光损伤鼠视网膜片培养上清液诱导 MSCs 分化为视网膜样细胞的研究

目的：应用大鼠视网膜片光损伤后的培养上清液,在体外诱导大鼠骨髓间充质干细胞（ mesenchymal stem cells , MSCs）成为视网膜样细胞的可能性。 方法：贴壁筛选法分离、培养大鼠MSCs ,流式细胞仪对其细胞纯度鉴定。取材大鼠视网膜神经上皮层作为视网膜片,常规石蜡切片HE染色鉴定各层组织完整性。电镜观察大鼠视网膜片光损伤程度。制备3种诱导分化大鼠MSCs的条件培养液。3种条件培养液均培养诱导至第3代大鼠MSCs 7～8d,用RT-PCR检测视紫红质（Rhodopsin）、神经元特异性烯醇化酶（neuron-specific enolase,NSE）、胶质纤维酸性蛋白（ glial fibrillary acidic protein ,GFAP）等视网膜细胞标志物在诱导后细胞中的表达情况。 结果：HE染色显示大鼠视网膜片取材结构完整,电镜显示大鼠视网膜片光损伤后结构损伤严重。 RT-PCR鉴定：条件培养液诱导大鼠MSCs 7～8d,条件培养液Ⅰ组Rhodopsin （0．3915±0．00644）、NSE （0．2019±0．00682）、GFAP （0.1972±0．00211）,条件培养液Ⅱ组Rhodopsin（0．0983±0．00319）、NSE （0．1048±0．00323）、GFAP （0．1040±0.00254）,条件培养液Ⅲ组Rhodopsin（0．0044±0．00126）、NSE（0．0498±0．00149）、GFAP（0．0467±0．00333）,组间差异有统计学意义。 结论：光损伤大鼠视网膜片培养上清液可诱导大鼠MSCs分化为视网膜样细胞,为干细胞治疗视网膜变性疾病提供新思路。     

视黄酸联合视网膜细胞培养上清液对胚胎干细胞体外分化的诱导作用

目的：探讨经过初步诱导的拟胚体在视黄酸和视网膜细胞培养上清液作用下的分化特征。方法：将胚胎干细胞(embryonic stem cells,ESC)自液氮中复苏、培养，传1代后进行拟胚体(embry-onic bodies,EB)培养。3天半的EB离心重悬后不作消化，使用视黄酸、视黄酸加鼠视网膜胶质细胞和神经细胞培养上清液、视黄酸加人视网膜色素上皮培养上清液、视黄酸加人胎儿视网膜胶质细胞培养上清液(分别记为A、B、C、D组)等进行诱导。观察诱导过程中形态学改变，培养3周时使用免疫细胞化学检测Nestin、GFAP、cytokeratin、MAP-2、rhodopsin等在诱导后细胞中的表达情况。结果：(1)形态学改变：4种条件下的早期改变基本相同，均可见多种形态的细胞；3周后：拟胚体结构基本已散开，A组：见多种形态的细胞，细胞边界欠清，饱满度下降；B组：细胞以透明度较高的圆形细胞为主；C组：拟胚体及拟胚体周围的细胞中出现了含有明显色素的细胞；D组诱导：细胞胞体较大，形态结构较为单一；(2)免疫细胞化学：4种条件下均表现为大部分细胞MAP—2阳性，未见Nestin阳性细胞；此外，A组中，未见GFAP、Cytokeratin、Rhodopsin阳性细胞；B组中，可见GFAP、Cytokeratin、Rhodopsin阳性细胞；C组仅见Cytokeratin阳性细胞；D组仅见GFAP阳性细胞。结论：在KSC的次级诱导中，视黄酸可以诱导大部分细胞成为神经细胞，多种视网膜细胞的上清液在诱导中具有一定的作用，ESC能被诱导形成与上清液来源细胞相关的细胞。眼科学报2003；19：122-125     

骨髓间充质干细胞在视网膜色素变性大鼠视网膜下的分化

目的 研究骨髓间充质干细胞(MSCs)在视网膜色素变性(RP)大鼠体内的分化. 方法 Lewis大鼠腹腔注射3%NaIO3 100mg/kg,建立大鼠RP模型,将体外培养的MSCs植入视网膜下腔,用免疫荧光标记法对MSCs进行追踪,并观察术后第1、2、3、4、5周MSCs在该微环境中的分化.结果 术后第1周即可见MSCs位于视网膜色素上皮(RPE)层与光感受器细胞层,但全角蛋白(PCK)及Rhodopsin标记阴性,第3周开始可见MSCs在体内表达PCK及Rhodopsin.结论 MSCs植入RP模型大鼠视网膜下腔后可存活,主要分布于RPE层和视锥、视杆细胞层,并表达RPE细胞和光感受器细胞的表面标志.     

