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。     

人脐带间充质干细胞对DR大鼠视网膜神经节细胞凋亡的影响及对p38MAPK通路的调控作用

目的:探讨人脐带间充质干细胞(hUC-MSC)对糖尿病视网膜病变(DR)模型大鼠视网膜神经节细胞(RGCs)凋亡的影响及对p38丝裂原活化蛋白激酶(p38MAPK)通路的调节作用。方法:取SPF级8周龄SD雄性大鼠45只,采用链脲佐菌素(STZ)腹腔注射联合高糖高脂饮食建立DR大鼠模型,期间每周测量大鼠空腹血糖(FBG...     

骨髓间充质干细胞视网膜下移植对光损伤SD大鼠光感受器细胞凋亡的影响

目的研究骨髓间充质干细胞(BMSC)视网膜下移植对光损伤SD大鼠光感受器细胞凋亡的影响及可能的机制。方法24只SD大鼠分为正常组,光损伤组,PBS注射组,BMSC视网膜下移植组。除正常组外大鼠接受绿光照射24h,光照后10 d手术,术后14 d或光照后24 d眼球摘除,制作冰冻切片,行HE染色,免疫荧光和Tunel凋亡检测。对各组外核层层数及凋亡细胞百分数行方差分析。结果BMSC移植组与PBS注射组及光损伤组相比,外核层层数显著增加,凋亡细胞百分比显著减少。BMSC在视网膜下腔表达Nestin,不表达MAP2、Rhodopsin、Calretin in和CD11b;表达bFGF,不表达BDNF和CNTF。结论BMSC视网膜下移植可抑制光损伤大鼠光感受器的凋亡,可能是通过其分泌的bFGF发挥作用。     

大鼠骨髓间充质干细胞视网膜下移植观察

目的：鉴定骨髓间充质干细胞（mesenchymal stem cells,MSCs)在体外不诱导的条件下视网膜下移植后的定位。方法：体外培养雄性大鼠MSCs，直接作为细胞供体视网膜下移植于成年雄性大鼠视网膜下，4周后将动物处死，取眼球做石蜡切片，用Y染色体鉴定，为做进一步验证，将MSCs用重组腺相关病毒AAV－gfp感染后移植，分别于4、8周取动物眼球作冰冻切片，于荧光显微镜下做绿色荧光蛋白（green fluorescence protein，GFP）表达观察。结果：培养的MSCs集落生长迅速，均一性好；Y染色体原位杂交鉴定表明来源于MSCs的阳性细胞融合入了原来的视网膜结构，在光学显微镜下可分布于视锥，视杆细胞层，双极细胞层及节细胞层；在荧光显微镜下可见GFP标记的阳性细胞存在，分布于视网膜色素上皮层、视锥，视杆细胞层，双极细胞层及节细胞层，细胞形态与结构同周围的视网膜相似；2种标记方法检测到的视网膜结构完整，未见到玫瑰花结样结构。结论：MSCs可在视网膜下移植后4周与原视网膜结构相融合，2种方法检测到的阳性细胞分布于视网膜色素上皮层，视细胞层，双极细胞层及节细胞层。     

骨髓间充质干细胞的视网膜保护研究进展

骨髓间充质干细胞( bone marrow mesenchymal stem cells, BMSCs)富集于骨髓,可跨胚层分化,是一种具有多向分化潜能的成体干细胞。 BMSC易于分离培养,可高效扩增和表达,具有组织修复能力。由于其具有免疫调节能力,能分泌神经营养因子,使BMSC可以作为移植治疗视网膜疾病的种子细胞。本文将对BMSCs的视网膜保护研究进展作一综述。     

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.     

