视网膜色素上皮细胞诱导骨髓间充质干细胞分化为神经样细胞

目的：检测视网膜色素上皮细胞对骨髓间充质干细胞分化的调节作用。方法：体外培养人骨髓间充质干细胞（BMSC）和视网膜色素上皮（RPE）细胞。RPE细胞采用紫外线照射处理,而后与CFSE标记的BMSC共培养14d。并在共培养体系中加入牛眼视网膜提取物（BRE）以研究视网膜成分对此分化过程的影响。采用NSE,Nestin和GFAP抗体标记检测BMSC分化前后的表达特征。结果：BMSC在与RPE细胞共培养后,能够分化成为神经样细胞,并表达神经性细胞的特异性标记NSE．Nestin和GFAP。BRE能够显著促进共培养体系中BMSC向神经样细胞的分化。结论：RPE和BRE能够诱导BMSC分化为神经样细胞。     

视网膜色素上皮细胞诱导骨髓间充质干细胞分化为神经样细胞

目的:检测视网膜色素上皮细胞对骨髓间充质干细胞分化的调节作用。方法:体外培养人骨髓间充质干细胞(BMSC)和视网膜色素上皮(RPE)细胞。RPE细胞采用紫外线照射处理,而后与CFSE标记的BMSC共培养14d。并在共培养体系中加入牛眼视网膜提取物(BRE)以研究视网膜成分对此分化过程的影响。采用NSE,Nestin和GFAP抗体标记检测BMSC分化前后的表达特征。结果:BMSC在与RPE细胞共培养后,能够分化成为神经样细胞,并表达神经性细胞的特异性标记NSE,Nestin和GFAP。BRE能够显著促进共培养体系中BMSC向神经样细胞的分化。结论:RPE和BRE能够诱导BMSC分化为神经样细胞。     

睫状缘色素细胞与骨髓间充质干细胞体外共培养构建视网膜干细胞的初步研究

目的 探索睫状缘色素细胞(pigmented cells from the ciliary margin,PCM)与骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM)体外共培养构建视网膜干细胞的可行性.方法 按照组织贴壁培养方法分别分离培养原代大鼠BM和PCM,再将二者进行直接共培养(1∶2),观察细胞生长情况;MTT方法进行增殖能力检测;在促神经分化诱导液中诱导21 d后行免疫荧光染色,观察视网膜干细胞相关分子标记物包括视杆细胞Rho1 D4、双极神经元CHX10和Müller胶质细胞10E4的表达.结果原代分离培养的两种细胞生长状态良好;共培养后细胞总体的增殖活性虽然显著低于单纯BM,但比单纯PCM有了一定提升;诱导分化后单纯PCM视网膜干细胞相关分子标记物表达阳性率显著高于BM,而共培养后的细胞表达阳性率显著高于单纯PCM和BM.结论 采用BM与PCM共培养能够获得大量表达视网膜干细胞相关分子标记物的细胞群,有望成为视网膜干细胞来源,用于视神经损伤修复.     

骨髓间充质干细胞向视网膜细胞诱导分化的研究

骨髓间充质干细胞作为成体干细胞的一种,具备自我更新能力和多向分化潜能的特性,在适当条件下能分化为骨、肌肉、脂肪、神经等多谱系细胞.在不同诱导环境下,骨髓间充质干细胞具有向视网膜神经细胞、视网膜色素上皮细胞、光感受器细胞等视网膜细胞分化的潜能,为视网膜疾病的治疗提供新思路.     

抑制消减杂交技术筛选骨髓间充质干细胞与视网膜神经节样细胞差异表达基因

目的　抑制消减杂交技术筛选和克隆视网膜神经节(RGC)样细胞与骨髓间充质干细胞的差异表达基因。方法　提取RGC样细胞总RNA为检测子,骨髓间充质干细胞的总RNA为驱动子,进而用抑制消减杂交技术方法获得消减杂交产物。结果　本消减文库共挑取克隆120个,经聚合酶链式反应（PCR）鉴定,其中含有插入片段的阳性克隆有20个。在这些阳性克隆中,PCR扩增片段大小分布于250~1500 bp。共得到5个差异基因片段,这些差异表达基因与视觉信号转导、神经细胞再生等功能有关。结论　抑制消减杂交技术有效地克隆了骨髓间充质干细胞诱导前后差异表达的基因。     

