大鼠骨髓间充质干细胞视网膜下移植的短期观察

目的探索骨髓间充质干细胞（MSCs）视网膜移植的可行性。方法采用贴壁筛选法分离、培养SD大鼠骨髓MSCs，将溴脱氧尿嘧啶（BrdU）标记的大鼠MSCs悬液经玻璃体腔注入大鼠视网膜下腔。术后14d处死大鼠，取新鲜眼球，连续冰冻切片，经BrdU单抗、FITC标记二抗和Rhodopsin单抗、Cy3标记二抗免疫荧光双重标记后，荧光显微镜下观察。结果荧光显微镜下可见移植后的MSCs主要分布于视网膜色素上皮层和视锥、视杆细胞层，未表达视网膜光感受器细胞的特异性抗原视紫红质。结论移植14d后MSCs可在视网膜下腔存活，主要分布于视网膜色素上皮层和视锥、视杆细胞层，未分化成视网膜光感受器细胞。     

鼠光感受器细胞层移植的方法及观察

目的 研究视锥和视杆细胞与周围组织间在解剖结构及是生理等方面的关系。方法 取２０只同龄Ｗｉｓｔａｒ和ＲＣＳ鼠作为供体和受体,利用视见解发削技术取得视锥及视杆细胞层,并植入ＲＣＳ鼠眼视网膜下。术后２周时摘除术眼,于光镜下观察交摄影。结果 术后２周,在视网膜完全复位的受体眼,移植的纯视锥和视杆细胞成活,可见移植细胞位于网膜下腔的视网膜色素上皮（ｒｅｔｉｎａｌ ｐｉｇｍｅｎｔ ｅｐｉｔｈｅｌｉｕｍ,ＲＰ     

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.     

鼠视网膜感光细胞移植术后超微结构观察

目的 以遗传性视细胞变性动物ＲＣＳ鼠为受体,Ｗｉｓｔａｒ鼠为供体,观察纯视锥、杆细胞移植术后ＲＣＳ鼠视网膜外层超微结构的变化。方法 应用视网膜板层切削技术或准分子激光技术制备供体层状视细胞;视网膜板层外路移植技术,获得视网膜移植动物模型。术后２周及４周时处死动物,眼球常规切片后于光镜及透射电镜下观察受体视网膜。结果 移植的视细胞大多成层排列于受体鼠的视网膜色素上皮层及内颗粒层之间。重新出现的外网状     

小鼠实验性视网膜脱离后光感受器细胞的凋亡

目的 研究小鼠实验性视网膜脱离后光感受器细胞的凋亡情况。 方法 将成年C57Bl/6J小鼠36只分为2组：实验组小鼠18只左眼视网膜下注射1.4%透明质酸钠造成视网膜脱离，对照组小鼠18只左眼仅作巩膜穿刺。分别于手术后1、3、7和28 d摘除眼球，视网膜切片进行组织化学、免疫荧光染色，共聚焦显微镜检查。抗视锥和抗视杆细胞的抗体分别标记视锥和视杆细胞，dUTP缺口末段标记法(TUNEL)标记凋亡细胞。通过计数存活和凋亡的视锥和视杆细胞来定量光感受器细胞的凋亡和细胞丢失。 结果 凋亡细胞只存在于脱离部分视网膜的外核层，凋亡细胞在视网膜脱离后1 d即可检测得到，3 d时达到高峰，7 d后陡然减少。视网膜脱离后视杆和视锥细胞的死亡呈现同样的时程。 结论 凋亡是视网膜脱离后光感受器细胞死亡的主要病理改变。 (中华眼底病杂志, 2006, 22: 124-127)     

斑马鱼视网膜感光细胞的发育及视蛋白的表达

目的研究斑马鱼视网膜感光细胞的发育和视蛋白的表达，明确利用斑马鱼研究视网膜和感光细胞的可行性。方法制备斑马鱼视网膜感光细胞视蛋白（视杆细胞视紫质、视锥细胞紫外线视蛋白和视锥细胞蓝色视蛋白）的RNA探针。收集受精后72，96，120h的斑马鱼眼球组织进行切片、电镜观察和整体原位杂交。结果斑马鱼的神经视网膜分层排列，包括3个细胞层和2个丛状层。随时间发展，视网膜的发育逐渐成熟，3个细胞层的细胞排列更加整齐，感光细胞的外节盘发育更加成熟，3种视蛋白的表达逐渐增强，范围扩大。结论利用斑马鱼进行视网膜和感光细胞的研究是可行的，视蛋白可作为感光细胞的标记。     