水蛭提取液对人视网膜母细胞瘤细胞的抑制作用

目的：研究水蛭提取液对人视网膜母细胞瘤WERI-RB-1细胞的抑制作用。

方法：采用不同浓度水蛭提取液(0.02、0.04、0.08、0.16U/mL)作用于体外培养的WERI-RB-1细胞0、24、48、72h,经CCK-8法筛选最佳药物干预浓度和时间进行后续实验。将体外培养的WERI-RB-1细胞分为对照组(正常培养基培养)和实验组(含水蛭提取液培养基培养),采用流式细胞仪检测药物对细胞周期和细胞凋亡的影响,Transwell侵袭实验检测药物对细胞侵袭能力的影响。

结果：根据CCK-8法检测结果选择0.04、0.08U/mL水蛭提取液作用48h为最佳干预条件进行实验。水蛭提取液干预的细胞主要阻滞在G2/M期,其中0.04、0.08U/mL实验组处于G2/M期的阳性细胞率分别为(12.59±5.36)%、(14.79±4.12)%,均明显高于对照组\〖(3.00±2.32)%,P<0.01\〗。水蛭提取液可诱导细胞凋亡,其中0.04、0.08U/mL实验组细胞凋亡率分别(37.91±3.44)%、(33.05±2.25)%,均明显高于对照组\〖(4.64±2.56)%,P<0.01\〗。Transwell侵袭实验检测结果显示,实验组Transwell小室下细胞数明显少于对照组,表明水蛭提取液能够抑制细胞侵袭。

结论：水蛭提取液在体外实验中能够抑制人视网膜母细胞瘤细胞的增殖、侵袭,并诱导细胞凋亡。     

视网膜神经节细胞无血清上清培养液诱导大鼠视网膜干细胞的分化

目的　探讨视网膜神经节细胞无血清上清培养液对视网膜干细胞分化的影响。方法　分离大鼠视网膜干细胞和视网膜神经节细胞;采用免疫荧光法鉴定体外培养的大鼠视网膜干细胞与视网膜神经节细胞,视网膜干细胞以Nestin抗体进行鉴定,视网膜神经节细胞以Thy-1抗体进行鉴定;以视网膜神经节细胞无血清上清培养液培养视网膜干细胞,以不加入条件培养液培养的视网膜干细胞为对照组,收集分化细胞,采用qPCR法检测Nestin、Pax6、Thy-1及Brn-3的基因表达。结果　培养的视网膜干细胞Nestin抗体染色阳性,视网膜神经节细胞Thy-1抗体染色阳性。培养视网膜干细胞72 h后,与对照组相比,无血清上清培养液组细胞Nestin和PAX6基因相对表达量降低,差异均有统计学意义（均为P<0.000 1）;Thy-1和Brn-3基因相对表达量升高,差异均有统计学意义（均为P<0.05）。结论　视网膜神经节细胞无血清上清培养液能够诱导视网膜干细胞分化为视网膜神经节样细胞。     

SD大鼠视网膜神经细胞培养上清液对胚胎干细胞体外分化的诱导作用

目的 探讨视网膜神经细胞培养的上清液对胚胎干细胞（embryonic stem cells, ES）体外分化的诱导作用。 方法 收集SD大鼠视网膜神经细胞培养上清液，抽滤后按2∶3比例与DMEM培养液混合，用该混合液进行ES 细胞的诱导分化，每天倒置相差显微镜观察ES细胞的生长及分化情况,对诱导分化后的细胞进行神经丝蛋白(nellcofilament protein，NFP)免疫组织化学检查。 结果 加入了视网膜神经细胞培养上清液的ES细胞生长出类似神经细胞突起样结构，NFP免疫组织化学染色阳性。 结论 SD大鼠视网膜神经细胞培养的上清液具有诱导ES细胞向神经细胞分化的作用。 (中华眼底病杂志, 2002, 18: 134-136)     

Caspase-1对氧诱导视网膜病变中小胶质细胞参与视网膜新生血管生成的促进作用及其机制

目的：探讨Caspase-1对小鼠氧诱导视网膜病变(oxygen-induced retinopathy,OIR)中小胶质细胞参与视网膜新生血管生成的作用及其机制。