色素上皮衍生因子基因修饰的脐带间充质干细胞对糖尿病大鼠视网膜组织病理改变的影响

目的　观察玻璃体内注射色素上皮衍生因子基因修饰的人脐带间充质干细胞（pigment epithelial-derived factor gene-modified human umbilical cord mesenchymal stem cells,PEDF-MSCs）对糖尿病大鼠视网膜组织病理结构改变的影响。方法　组织块培养法获取人脐带间充质干细胞（human umbilical cord mesenchymal stem cells,hUCMSCs）。获取的hUCMSCs表达CD105、CD73和CD90,不表达CD34、CD45、CD11b、CD19和HLA-DR。用慢病毒载体以感染复数值为50对其转染。利用链脲佐菌素腹腔注射途径诱导糖尿病大鼠模型,实验动物分为四组:正常对照组（N 组）、实验对照组糖尿病注射PBS组（D1组）、糖尿病注射hUCMSCs治疗组（D2组）、糖尿病注射PEDF-MSCs治疗组（D3组）。造模成功后3个月进行干预治疗,N组不予特殊治疗。玻璃体内注射2周后利用荧光显微镜观察PEDF-MSCs在大鼠眼内表达情况。干预治疗2周后进行HE染色观察各层视网膜结构,及各组视网膜总厚度变化。结果　玻璃体内注射后2周,荧光显微镜下观察到D2组大鼠玻璃体内簇状排列的红色荧光,视网膜中未发现明显红色荧光;D3组大鼠玻璃体内簇状排列的绿色荧光,视网膜中未发现明显绿色荧光。HE染色显示,N组大鼠的视网膜各层结构完整,层次清晰,细胞排列整齐,染色均匀;D1组大鼠视网膜神经纤维层（nerve fiber layer,NFL）出现明显水肿、血管扩张,内丛状层（innerplexiform layer,IPL）结构疏松,内核层（inner nuclear layer,INL）细胞排列紊乱;D2组NFL水肿减轻;D3组NFL水肿明显减轻,INL与外核层（outer nuclear layer,ONL）层细胞排列规整。视网膜总厚度N组为（103.82±4.15）μm、D1组为（138.86±4.71）μm、D2组为（131.17±3.89）μm、D3组为（112.24±4.22）μm;各组间差异均有统计学意义（均为P＜0.05）。结论　PEDF-MSCs可较长时间在糖尿病大鼠玻璃体内存活且持续表达。玻璃体内注射 PEDF-MSCs能明显改善糖尿病大鼠视网膜损伤,减轻视网膜水肿。     

人牙周膜干细胞、脐带间充质干细胞对体外培养角膜缘干细胞的影响

目的　比较人牙周膜干细胞（ｈｕｍａｎｐｅｒｉｏｄｏｎｔａｌｌｉｇａｍｅｎｔｓｔｅｍｃｅｌｌｓ,ＨＰＤＬＳＣｓ）、人脐带间充质干细胞（ｈｕｍａｎｕｍｂｉｌｉｃａｌｃｏｒｄｍｅｓｅｎｃｈｙｍａｌｓｔｅｍｃｅｌｌｓ,ＨＵＣＭＳＣｓ）作为饲养层培养角膜缘干细胞（ｌｉｍｂａｌｓｔｅｍｃｅｌｌｓ,ＬＳＣｓ）的优越性,从而筛选出扩增ＬＳＣｓ的最佳饲养层。方法　体外分离培养ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ,诱导细胞成脂、成骨分化,并检测细胞表面标志物的表达;将兔ＬＳＣｓ与ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ、ＮＩＨ-３Ｔ３共培养和无饲养层单独培养,比较各组克隆形成率,免疫荧光比较各组ＬＳＣｓ克隆团ＡＢＣＢ５、ＩＰＯ１３、ＣＫ３／１２的表达情况。结果　体外培养ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ均可向脂肪、成骨细胞分化,两种细胞均高表达间充质来源的表面标志ＣＤ９０,不表达ＣＤ４５;三组饲养层与兔ＬＳＣｓ共培养均可形成小克隆团, ＨＰＤＬＳＣｓ组、ＨＵＣＭＳＣｓ组、ＮＩＨ-３Ｔ３组、无饲养层组克隆形成率分别为（４．９０±０．９６）％、（４．１０±０．５６）％、（４．６７±０．７６）％、（０．８３±０．３５）％,四组间总体比较差异有统计学意义（Ｆ＝２２．０４７,Ｐ＝０．００）;ＨＰＤＬＳＣｓ组、ＨＵＣＭＳＣｓ组与ＮＩＨ-３Ｔ３组的克隆形成率差异均无统计学意义（均为Ｐ＞０．０５）;ＨＰＤＬＳＣｓ组、ＨＵＣＭＳＣｓ组、ＮＩＨ-３Ｔ３组与无饲养层组间差异均有统计学意义（均为Ｐ＜０．０１）。免疫荧光化学检测三种饲养层ＬＳＣｓ标志物ＡＢＣＢ５、ＩＰＯ１３呈高表达,ＣＫ３／１２呈低表达。结论　ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ均可作为替代饲养层用以培养ＬＳＣｓ。     

间充质干细胞治疗视网膜疾病的潜能及影响因素

作为干细胞家族的一员,其对损伤组织的修复作用已得到证实。虽然具体作用机制尚不完全明确,在间充质干细胞治疗视网膜疾病中有巨大潜能。现阶段不同的实验所得的结果相差较大,对间充质干细胞的研究任重而道远。综合分析其可能的作用机制和影响因素,对进一步探索间充质干细胞有重要意义。     

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。     

Protective effect of human umbilical cord mesenchymal stem cell-derived exosomes on rat retinal neurons in hyperglycemia through the brain-derived neurotrophic factor/TrkB pathway