玻璃体内注射他克莫司对异种移植的视网膜色素上皮细胞存活的影响

目的 观察少量、多次玻璃体内注射免疫抑制剂他克莫司（FK506）对移植到兔眼视网膜下间隙内的人视网膜色素上皮（retinal pigment epithelium, RPE）细胞存活情况的影响。 方法 采用绿色荧光蛋白（green fluorescent protein, GFP）逆转录病毒感 染人RPE细胞。将50 μl（4×10³个/μl）表达GFP的人RPE细胞悬液注射到18只白兔及1 0只灰兔双眼视网膜下间隙，手术后所有兔左眼玻璃体内注射5 μl FK506（5 μg/ μl），每周1次，连续5周，然后间周一次至20周;右眼不注射作为对照。眼球壁铺片倒置荧光 显微镜观察移植细胞的存活状态。 结果 白兔于移植后1、2、3、4 、6、10、11、14、18、20、23、24、25、33、54周，灰兔于移植后4、5、6、7、14、18、20、26周双眼视网膜下均可见表达的GFP细胞，但移植术后1~14周内玻璃体内注射了FK506的左眼视网膜下RPE-GFP细胞形态、细胞膜的完整程度均比未注射的右眼好。18周后，7只白兔和3只灰兔双眼视网膜下移植细胞的状态差异不明显。眼球切片苏木素-伊红（hematoxylin-eosin, HE）染色观察结果显示，移植术后1~6周，6只白兔和3只灰兔右眼脉络膜小血管周围可见灶性或弥散的淋巴细胞，应用了FK506的左眼淋巴细胞的浸润明显减少。 结论 移植术后早期玻璃体内使用小剂量免疫抑制剂可减轻局部炎症反应，有利于维持视网膜下间隙移植的异种细胞的存活。 （中华眼底病杂志，2003，19：333-404）     

胚胎干细胞源性视网膜前体细胞在rd鼠视网膜下腔移植后的分化

目的 研究绿色荧光蛋白标记的胚胎干细胞(green fluorescent protein-embryonic stem cells,GFP-ESC)源性视网膜前体细胞在rd小鼠视网膜下腔移植后的存活分化情况.方法 将GFP-ESC 源性视网膜前体细胞移植到伴有视网膜感光细胞变性和继发性视网膜神经节细胞变性的rd 小鼠视网膜下腔,移植后1周、4周、8周取材行免疫荧光细胞化学检测,观察移植细胞存活、分化情况.对移植细胞进行核型分析,并接种裸鼠眼内观察是否成瘤,评价其安全性.结果 GFP-ESC 源性视网膜前体细胞移植眼内可存活、整合到宿主变性视网膜层间.1周时 GFP 阳性细胞移行于外层视网膜并呈视杆细胞标志抗原rhodop-sin阳性:4周时整合于内层视网膜,共表达成熟神经元标志抗原神经丝蛋白-200、微管相关蛋白-2,突触标志抗原synaptophysin检测阳性;8周时移至邻近玻璃体腔形成节细胞样结构,表达节细胞标志抗原Thy1.1;移植细胞维持正常二倍体核型,术后8周未见排斥反应及瘤性增殖.结论 GFP-ESC源性视网膜前体细胞在变性视网膜中可存活、整合,并有优先向变性的感光细胞及.神经节细胞类型分化的倾向,有望为青光眼等视神经视网膜病变的细胞移植治疗提供安全有效的种子细胞.     

视网膜下间隙移植经基因修饰的视网膜色素上皮细胞对大鼠光感受器变性及视功能的保护作用

目的 以RCS大鼠作模型,研究经基因修饰的永生化视网膜色素上皮细胞(RPE)视网膜下移植对光感受器变性的保护作用。方法 在绿色荧光蛋白基因逆转录病毒感染的基础上,利用脂质体介导节状神经生长因子(CNTF)表达质粒转移,修饰成人RPE细胞系CRL-2302。将1×105个表达绿色荧光蛋白(GFP)或GFP及CNTF的RPE细胞移植到4～5周龄RCS大鼠右眼视网膜下间隙,左眼不移植或注射。PBS作为对照。术后2、4、6、8、10和12周作荧光显微镜、光镜、电镜及电生理检查。结果 荧光显微镜观察,术后1周移植的人RPE细胞在RCS大鼠视网膜下间隙已扩散到几乎整个眼底,但随时间延长移植的细胞逐渐减少,术后6周仅残留少量移植细胞。光镜及电镜观察显示移植眼保留的光感受器数量明显较对照眼多,凋亡细胞则较对照跟少。此外,移植眼宿主RPE细胞形态较正常,并可见吞噬小体。视网膜电图(ERG)检查结果表明部分移植眼视网膜功能明显较对照眼好。结论 经过基因修饰的RPE细胞移植可延缓RCS大鼠视网膜光感受器变性,为治疗视网膜变性提供了新的途径。(中华眼科杂志,2004,40:552-556)     