大鼠骨髓间充质干细胞分离培养方法的比较

目的:探讨体外分离培养高纯度、高活力大鼠骨髓间充质干细胞(MMSC)的方法,为眼科组织工程提供种子细胞。方法:取清洁级SD大鼠,改良贴壁筛选法分离培养MMSC,采用MTT法检测MMSC活性,流式细胞术检测MMSC纯度。对比改良贴壁筛选法与密度梯度离心法,剪去干骺端法与骨干中间剪断法,不同月龄大鼠MMSC活性和纯度(CD44 CD34-,CD90 CD34-)的差别。结果:改良贴壁筛选法比密度梯度离心法获取的MMSC细胞数量多,细胞纯度差异无明显统计学意义。剪去干骺端法比骨干中间剪断法获取的MMSC细胞数量少,细胞纯度差异无明显统计学意义。在相同接种密度条件下,MMSC的增殖活力随大鼠年龄的增长而下降。结论:改良贴壁筛选法是一种简便易行的培养高活力、高纯度MMSC的方法。     

小分子化合物在干细胞诱导分化为视网膜色素上皮细胞中的应用

视网膜色素上皮细胞的变性、坏死将导致患者的视力丧失。由于视网膜色素上皮细胞不能再生,移植健康的视网膜色素上皮细胞已成为视网膜变性性疾病最具有前景的治疗策略。随着化学、分子生物学和再生医学的发展,人们发现越来越多的小分子化合物通过调控某些特定的信号通路,能够操纵干细胞的分化方向。本文主要对小分子化合物在体外诱导干细胞定向分化为视网膜色素上皮细胞的应用作一综述。     

Differentiation of human olfactory mucosa mesenchymal stem cells into photoreceptor cells in vitro

AIM: To investigate whether the human olfactory mucosa mesenchymal stem cells (OM-MSCs) can differentiate into photoreceptor cells in vitro. METHODS: Through the olfactory mucosa adherent method, olfactory mucosa was isolated, cultured and identified in vitro among mesenchymal stem cells. The cell surface markers were analyzed by flow cytometry, induced to differentiate into retinal photoreceptor cells in vitro, and the expression of rhodopsin was observed and identified by Immunofluorescence and Western blot methods. RESULTS: OM-MSCs from human were spindle cell-based, and showing radial colony arrangement. OM-MSCs were negative for CD34, CD45 and CD105, but positive for CD73 and CD90. Following induction, a strong positive reaction was produced by photoreceptor specific marker rhodopsin in the cells.     

经角膜基质细胞诱导的大鼠骨髓间充质干细胞在人羊膜上构建角膜上皮移植片

目的:研究将骨髓间充质干细胞(mesenchymal stem cells,MSC)诱导分化后在羊膜表面培养构建角膜上皮移植片的可行性。方法:取SD大鼠骨髓间充质干细胞和角膜基质细胞,分别培养并传2代后进行共培养,取共培养7d后的MSC覆载于新鲜人羊膜,再培养7d。对MSC及诱导后MSC进行免疫荧光染色和扫描电镜检查;对覆载细胞的羊膜进行HE染色及免疫荧光染色。结果:MSC在体外培养条件下贴壁生长,免疫荧光染色CD29和CD44阳性,CK12阴性。经角膜基质细胞诱导分化后,MSC细胞CK12染色转为阳性。诱导后MSC接种到羊膜表面,迅速贴壁生长,组织学特性无明显改变,CK12染色仍为阳性。结论:经角膜基质细胞诱导的MSC表现出角膜上皮细胞特征,在羊膜上生长后保持不变。     

视网膜细胞联合bFGF体外诱导大鼠骨髓间充质干细胞向神经样细胞的分化

目的 探讨大鼠骨髓间充质干细胞（BMSC）在体外分化为神经样细胞的有效途径,从而解决体外诱导分化效率低及存活状况不佳等问题.方法 采用密度梯度离心法和贴壁筛选法分离BMSC,免疫组化检测CD31、CD44、CD45、CD105表达并对细胞进行鉴定.按照诱导方式的不同分为3组：视网膜细胞＋碱性成纤维细胞生长因子（bFGF）组、bFGF组、不加诱导液组,分别诱导大鼠BMSC向神经样细胞分化.于诱导后第3、第7、第14天分别进行细胞形态学观察;并采用免疫细胞化学法检测神经微管蛋白-βⅢ（Tui1）、神经元特异性烯醇化酶（NSE）和胶质纤维酸性蛋白（GFAP）的表达,分析阳性细胞率：采用MTT法检测诱导后BMSC增殖状况,比较细胞存活率.相同时间组间比较用两独立样本t检验,组内不同时间比较用单因素方差分析.结果 形态学观察：视网膜细胞＋hFGF组诱导BMSC 12 h后出现形态变化,逐步形成典型的神经样细胞：bFGF阳性对照组也可见形成突起结构,但无典型的神经样细胞形态.免疫组化检测：处理组诱导3 d后即可检测到阳性细胞,随着诱导时间的延长.Tui1、NSE和GFAP阳性细胞率增加,与阳性对照组比较差异有统计学意义（P〈0.01）;阴性对照组未发现阳性细胞.存活率：各组BMSC存活率随着时间延长逐渐下降,但不同诱导方法对BMSC存活无显著影响.结论 模拟视网膜微环境配合bFCF能够诱导大鼠BMSC分化为神经样细胞并继续存活.     