AIM: To explore whether human umbilical cord mesenchymal stem cell (hUCMSC)-derived exosomes (hUCMSC-Exos) protect rat retinal neurons in high-glucose (HG) conditions by activating the brain-derived neurotrophic factor (BDNF)-TrkB pathway.METHODS: hUCMSC-Exos were collected with differential ultracentrifugation methods and observed by transmission electron microscopy. Enzyme-linked immunosorbent assays (ELISAs) was used to quantify BDNF in hUCMSC-Exos, and Western blot was used to identify surface markers of hUCMSC-Exos. Rat retinal neurons were divided into 4 groups. Furthermore, cell viability, cell apoptosis, and TrkB protein expression were measured in retinal neurons.RESULTS: hUCMSCs and isolated hUCMSC-Exos were successfully cultured. All hUCMSC-Exos showed a diameter of 30 to 150 nm and had a phospholipid bimolecular membrane structure, as observed by transmission electron microscopy. ELISA showed the BDNF concentration of hUCMSCs-Exos was 2483.16±281.75. hUCMSCs-Exos effectively reduced the apoptosis of retinal neuron rate and improved neuron survival rate, meanwhile, the results of immunofluorescence verified the fluorescence intensity of TrKB in neurons increased. And all above effects were reduced by treated hUCMSCs-Exos with BDNF inhibitors. hUCMSC-Exos effectively reduced the apoptosis rate of retinal neurons by activating the BDNF-TrkB pathway in a HG environment.CONCLUSION: In the HG environment, hUCMSC-Exos could carry BDNF into rat retinal neurons, inhibiting neuronal apoptosis by activating the BDNF-TrkB pathway.     

Protective effects of umbilical cord mesenchymal stem cell exosomes in a diabetic rat model through live retinal imaging

AIM: To assess the protective effect of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Exs) in a diabetic rat model by using a variety of retinal bioassays. METHODS: hucMSCs were subjected to differential ultracentrifugation for the collection of exosomes, and transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) using a NanoSight analysis system and Western blotting (WB) were used to analyze the expression of surface marker proteins such as CD63, CD9 and Calnexin. Streptozotocin (STZ) was injected into the intraperitoneal cavity to establish a diabetic model. Rats were divided into a normal group, diabetic group and hucMSC-Ex group. Fundus fluorescein angiography (FFA), optical coherence tomography (OCT) and other live imaging methods were used to observe the fundus of the rats. Finally, the eyeballs of rats from each group were collected for hematoxylin-eosin (HE) staining to further analyze the retinal structure. RESULTS: Through TEM, NTA and WB, we successfully isolated hucMSC-Exs. Subsequent FFA and OCT confirmed that hucMSC-Exs effectively prevented early retinal vascular damage and thickening of the retina. Finally, HE staining of rat retinal sections revealed that exosomes effectively alleviated retinal structure disruption caused by diabetes. CONCLUSION: hucMSC-Exs have a protective effect on the retina in diabetic rat through FFA, OCT and HE staining.     

枸杞子提取物对光诱导视网膜损伤的保护作用

目的探索枸杞子提取物对人视网膜色素上皮(ARPE-19)细胞及C57BL/6J小鼠视网膜光诱导损伤的保护作用。方法 ARPE-19细胞分为正常细胞对照组,光诱导细胞损伤组,细胞低、中、高剂量组(0.1 g·L^-1、0.5 g·L^-1、1.0 g·L^-1枸杞子提取物+光诱导细胞损伤),测定各组细胞活力以及细胞内活性氧(ROS)含量的变化。40只C57BL/6J小鼠随机分为正常动物对照组,光诱导动物损伤组,动物低、中、高剂量组(280 mg·kg^-1、370 mg·kg^-1、460 mg·kg^-1枸杞子提取物+光诱导动物损伤组),每组8只。各剂量枸杞子提取物干预组小鼠在6周龄开始给予枸杞子提取物灌胃,8周后再给予10 000 lux光照射24 h;光照结束后ERG评估各组小鼠视网膜功能,OCT检测视网膜外核层(ONL)厚度,FFA检测视网膜血管渗漏情况,HE染色后对视网膜ONL细胞进行计数,同时检测血清中丙二醛(MDA)含量和超氧化物歧化酶(SOD)、谷胱甘肽...     

