peking, China).To collect the HRPE cells by the trypsin in sterile circumstance.RPE cell origin was confirmed by positive cytokeration immunocytochemical analysis.(2) MSC culture and lablingMSC isolation and culture by density gradient centrifugalization and adher ence sieving from human bone. MSC were tested by immunocytochemical analysis.(3) Establishing indirect co-culture system of HRPE and MSCIndirect co-culture was established using millicell culture plate inserts with 0.4 urn pore (Millipore, Oxford, UK). A cell suspension containing HRPE cells was added to the upper membrane surface. Inserts were positioned in the cult ure wells, which pre-seeded with MSCs, with 1:1 mix of growth medium for the two cell types used. We observing the change of cells morphology by microscope.2. Differentiation protocols(1) Pigment epithelial-like cells Immunocytochemical analysisPerforming the cytokeration immunocytochemical analysis on pigment epithet lial-like cells.(2) Reverse transcriptase-PCR was used to examine expression of RPE65 and Mertk①Extracting the total RNA: the total RNA of three cells was extracted using the TRIZOL reagent.②Primer design:According RPE65 and Mertk gene order which NCBI published,I designed primers by Primer 3 software internet.③PCR:After adding different primer,I take 25μl PCR product to analysis by gel imaging system. 3. Assay of pigment epithelial-like cells Phagocytosis(1) Double Fluorescent Vital Assay of PhagocytosisROS was isolated from human donor eyes by a discontinuous sucrose gradient in a dark room according to the method of Papermaster et al.MSCs and rod outer segments (ROS) were labled by sulforhodamine (SR) (Invitrogen cor, California, U.S.A.) and fluorescein isothiocyanate (FITC) (Sigma-aldrich, Saint Louis, U.S.A.) respectively. Phagocytosis was observe by Double Fluorescent Vital Assay according to Margaret.Briefly, under fluorescence microscopy, ingested ROS was shown by yellow fluorescence, which was a mixture of red (SR labled) and green (FITC labled), ROS just bounding to MSCs surface only revealed green.(2) Assay of pigment epithelial-like cells Phagocytosis by TEMHRPE were incubated with ROS for 24 hours,removed liquid.The sample were fixed by 2.5% glutaric dialdehyde for transmission electron microsco pe(TEM).Result1. Establishing indirect co-culture system of HRPE and MSC in vitro(1) HRPE culture and lablingThe just digested HRPE cells were round,and contained large quantity of pigments, but those pigments in the RPE cells decreased along with the cell division, Phase-contrast examination of living cells were performed with a photomicro scope. RPE cell origin was confirmed by positive cytokeration immunocytochemical analysis.(2) Bone MSCs isolation, culture and lablingAfter culturring for primary MSC 24 hours, the cultured cells were characteriz ed by spindle-shaped appearance and prolifered rapidly. MSCs cytoplasm were stained positively by CD44 monoclonal antibody.MSCs were positive in nucleus with P63 monoclonal antibody.(3) indirect co-culture system of HRPE and MSC in vitro protocolsHRPE were characterized by spindle-shaped appearance,filled with pigm ents.MSCs were characterized by spindle-shaped appearance and prolifered rapidly,after 7 days,part of MSCs of spindle-shaped appearance changed to polygon-shaped gradually, filled with typical pigments;After 10 days ,more and more MSCs showed the same change. The differentiated rate were 45±5.12%.2. Differentiation protocols(1) Immunocytochemical analysisThrough microscope, I observed that MSCs were differentiated pigment epithelial-like cells.Differentiated pigment epithelial-like cells showed in green fluorescence,which indicated the cell were positive stain for pan- cytokeratin.(2) Reverse transcriptase-PCR was used to examine expression of RPE65 and Mertk.I detect RPE65 and Mertk mRNA expression of pigment epithelial-like cells by RT-PCR, the result show RPE and pigment epithelial-like cells have mRNA expression,but MSCs have no expression.3. Assay of pigment epithelial-like cells phagocytosis(1) Double Fluorescent Vital Assay of PhagocytosisAfter added HRPE cells’ supernatant for 90 min, ROS were found in pigment epithelial-like cells surface , the phagocytosis reach the saturation. MSC can’ t adhere and phagocytosis ROS.(2) Assay of pigment epithelial-like cells Phagocytosis by TEMIt can indicate the pigment epithelial-like cells’ ultramicrostructure by TEM. which have Phagolysosome including ROS.Conclusion1.MSC can be differentiated epithelial-like cells in indirect co-culture system of HRPE.2.These MSCs-converted pigment epithelial cells showed some of the charac teristics of epithelioid cell. 3.These MSCs-converted pigment epithelial cells showed some of the characteristics of HRPE.4.These MSCs-converted pigment epithelial cells have the function of phagocytosis ROS.更多还原