RNAi介导Caspase-3基因及其抑制LPS诱导肾脏上皮细胞凋亡的研究

目的：探讨RNA干扰Caspase-3基因及抑制LPS诱导的肾脏上皮细胞凋亡的作用效果。方法：构建针对大鼠Caspase-3基因编码区的短发夹状RNA（shRNA）真核表达载体质粒pRNAT—U6．1-Caspase-3shRNA，用电穿孔法转染RK3E细胞，经G418筛选后，形成稳定的表达Caspase-3shRNA的细胞系。实验分为3组：①正常对照组：未转染的RK3E细胞；②阴性对照组：转染空载体pRNAT-U6．1的RK3E细胞系；③实验组：转染Caspase-3shRNA的RK3E细胞系。经脂多糖（LPS）孵育12小时后，流式细胞仪检测各组细胞凋亡率，RT-PCR检测mRNA的表达，以及各组Caspase-8蛋白的活性。结果：与正常对照组和阴性对照组相比，实验组的细胞凋亡率显著降低（P〈0．01），Caspase-3的mRNA水平和蛋白活性显著降低（P〈0．01）。结论：针对Caspase-3的特异性短发夹RNA可以明显引起靶基因的沉默，进而抑制LPS诱导的肾脏上皮细胞凋亡的发生。     

玻璃体注射腺相关病毒2和慢病毒转染大鼠视网膜的比较

目的通过玻璃体注射重组腺相关病毒2载体(r AAV2)和慢病毒载体(LV),比较两者转染成年大鼠视网膜的异同。方法实验组大鼠60只,每组12只玻璃体内分别注射重组腺相关病毒2载体-增强绿色荧光蛋白(r AAV2-EGFP)、重组腺相关病毒2载体-神经突起素-增强绿色荧光蛋白(r AAV2-neuritin-EGFP)、慢病毒载体-红色荧光蛋白(LV-RFP)和慢病毒载体-神经突起素-红色荧光蛋白(LV-neuritin-RFP),对照组注射等量的生理盐水,4周后取材,采用免疫荧光和CTB-FITC检测2种病毒载体在视网膜中转染的细胞及转染率,然后分别采用Real-time PCR和Western blotting检测神经突起素(neuritin)在视网膜中的表达变化。结果 r AAV2能够转染约70%视网膜节细胞(RGCs),LV主要转染色素上皮细胞,RGCs转染率仅为30%;r AAV2-neuritin-EGFP组神经突起素mRNA表达约是r AAV2-EGFP组和对照组的16倍,蛋白表达约为3倍;LV-neuritin-RFP组神经突起素mRNA表达约是LVRFP组和对照组的5.5倍,蛋白表达约为1.7倍。结论 r AAV2玻璃体注射后,转染大部分RGCs且神经突起素表达量高于LV,LV主要转染色素上皮细胞,提示基因治疗眼科疾病和损伤时,涉及到转染RGCs时应采用r AAV2载体,转染色素上皮时宜采用LV载体。     

血管内皮生长因子基因转染的心肌细胞移植对心肌梗死大鼠心功能的影响

目的：探讨腺病毒介导血管内皮生长因子165基因（AdVEGF165）转染乳鼠心肌细胞后血管内皮生长因子（VEGF）的表达及移植于大鼠急性心肌梗死（MI）模型后对心功能的影响。方法:体外培养新生大鼠心室肌细胞、标记BrdU、共培养法转染AdVEGF165基因；收集培养液上清，ELISA法检测转染细胞VEGF的表达。结扎同种大鼠前降支建立心肌梗死模型，4周后将心肌梗死大鼠随机分为4组，分别注射移植转染心肌细胞(组Ⅰ)、单纯心肌细胞（组Ⅱ）、AdVEGF165（组Ⅲ）和DMEM培养基（组Ⅳ）。超声心动图检测移植前及移植4周后的心功能。处死大鼠，留取心脏标本作HE病理染色及免疫组化检测，并计数血管密度。结果:AdVEGF165基因转染的心肌细胞表达VEGF高于对照组(P<0.01)；超声检测心功能提示转染细胞组(组Ⅰ)心功能障碍轻于其它3组（P<0.01）；免疫组化检测显示，移植细胞在移植区存活；HE染色血管计数显示转染组(组Ⅰ)有更多的新生血管形成（P<0.01） 。结论：AdVEGF165基因转染心肌细胞后表达分泌VEGF增加，可促进梗死区新生血管形成，改善心肌血供，有利于移植细胞的存活，能更好地减轻心功能障碍。     

过氧化物酶增殖体激活受体-β在大鼠急性肺损伤中的作用

目的探讨过氧化物酶增殖体激活受体-β（PPARβ-）在急性肺损伤（ALI）发生发展中可能的作用,以期从新的视角揭示ALI/ARDS的发病机理,并为ALI/ARDS的防治提供理论基础。方法采用颈静脉注射脂多糖（LPS）的方法复制大鼠ALI模型,随机分为对照组和LPS组。半定量RT-PCR方法测定肺组织中PPARβ-mRNA的表达水平;免疫组织化学染色法测定肺组织PPARβ-蛋白表达水平;同时测定动脉血气分析、肺组织湿/干（W/D）比值、肺组织MPO活性、光镜观察肺组织病理变化。结果LPS组大鼠PaO2显著降低（P〈0.01）,肺组织W/D比值、肺组织髓过氧化物酶（MPO）活性以及肺组织的病理积分均显著升高（P〈0.05）。免疫组化显示对照组大鼠肺组织可表达PPARβ-蛋白,主要位于肺泡上皮细胞及肺间质细胞;与对照组相比,LPS组大鼠肺组织PPARβ-蛋白的表达均显著升高（P〈0.05）,分布在肺泡上皮细胞及炎性细胞。RT-PCR结果显示对照组肺组织表达一定量的PPARβ-mRNA,与对照组比较,LPS组大鼠肺组织PPARβ-mRNA的表达量显著升高（P〈0.05）。结论正常大鼠肺泡上皮细胞及间质细胞存在PPARβ-,在ALI形成过程中PPARβ-蛋白和mRNA表达均显著增加,提示PPARβ-可能与ALI的炎症反应相关。     