In vitro induction and differentiation of umbilical cord mesenchymal stem cells into neuron-like cells by all-trans retinoic acid

AIM: To determine the optimal concentration for inducing the differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) into neuron-like cells, although it is understood that all-trans retinoic acid (ATRA) regulates cell proliferation in the nervous system by modulating the balance between mitosis and apoptosis. METHODS: The abilities of ATRA to promote apoptosis as well as neural differentiation were assessed in cultured hUC-MSCs by morphological observation, MTT assay, annexin V-FITC/PI flow cytometry and immunocytochemistry. RESULTS: The data showed that low concentrations of ATRA (0.5 μmol, 0.25 μmol) had no effect on the number of cells. However, treatment with 1.0 μmol or 2.0 μmol ATRA induced a 24.16% and 52.67% reduction in cell number, respectively, compared with vehicle-treated cultures. Further, 4.0 μmol ATRA had a potent effect on cell number, with almost no adherent cells recovered after 24h. We further showed that 0.5 μmol ATRA caused these cells to express characteristic markers of neuronal progenitor cells. CONCLUSION: Taken together, we conclude that ATRA has a dose-dependent influence on the neural differentiation and apoptosis of hUC-MSCs. These findings have implications on the use of ATRA-differentiated hUC-MSCs for the study of neural degeneration diseases.     

人脐带间充质干细胞移植治疗角膜碱烧伤的实验研究

目的:研究人脐带间充质干细胞(hUCMSCs)移植治疗兔角膜碱烧伤的疗效,分析多形核中性白细胞(PMNs)的浸润和血管内皮生长因子(VEGF)的表达变化。方法:将75只健康日本白兔随机分成A、B、C组,每组25只,均建立右眼角膜碱烧伤模型。A组兔右眼角膜碱烧伤后立即行角膜病灶区羊膜负载hUCMSCs覆盖术;B组兔右眼角膜碱烧伤后立即行角膜病灶区羊膜覆盖术;C组兔右眼角膜碱烧伤后未进行处理。角膜碱烧伤后3、7、14、21、28d,裂隙灯下观察实验兔角膜恢复情况并照相,对角膜新生血管(CNV)生长情况进行评分,并分离角膜组织制作病理切片,通过苏木素-伊红(HE)染色观察PMNs浸润情况,采用免疫组化染色法测定VEGF的表达。结果:角膜碱烧伤后14d时,A组CNV较B组生长明显缓慢,A组病灶区周围CNV生长评分明显低于B组(P<0.05)。碱烧伤后3d时,角膜PMNs数量增加,浸润角膜基质层,7d时稍有下降,14d时达到峰值,后渐进性下降,碱烧伤后早期浸润在病灶区角膜基质内,后期浸润范围与溃疡面积相同,碱烧伤后各时间点A组和B组角膜PMNs密度均明显低于C组(P<0.05),且1...     

诱导人脐带间充质干细胞分化为角膜上皮样细胞的研究

背景眼表疾病导致的角膜盲已成为全球致盲性角膜疾病中的主要原因之一。随着组织工程技术的发展和进步,组织工程角膜为眼表疾病的治疗开辟了新的途径。目的观察体外培养的人脐带间充质干细胞（UC—MSCs）移植到兔角膜基质后的分化发育情况,探讨人UC-MSCs分化为角膜上皮细胞以及治疗兔角膜损伤的可行性。方法获取人脐带组织,采用Ⅳ型胶原酶消化法分离纯化人UC—MSCs并传代,取第3代细胞用于扩增和实验。流式细胞仪检测细胞的免疫表型及诱导成骨分化鉴定。24只新西兰大白兔按随机数字表法随机分为2个组,将人UC—MSCs接种于去上皮的猪角膜基质上,培养4d后行实验组兔左眼板层角膜移植;对照组以相同的手术方法单纯移植去上皮猪角膜基质。术后对角膜定期行活体激光共焦显微镜检查,并分别于术后2、4、8周摘除各组实验眼行组织病理学和免疫荧光检查,评价移植到兔角膜基质的人UC-MSCs的存活、分化以及移植局部的反应等;应用免疫荧光技术检测移植后角膜上皮细胞中角蛋白3（CK3）、CKl2以及转运蛋白G超家族成员（ABCG2）的表达。结果消化培养的人UC—MSCs呈圆形,细胞胞体较大,贴壁后细胞呈长梭形。培养获得的人UC—MSCs的细胞表型CD105^＋／CD29^＋／CD44^＋／CD34^-／CD45^-,并可诱导分化为成骨细胞。实验组人ISC—MSCs接种到去上皮猪角膜基质后贴附良好、生长迅速,术后植片在植床上存活良好,种植了人UC-MSCs的去上皮猪角膜移植到兔眼,可见实验组受体角膜较对照组透明,未见明显新生血管,在活体共焦显微镜下可见新生的角膜上皮样细胞,未发生免疫排斥反应。免疫荧光检测可见在重建的角膜上皮层检测到CK3及CKl2的阳性表达,而未见ABCG2的表达。结论将种植了人UC—MSCs的猪角膜基质移植到损伤的兔角膜后,人UC—MSCs可以存活、增生并分化为角膜上皮样细胞,可用于修复甚至重建损伤的角膜表层。