方法：随机将12只7日龄(P7)C57BL/6J小鼠分为正常组、OIR组和OIR+VX-765组,正常组在正常氧环境中饲养,其余两组构建OIR模型; P12～P16,OIR+VX-765组和OIR组分别每天腹腔注射Caspase-1抑制剂VX-765(4mg/kg)和等量0.4%聚乙二醇(VX-765溶剂); 于P17制作视网膜铺片行Lectin染色,比较三组间视网膜无血管区和新生血管区面积的大小; 采用免疫荧光染色法观察视网膜组织中Caspase-1的表达和活化小胶质细胞的分布。培养小胶质细胞BV-2细胞分为对照组、缺氧组及抑制剂组,抑制剂组和缺氧组分别经VX-765和0.4%聚乙二醇预处理3h后,缺氧条件下培养24h; 对照组常规培养相同时间。通过Western blot检测Caspase-1、p20(Caspase-1活化形式)、IL-1β和VEGF的蛋白表达变化; 用各组BV-2细胞培养上清液作为条件培养基,刺激培养血管内皮细胞RF/6A,进行管腔形成和细胞迁移实验,并比较各组间的差异。

结果：P17正常组小鼠视网膜血管化完全,未见明显无血管区及视网膜新生血管; OIR组视网膜无血管区和新生血管面积百分比分别为16.58%±1.14%、4.00%±0.41%; OIR+VX-765组两者明显减少,分别为12.23%±1.02%和2.16%±0.52%(P<0.01)。免疫荧光染色结果显示,Caspase-1在正常小鼠视网膜组织中表达较弱,在OIR小鼠中主要在神经节细胞层和内丛状层有明显的阳性表达,并与活化的小胶质细胞有明确的共定位。Western blot检测结果显示,缺氧处理的培养小胶质细胞BV-2中Caspase-1、p20、IL-1β和VEGF蛋白表达明显提高,而Caspase-1抑制剂则可明显下调p20、IL-1β和VEGF的蛋白表达水平(P<0.05)。管腔形成和细胞迁移实验结果显示,RF/6A细胞经缺氧组BV-2培养上清液处理后,管腔形成长度和细胞迁移数目分别为271±12和347±34个,而加入抑制剂后,二者明显减少,分别为171±22和212±27个(P<0.05)。

结论：在小鼠OIR中,Caspase-1能够调节小胶质细胞促进视网膜新生血管的生成,其作用机制可能与Caspase-1活化小胶质细胞中其下游炎性效应分子IL-1β,并释放VEGF相关。     

氢对氧化应激诱导的视网膜衰老的保护机制

目的：探讨氢对氧化应激诱导的视网膜衰老的保护机制。

方法：将小鼠随机分为三组：对照组、模型组(NaIO₃处理组)和治疗组(H₂水灌胃组)。模型组通过鼠尾静脉注射NaIO₃溶液建立视网膜氧化应激损伤模型； 对照组小鼠注射生理盐水； 治疗组予富含H₂的饮用水灌胃后造模。利用SA-β-gal染色检测视网膜衰老情况。收集小鼠视网膜，western-blot检测DNA损伤反应相关蛋白ATM、NF-κB蛋白、细胞周期蛋白D1(Cyclin D1)和DNA修复相关蛋白HMGB1的表达。

结果：SA-β-gal染色显示，治疗组蓝绿色沉淀较模型组相比减少。western-blot结果显示治疗组中DNA损伤反应相关蛋白ATM、NF-κB、Cyclin D1相对表达量(0.10±0.009、0.32±0.01、0.19±0.002)较模型组(0.77±0.08、0.70±0.02、0.36±0.01)均显著降低，差异均具有统计学意义(均P<0.01)； 而治疗组中DNA修复相关蛋白HMGB1相对表达量(0.927±0.06)较模型组(0.383±0.07)显著升高，差异有统计学意义(P<0.01)。