锂对视网膜色素变性小鼠光感受器细胞凋亡的保护作用

目的 研究锂在视网膜色素变性小鼠模型上对光感受器细胞凋亡的神经保护作用.方法 实验研究.FVB/NJ视网膜色素变性小鼠出生后立刻用含锂的食物喂养,7和14 d时摘除眼球做视网膜冰冻切片,同时取血测血清锂浓度.HE、TUNEL和视杆细胞免疫荧光染色进行视网膜组织形态、光感受器细胞凋亡分析.结果 光镜下可见,对照组外核层可见TUNEL阳性细胞,出生后14 d外核层厚度和细胞层数(3或4层)明显低于7 d时的厚度(9或10层),而锂喂养组出生后14 d外核层的厚度和细胞层数明显高于对照组,与出生7 d时的外核层厚度及细胞层数无明显差异.结论 锂能够保护视网膜色素变性小鼠光感受器细胞免于凋亡.(中华眼科杂志,2008,44:248-252)     

Differentiation of rat mesenchymal stem cells transplanted into the subretinal space of sodium iodate-injected rats

Purpose: The differentiation of rat bone marrow mesenchymal stem cells (MSCs) was investigated in a retinal pigment epithelium (RPE) damage model induced by the administration of sodium iodate. Methods: Cultured rat MSCs were transfected with enhanced green fluorescent protein and transplanted into the subretinal space of rats injected 4 days earlier with sodium iodate. Immunofluorescence analysis was performed 5 weeks later. Results: The transduction efficiency was 99.9%. Viable MSCs were detected 5 weeks after transplantation, mainly in the subretinal space. The cells expressed pan‐cytokeratin, glial fibrillary acidic protein and rhodopsin. Conclusions: Bone marrow MSCs transplanted into the subretinal space of sodium iodate‐injected rats have the ability to differentiate into RPE, photoreceptor and glial lineage cells.     

Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats

PURPOSE: To determine the potential of adenovirally transduced bone marrow stromal cells (BMSCs) to differentiate into retinal pigment epithelial-like cells and to evaluabe possible rescue effects after transplantation into the retinas of Royal College of Surgeons (RCS) rats. METHODS: Through a high-capacity adenoviral vector expressing either green fluorescent protein (GFP) or pigment epithelial-derived factor (PEDF), rat MSCs were transduced in vitro before subretinal transplantation into Wistar rats or, alternatively, RCS rats. Two months after cell injection, the rats were killed and the eyes enucleated. The eyes were then investigated light microscopically or processed for electron microscopic investigations. Cell differentiation and integration were analyzed immunocytochemically using antibodies against cytokeratin and the tight junction protein ZO-1. Electroretinography was performed 16 days after injection of cells, to check whether a functional rescue could be detected. RESULTS: In vitro experiments in cocultured human MSCs and human RPE cells showed that MSCs adopted RPE-like characteristics. In grafting experiments, some rat MSCs integrate into the host RPE cell layer of Wistar and RCS rats, indicated by their hexagonal morphology. Subretinally transplanted cells express the epithelial marker cytokeratin and establish tight junctions with the host RPE cells. Furthermore, rescue effects can be demonstrated after grafting of vector-transduced and nontransduced MSCs in semithin sections of dystrophic retinas. Ultrastructurally, MSCs can be detected on top of host RPE and in close contact with photoreceptor outer segments phagocytosing rod outer segments. CONCLUSIONS: Taken together, these results raise the possibility that MSCs have the potency to replace diseased RPE cells and deliver therapeutic proteins into the subretinal space to protect photoreceptor cells from degeneration.     

Characteristics of embryonic retina transplanted to rat and rabbit retina

When aggregates of immature retina are transplanted to lesioned adult retina, the donor cells reorganize themselves into folded sheets and rosettes. With the exception of retinal ganglion cells and an inner limiting membrane, most cell types and all layers develop, corresponding to the normal retina. The transplants can integrate with the adult host retina without the presence of glial barriers. Mouse and human donor retinas have been transplanted to immunosuppressed rat hosts with long-term survival. Transplants of embryonic retina cografted with RPE can contain an apparent ganglion cell layer and an inner limiting membrane, which are not observed in transplants of retina alone. Embryonic rat donor retinas can be transplanted successfully after long-term storage in liquid nitrogen.     