大鼠骨髓间充质干细胞体外可诱导分化为角膜上皮细胞

目的:探讨骨髓间充质干细胞 (mesenchymal stem cell,MSC) 分化为角膜上皮细胞的可塑性及其重建角膜上皮的可能性.方法:用密度梯度离心法结合贴壁培养法分离纯化大鼠骨髓MSC,经体外与角膜基质细胞共培养诱导分化,免疫荧光法检测角膜上皮细胞特异标志物K12的表达.结果:体外培养的大鼠骨髓MSC表现出很强的增殖潜能,原代培养的骨髓MSC CD29免疫荧光染色阳性,CD34和CD45为阴性,符合骨髓MSC的特征.MSC与角膜基质细胞共培养1wk后大部分细胞分化为间质细胞,少部分细胞形态上相对偏小,免疫荧光检测这部分细胞表达角膜上皮细胞特异性标志角蛋白K12.结论:体外培养的MSC在角膜基质细胞的诱导下可横向分化为角膜上皮细胞.     

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.     

内皮素对人视网膜色素上皮细胞黏附的影响

目的 探讨内皮素-1(endothelin-1,ET-1)对人视网膜色素上皮细胞(retinal pigment epithelium,RPE)黏附作用的影响.方法 用RPE黏附实验和放射免疫测定法检测不同浓度ET-1对RPE黏附的影响和6-Keto-PGFal的浓度变化.结果 10-11～10-mol·L-1～ET-1对RPE细胞黏附力呈剂量依赖性增加,而RPE细胞黏附力随着ET-1浓度的进一步增加(10-9～10～mol·L-1)呈剂量依赖性下降(F=6.352,P＜0.01);黏附实验上清液中6-Keto-PGFα1含量随着ET-1浓度增加呈浓度依赖性增加(F=33.929,P＜0.01).结论 ET-1对RPE有促进黏附作用,ET-1刺激后6-Keto-PGFα1含量上升,与RPE细胞对ET-1作出的反应有关,并抑制ET-1作用.     

骨髓间充质干细胞在视网膜移植领域的研究进展

临床上对于视网膜退行性疾病的治疗仍然只停留在改善循环、营养神经等支持治疗阶段,缺少特效的治疗方法,骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)是具有较强自我复制功能和多向分化潜能的一类细胞,人们开始通过尝试不同的分离、诱导、移植等手段,以实现其向视网膜细胞的转化,并最终实现体内的替代治疗。我们就BMSCs在视网膜移植领域的研究进展进行综述,以期为进一步的研究提供参考。     

缺氧诱导体外培养人视网膜色素上皮细胞凋亡

目的:了解缺氧条件下体外培养人视网膜色素上皮(retinal pigment epithelium,RPE)细胞的凋亡情况。方法:将培养的人RPE细胞置于含10mL/LO2、50mL/LCO2和940mL/LN2的培养箱内建立缺氧模型。于缺氧后1,3,6,12,24h,利用扫描电镜、透射电镜、脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucle-otidyl transferase mediated nick end labeling,TUNEL)技术和流式细胞仪测定RPE细胞凋亡水平。结果:常氧状态下RPE细胞生长良好,几乎没有凋亡(TUNEL法测凋亡指数为1.2)。缺氧条件下,RPE细胞发生了不同程度的凋亡,缺氧后3h凋亡水平达峰值(凋亡指数为34.43)。结论:缺氧可导致体外培养的人RPE细胞凋亡,提示RPE凋亡可能参与了某些缺血缺氧性眼病的发生。     

Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration

Because there is no effective treatment for this retinal degeneration, potential application of cell-based therapy has attracted considerable attention. Several investigations support that bone marrow mesenchymal stem cells (MSCs) can be used for a broad spectrum of indications. Bone marrow MSCs exert their therapeutic effect in part by secreting trophic factors to promote cell survival. The current study investigates whether bone marrow MSCs secrete factor(s) to promote photoreceptor cell survival and whether subretinal transplantation of bone marrow MSCs promotes photoreceptor survival in a retinal degeneration model using Royal College of Surgeons (RCS) rats. In vitro, using mouse retinal cell culture, it was demonstrated that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that the secreted factor(s) from the MSCs promote photoreceptor cell survival. In vivo, the MSCs were injected into the subretinal space of the RCS rats and histological analysis, real-time RT-PCR and electrophysiological analysis demonstrated that the subretinal transplantation of MSCs delays retinal degeneration and preserves retinal function in the RCS rats. These results suggest that MSC is a useful cell source for cell-replacement therapy for some forms of retinal degeneration.     