结论：氢可以通过抑制氧化应激诱导的DNA损伤减弱视网膜衰老。     

永生化胚胎视网膜干细胞的体外诱导分化

目的：研究R71882永生化视网膜干细胞系细胞的体外诱导分化。方法：对R71882细胞系细胞进行体外培养扩增，利用含有表皮生长因子(epidermal growth factor，EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)及白血病抑制因子(leukemia inhibitory factor，LIF)等生长因子的DMEM／F12培养液对第43代细胞进行体外诱导分化，免疫荧光细胞化学鉴定诱导后细胞的抗原表达。结果：诱导后的R71882细胞系细胞增殖速度减慢，细胞表达微管相关蛋白2(microtubule-associated protein 2，MAP2)、胶质源性纤维酸性蛋白(glial fibrillary acid protein，GFAP)、蛋白激酶C-α(protein kinase C-α，PKC-α)、Thy1．1及Calretinin，同时部分细胞仍表达巢蛋白(Nestin)，但未见视紫红质(Rhodopsin)表达。结论：R71882细胞是一种多能视网膜干细胞。在EGF及bFGF等多种生长因子的共同作用下，该细胞系中部分细胞仍能表达未分化细胞标记物Nestin，部分细胞开始向神经元、神经胶质细胞及双极细胞、神经节细胞和无长突细胞等视网膜终末细胞方向分化。     

基因修饰诱导骨髓间充质干细胞分化治疗视神经损伤

目的：探讨基因修饰诱导骨髓间充质干细胞分化治疗视神经损伤的效果。

方法：将带有大鼠睫状神经营养因子(CNTF)编码序列的慢病毒转染进入从大鼠股骨中分离出的骨髓间充质干细胞。将采用钳夹视神经方法构建的视神经损伤模型大鼠随机分为对照组和研究组,分别于造模成功后第4、7、10、13d,研究组术眼玻璃体腔内注入经转染的骨髓间充质干细胞悬浮液,对照组术眼玻璃体腔注入等量生理盐水。造模成功后第14d,观察两组大鼠的光反射情况,视网膜神经节细胞数量及胶质原纤维酸性蛋白(GFAP)和Caspase-3蛋白表达水平。

结果：研究组大鼠光反射恢复率明显高于对照组(83% vs 25%,P<0.05),视网膜细胞结构相对整齐,可见少量空泡,而对照组大鼠视网膜细胞结构欠整齐,可见明显空泡,视网膜神经节细胞数明显减少,且对照组大鼠视网膜GFAP和Caspase-3蛋白表达水平均高于研究组。

结论：基因修饰诱导骨髓间充质干细胞分化能够有效治疗大鼠视神经损伤。     

Composition of the Inner Nuclear Layer in Human Retina

PurposeThe purpose of this study was to measure the composition of the inner nuclear layer (INL) in the central and peripheral human retina as foundation data for interpreting INL function and dysfunction.MethodsSix postmortem human donor retinas (male and female, aged 31–56 years) were sectioned along the temporal horizontal meridian. Sections were processed with immunofluorescent markers and imaged using high-resolution, multichannel fluorescence microscopy. The density of horizontal, bipolar, amacrine, and Müller cells was quantified between 1 and 12 mm eccentricity with appropriate adjustments for postreceptoral spatial displacements near the fovea.ResultsCone bipolar cells dominate the INL a with density near 50,000 cells/mm² at 1 mm eccentricity and integrated total ∼10 million cells up to 10 mm eccentricity. Outside central retina the spatial density of all cell populations falls but the neuronal makeup of the INL remains relatively constant: a decrease in the proportion of cone bipolar cells (from 52% at 1 mm to 37% at 10 mm) is balanced by an increasing proportion of rod bipolar cells (from 9% to 15%). The proportion of Müller cells near the fovea (17%) is lower than in the peripheral retina (27%).ConclusionsDespite large changes in the absolute density of INL cell populations across the retina, their proportions remain relatively constant. These data may have relevance for interpreting diagnostic signals such as the electroretinogram and optical coherence tomogram.     

Expression patterns of calretinin, calbindin and parvalbumin and their colocalization in neurons during development of Macaca monkey retina

The developmental expression of calbindin (CalB), calretinin (CaR) and parvalbumin (PV) was followed in Macaca monkey retina using single and double immunolabeling to identify which proteins provide distinctive labels for specific cell types and to clarify the role of these proteins during development. Ganglion cells (GC) expressed PV at fetal day (Fd)55 and CaR and CalB by Fd85. CaR was downregulated after birth. Separate subsets of amacrine (AM) cells expressed CaR and CalB at Fd65-70. After Fd115, many CaR+ AM coexpressed CalB. After Fd120 a few AM expressed PV and these added CaR and CalB after birth. A subset of horizontal cells (HZ) expressed CaR and CalB at Fd70. Slightly later all HZ express PV and CaR while the early subset is CalB+/PV+/CaR+. CaR downregulates in all HZ after birth. The DB3 cone bipolar cells (BP) under the HZ label for CalB by Fd90-110 while a probable OFF BP cell body just above the AM layer becomes CaR+ near birth with labeling increasing after birth. All cones outside of the fovea label for CalB by Fd125. Foveal cones, rods, most BP and Müller glia do not label for these proteins at any age. The complex patterns of up- and down-regulation found in Macaca retina are similar to previous reports of expression in human retina, but in many instances are quite different than earlier reports of CaR, CalB and PV expression patterns in monkey central visual centers. This makes it highly likely that each protein plays a specific but undetermined role(s) in each visual center, and that its expression is controlled at a given stage of retinal development by multiple intrinsic and extrinsic factors.     