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（MSC）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30g/L NaIO₃ 100mg/kg,建立大鼠RP模型,将体外培养的MSC植入视网膜下腔,用免疫荧光标记的方法对MSC进行追踪,并观察术后第1,2,3,4,5wk MSC在该微环境中的分化。结果:术后第1wk即可见MSC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3wk开始可见MSC在体内表达PCK及rhodopsin。结论:MSC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。     

Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate

PURPOSE: To characterize the potential of newborn retinal stem cells (RSCs) isolated from the radial glia population to integrate the retina, this study was conducted to investigate the fate of in vitro expanded RSCs transplanted into retinas devoid of photoreceptors (adult rd1 and old VPP mice and rhodopsin-mutated transgenic mice) or partially degenerated retina (adult VPP mice) retinas. METHODS: Populations of RSCs and progenitor cells were isolated either from DBA2J newborn mice and labeled with the red lipophilic fluorescent dye (PKH26) or from GFP (green fluorescent protein) transgenic mice. After expansion in EGF+FGF2 (epidermal growth factor+fibroblast growth factor), cells were transplanted intravitreally or subretinally into the eyes of adult wild-type, transgenic mice undergoing slow (VPP strain) or rapid (rd1 strain) retinal degeneration. RESULTS: Only limited migration and differentiation of the cells were observed in normal mice injected subretinally or in VPP and rd1 mice injected intravitreally. After subretinal injection in old VPP mice, transplanted cells massively migrated into the ganglion cell layer and, at 1 and 4 weeks after injection, harbored neuronal and glial markers expressed locally, such as beta-tubulin-III, NeuN, Brn3b, or glial fibrillary acidic protein (GFAP), with a marked preference for the glial phenotype. In adult VPP retinas, the grafted cells behaved similarly. Few grafted cells stayed in the degenerating outer nuclear layer (ONL). These cells were, in rare cases, positive for rhodopsin or recoverin, markers specific for photoreceptors and some bipolar cells. CONCLUSIONS: These results show that the grafted cells preferentially integrate into the GCL and IPL and express ganglion cell or glial markers, thus exhibiting migratory and differentiation preferences when injected subretinally. It also appears that the retina, whether partially degenerated or already degenerated, does not provide signals to induce massive differentiation of RSCs into photoreceptors. This observation suggests that a predifferentiation of RSCs into photoreceptors before transplantation may be necessary to obtain graft integration in the ONL.     

Differentiation of mesenchymal stem cell in the microenviroment of retinitis pigmentosa

AIM: To access the differentiation of rat mesenchymal stem cell (MSC) in the microenvironment of retinal degeneration induced by the administration of sodium iodate. METHODS: In-vitro cultured Lewis rat MSC were injected into the sub-retinal space of NaIO3 induced retinal degeneration rat eyes (30g/L NaIO3 100mg/kg). To observe the trace and differentiation of MSC by immuno-fluorescent method successively in 5 weeks after the surgery. RESULTS: The majority of the transplanted cells stay in retinal pigment epithelium layer and cones & rods layer. From the 2nd week after transplantation, the engrafted MSC express PCK and rhodopsin under fluorescent microscope. CONCLUSION: MSC can survive mainly in the outer layer of retina in the microenvironment of retinal degeneration and differentiate forward the RPE cell and photoreceptor.     

N-cadherin expression in a rat model of retinal detachment and reattachment

PURPOSE: To observe the changes in N-cadherin expression in the retina after experimental retinal detachment (RD) and reattachment in the rat and to explore the role N-cadherin might play after RD. METHODS: Forty rat retinas were detached by transscleral injection of 1.4% sodium hyaluronate into the subretinal space. The eyes were enucleated at different time intervals (n = 5), followed by fixation, embedding, and sectioning. The differences in N-cadherin expression in the normal retina, detached retina, and spontaneously reattached retina were determined. Furthermore, an N-cadherin antagonist was injected in combination with 1.4% sodium hyaluronate into the subretinal space in another 10 eyes, in an attempt to demonstrate the role N-cadherin plays after RD. RESULTS: N-cadherin was not expressed in the RPE layer of the normal rat retina. After RD, intense immunolabeling of N-cadherin was seen in the RPE cells, the photoreceptors, and the outer limiting membrane (OLM). An increasing number of cytokeratin (CK)-positive cells likely to be RPE cells was found attached to the outer surface of the detached neural retina. Where the retina was reattached, the N-cadherin immunolabeling rapidly decreased. In eyes treated with an N-cadherin antagonist, the retinas appeared thinner than that in eyes without treatment, and the photoreceptor nuclei showed significantly loss. Moreover, CK-positive cells attached to the outer surface of the detached retina were markedly fewer in number. CONCLUSIONS: Increased expression of N-cadherin in the RPE cells, the photoreceptor cells, and the OLM of the retina after RD may contribute to RPE cell migration and photoreceptor survival. These changes could be reversed by retinal reattachment.