Modulation of retinal wound healing by systemically administered bone marrow-derived mesenchymal stem cells

Purpose

To evaluate whether systemically injected bone marrow-derived mesenchymal stem cells (MSCs) can be incorporated into neuroretinal tissues and play an important role in retinal wound healing in the laser-induced retinal trauma model.

Methods

Retinotomies were made by applying an Nd:YAG laser to rat retina. On the first day after the injuries, cell suspensions that were obtained from the same line of rat (containing 1 × 10⁶ green fluorescence protein [GFP]-marked bone marrow-derived MSCs) were injected through a tail vein in the experimental group and phosphate buffer solution (PBS) was injected in the same way in the control group. Fundus photographs were taken serially for fundus examination and eyeballs were enucleated for histological studies that were conducted at five and seven weeks after MSC and PBS injection. After the tissues were prepared, the retinotomy sites were observed with routine histological staining and confocal microscopy.

Results

Retinal detachment resolved in the experimental group, whereas it progressed in the control group. The retinotomy sites closed partially with identifiable GFP positive cells 5 weeks after MSC injection. At 7 weeks after MSC injection, complete healing without retinal detachment and plentiful GFP positive cells were observed at the transitional zone between damaged and normal retina.

Conclusions

Systemically administered GFP-marked MSCs may be incorporated into the neuroretinal tissues and play an important role in the wound modulation of physically damaged retinal tissues.     

联合共培养系统诱导骨髓间充质干细胞分化为视网膜色素上皮样细胞

目的 探讨添加脑源性神经生长因子（BDNF）、表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）的培养基联合人类视网膜色素上皮细胞（HRPECs）共培养对骨髓间充质干细胞（BMSCs）定向诱导分化的影响.方法 实验研究.实验分三组：HRPECs＋BDNF、EGF、bFGF＋BMSCs共培养组、BDNF、EGF、bFGF＋BMSCs共培养组和对照组（单独BMSCs）.第一组取第3代HRPECs接种在双层培养板的上层,将第3代人BMSCs接种于下层培养板中,在双层六孔板的每孔中加入混合有20 ng/mlbFGF、20 ng/ml EGF、20 ng/ml BDNF及10%胎牛血清（FBS）的DMEM-LG培养液（需做免疫细胞化学染色应同时在下层放入18 mm×18 mm盖玻片进行细胞爬片）.第二组取第3代人BMSCs接种在六孔培养板中,每孔中加入含20 ng/ml bFGF、20 ng/ml EGF、20 ng/ml BDNF及10%FBS的DMEM-LG培养液.第三组将第3代人BMSCs接种于六孔培养板中,加入10%FBS的DMEM-LG培养液.在倒置相差显微镜下观察细胞形态学变化.2周后停止培养,采用免疫细胞化学染色法和RT-PCR检测角蛋白18、RPE65蛋白存诱导细胞中的表达.数据采用Holm-Sidak法进行分析.结果 诱导2周后,第一组BMSCs细胞呈圆形、类圆形、不规则形、短棒状外观,细胞内有色素颗粒形成,其他两组没有类似改变.三组间免疫细胞化学染色法检测RPE65蛋白、角蛋白18,光密度值结果显示第一组和第二组间、第一组和第三组间差异有统计学意义（RPE65：t=37.416、36.236,P＜0.05;角蛋白18：t=38.611、37.532,P＜0.05）.而第二组和第三组间差异没有统计学意义（RPE65：t=1.180,P＞0.05;角蛋白18：t=1.079,P＞0.05）.RT-PCR检测相对mRNA表达量,结果显示第一组和第二组间、第一组和第三组间差异有统计学意义（RPE65/β-actin：t=176.110、174.820,P＜0.05;角蛋白18/β-actin：t=243.230、241.560,P＜0.05）.而第二组和第三组间差异无统计学意义（RPE65/β-actin：t=1.283.P＞0.05;角蛋白18/β-actin：t=1.670,P＞0.05）.结论 利用BDNF、EGF、bFGF联合HRPECs共培养可以使BMSCs分化为视网膜色素上皮样细胞.