角膜上皮干细胞定位及功能的研究进展

角膜上皮干细胞定位于角膜缘已被广泛认可,并被命名为角膜缘干细胞,且认为角膜缘干细胞在角膜上皮的自我更新和损伤修复中起重要作用.但近年来的一些研究却提出了与之相悖的观点:整个眼表层均含有寡能干细胞,且角膜缘干细胞在维持自我更新及稳态方面并未起到至关重要的作用.这些发现提示可能还有其他干细胞存在并维持着角膜上皮的自我更新,但也有人对这些新观点提出质疑.本文通过对最新研究的不同观点及证据的阐述,对近年来角膜上皮干细胞的定位及其功能等方面的进展作一综述.     

Neuroprotection of the Inner Retina Also Prevents Secondary Outer Retinal Pathology in a Mouse Model of Glaucoma

PurposeWe examined structural and functional changes in the outer retina of a mouse model of glaucoma. We examined whether these changes are a secondary consequence of damage in the inner retina and whether neuroprotection of the inner retina also prevents outer retinal changes.MethodsWe used an established microbead occlusion model of glaucoma whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to label rod and cone bipolar cells (BCs), horizontal cells (HCs), and retinal ganglion cells (RGCs), as well as synaptic components in control and glaucomatous eyes, to assess structural damage and cell loss. ERG recordings were made to assess outer retina function.ResultsWe found structural and functional damage of BCs, including significant cell loss and dendritic/axonal remodeling of HCs, following IOP elevation. The first significant loss of both BCs occurred at 4 to 5 weeks after microbead injection. However, early changes in the dendritic structure of RGCs were observed at 3 weeks, but significant changes in the rod BC axon terminal structure were not seen until 4 weeks. We found that protection of inner retinal neurons in glaucomatous eyes by pharmacological blockade of gap junctions or genetic ablation of connexin 36 largely prevented outer retinal damage.ConclusionsTogether, our results indicate that outer retinal impairments in glaucoma are a secondary sequalae of primary damage in the inner retina. The finding that neuroprotection of the inner retina can also prevent outer retinal damage has important implications with regard to the targets for effective neuroprotective therapy.     

树突状细胞在葡萄膜炎中的作用机制及临床应用前景

葡萄膜炎是主要以辅助性T细胞(Th)介导的自身免疫性疾病,是眼科常见的致盲眼病之一.树突状细胞(DCs)是目前已知的功能较强的专职抗原递呈细胞(APCs),能有效刺激初始型T细胞活化,是机体免疫应答的主要启动者,在自身免疫性疾病、移植排斥和调节免疫平衡中均发挥重要作用.DCs被认为是引起葡萄膜炎发病的起始细胞,可能是葡萄膜炎发生和发展的关键因素.近年来研究表明,DCs可以通过与多种免疫细胞相互作用,调节Th1、Th17与Th2的免疫平衡,诱导免疫耐受等,调节葡萄膜炎进程.DCs在葡萄膜炎中的作用逐渐受到重视.本文就DCs的生物学特性、DCs在葡萄膜组织中的生物学特点以及其在葡萄膜炎中的相关免疫调节机制和临床应用前景进行综述.     

A spatial analysis of on- and off-ganglion cells in the cat retina

Using reduced silver staining methods it was possible to stain all α-ganglion cells of the cat retina. The dendritic trees of α-cells are unistratified in either of two laminae within the inner plexiform layer. This stratification difference was shown physiologically to correspond to the on-off dichotomy. For all α-cells recorded, the dendritic field was covered by the corresponding receptive field centre. In addition the general shape of the receptive field centre corresponded to the shape of the dendritic field. The size of the dendritic tree was always smaller than the receptive field centre. The topographical distribution of on- and off-α cells could be studied. They were found to occur in about equal numbers. Both on- and off-α-cell perikarya form a regular lattice and both lattices are superimposed independently. The dendritic branches of neighbouring α-cells overlap and each retinal point is covered by the dendritic field of at least one on- and one off-α-cell.After horseradish peroxidase (HRP) injection into the lateral geniculate nucleus all β-cells were labelled. In this way it is shown that about 55% of all ganglion cells are β-cells. The mosaic of on- and off-β-cells was studied from the HRP-labelled material. It is commonly assumed that beta-cells are associated with the resolution of fine detail in the cat visual system. The mosaic of β-cells imposes some constraints and permits some predictions to be made with respect to the cat's visual discrimination.     

我国眼内干细胞研究现状与展望

干细胞为机体组织中的一个极小的未分化细胞亚群，具有增生、自我维持、自我更新、能产生大量具有功能的子代细胞。对胚胎干细胞、人成体视网膜干细胞及眼内肿瘤干细\胞研究发现，人胚胎及成体视网膜均存在有干细胞，眼内肿瘤如视网膜母细胞瘤、脉络膜黑色素瘤内也有肿瘤干细胞的存在；将不同干细胞移植到视网膜下或玻璃体腔，均能分化成神经元或视网膜结构。干细胞有望成为疾病发病机制及治疗研究突破的最新靶点。(中华眼底病杂志, 2007, 23: 83-86)     

Culture and identification of endothelial progenitor cells from human umbilical cord blood

AIM: To elucidate a simple method for isolating endothelial progenitor cells (EPCs) from human umbilical cord blood mononuclear cells and observe the endothelial cell-specific expression profile during proliferation and differentiation in vitro. · METHODS: Human umbilical cord blood were isolated by Percoll density gradient centrifugation from human cord blood and cultured in vitro. The adherent cells were then identified by immunohistochemical staining and flow cytometric analysis. CD34, vascular endothelial growth factor receptor-2 (VEGFR-2), EPCs specific antigen CD133, as well as endothelial cell specific markers CD31 and vWF were used. The cells were characterized by acetylated LDL(acLDL) up-taking and lectin binding by direct fluorescentstaining. · RESULTS: During culture, the attached cells exhibited spindle-shape in early stage, and gradually display endothelium- like cobblestone morphology with outgrowth. On day 7, flow cytometric analysis showed that the positive staining rate of attached cells for CD133, CD34 and VEGFR-2 were 17.8%±3.7%, 22.1%±4.4% and 81.5%±5.0%, respectively. While, immunohistochemical staining showed that the adherent cells were positive to CD31 and vWF at the rate of 92.7%±2.2% and 73.3%±4.2%, respectively. By direct fluorescentstaining, we observed that 83.0%±4.3% of the attached cells were double positive for DiI-acLDL and FITC-UEA-I. · CONCLUSION: EPCs can be separated from human cord blood under certain conditions in vitro. This observation may provide a basis for study of relationship between EPCs and retinal neovascularization, as well as further clinical application of EPCs in ischemic retinal lesions.     

人脐血内皮祖细胞体外培养和鉴定的研究(英文)

目的:阐述从人脐血单核细胞分离培养EPCs的方法,并观察EPCs体外增殖分化过程中内皮细胞特异性抗原的表达,为进一步研究EPCs在缺血性视网膜疾病的临床应用奠定基础。方法:采用密度梯度离心方法获得脐带血单个核细胞,体外进行诱导、分化和扩增,通过免疫组化和流式细胞仪分析等技术对脐血来源的EPCs进行鉴定,在我们的研究中主要分析CD34,血管内皮细胞生长因子受体-2(VEGFR-2),EPCs特异性抗原CD133,以及内皮细胞特异性抗原CD31和第八因子相关抗原(vWF)的表达情况。同时我们通过分析细胞摄取乙酰化低密度脂蛋白(acLDL)和结合植物凝集素的能力进一步鉴定细胞。结果:脐带血单个核细胞在培养早期主要为梭形细胞,逐渐呈现铺路石样外观;在细胞培养至第7d,贴壁细胞流式细胞仪分析显示,CD133, CD34和VEGFR-2的阳性率分别为17.8%±3.7%, 22.1%±4.4%和81.5%±5.0% ;免疫组化染色结果显示CD31、vWF的表达率分别为92.7%±2.2%和73.3%±4.2%;免疫荧光染色结果表明83.0%±4.3%的贴壁细胞DiI-acLDL和FITC-UEA-I染色均阳性。结论:本实验证明在体外特定的培养条件可以从脐带血单个核细胞中分离培养出EPCs,为进一步研究EPCs与视网膜新生血管的关系以及EPCs治疗缺血性眼底的临床研究奠定了基础。