人端粒酶反转录酶转染的骨髓间充质干细胞移植治疗大鼠糖尿病

背景：人端粒酶反转录酶是调控增殖及定向分化的首选生长因子之一,具有多重生物学效应。 目的：观察人端粒酶反转录酶表达的骨髓间充质干细胞移植治疗大鼠糖尿病的效果。 方法：体外培养SD大鼠骨髓间充质干细胞,经反转录病毒PLXSN 为载体介导人端粒酶反转录酶基因转染骨髓间充质干细胞,在转染前后用RT-PCR检测骨髓间充质干细胞人端粒酶反转录酶基因的表达。60只雌性SD大鼠中随机取15只作为正常对照组,一次性注射生理盐水,余45只按45 mg/kg的剂量注射链脲霉素建立糖尿病模型后,随机分为3组,分别通过大鼠尾静脉注射移植人端粒酶反转录酶基因转染的骨髓间充质干细胞0.2 mL、骨髓间充质干细胞0.2 mL、生理盐水0.2 mL。 结果与结论：转染48 h后发现,骨髓间充质干细胞有人端粒酶反转录酶mRNA的表达,且重点集中于胞核内。移植后14 d,糖尿病组大鼠空腹血糖维持在较高水平,且高于正常对照组(P < 0.05);与糖尿病组相比,各移植组大鼠空腹血糖水平显著下降(P < 0.05);与骨髓间充质干细胞移植组相比,人端粒酶反转录酶基因转染的骨髓间充质干细胞移植组大鼠空腹血糖水平显著下降(P < 0.05),接近正常对照组水平(P > 0.05)。结果提示人端粒酶反转录酶表达的骨髓间充质干细胞移植能有效治疗大鼠糖尿病。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

脑源性神经营养因子基因修饰的胚胎大鼠皮质神经干细胞及其在体外的分化

目的 探讨脑源性神经营养因子（BDNF）基因修饰大鼠脑皮质神经干细胞（NSCs）后BDNF的表达,并观察其对NSCs体外诱导分化的影响。 方法 体外分离、培养大鼠胚胎脑皮质神经干细胞并鉴定;构建BDNF基因重组质粒,非脂质体法转染NSCs,实验分为pcDNA3-1- BDNF组、pcDNA3-1组和未转染NSCs组。免疫细胞化学技术和RT-PCR检测转染后BDNF蛋白和mRNA的表达;在含5％胎牛血清的分化培养基中诱导分化,βIII微管蛋白、神经胶质纤维酸性蛋白（GFAP） 和突触素( SYP)免疫细胞化学染色对分化细胞进行鉴定,并观察pcDNA3-1-BDNF转染NSCs分化过程中SYP的表达变化。 结果 体外培养获得巢蛋白（nestin）阳性的NSCs。成功构建BDNF基因重组质粒,免疫细胞化学和RT-PCR检测均显示,与pcDNA3-1组和NSCs组比较,pcDNA3-1-BDNF组NSCs的BDNF表达明显增强（P<0.05）。pcDNA3-1组和NSCs组比较,无显著性差异（P＞0.05）。分化后第4天,各组细胞均可分化为Βiii-微管蛋白阳性和GFAP阳性细胞,pcDNA3-1-BDNF组可见少量SYP阳性表达细胞。分化后第7天,与pcDNA3-1组和NSCs组比较,pcDNA3-1-BDNF组NSCs分化为βIII微管蛋白阳性神经元的比例明显增高,有显著性差异（P<0.05）,SYP阳性细胞数增多,表达增强。 结论 BDNF转染NSCs具有体外分泌BDNF的能力,能够促进NSCs定向分化为神经元,SYP表达增强,可能在促进神经元之间的突触发生中发挥作用。     

坐骨神经损伤后长链非编码RNA的聚类分析及长链非编码RNA MX1对大鼠雪旺细胞迁移、增殖能力的影响

目的 探讨坐骨神经损伤后长链非编码RNA（LncRNA）差异表达的基因,以及LncRNA MX1对大鼠雪旺细胞迁移、增殖能力的影响。方法 选取10周龄无特定病原体级雄性SD大鼠24只,随机分为0 d（T0）、3 d（T1）、7 d（T2）和14 d（T3）4组,每组6只,建立坐骨神经损伤动物模型。分别于模型建立后第0、3、7、14天取相应组大鼠坐骨神经损伤处的残端组织提取RNA,制备RNA基因芯片进行Heatmap聚类分析,筛选出各个时间点发生差异表达的基因,并选择其中差异表达显著的基因LncRNA MX1。培养iCell-r030大鼠雪旺细胞,分为对照组、Control siRNA组（siRNA组）和LncRNA MX1 siRNA组（siRNA-MX1组）,使用相应试剂进行转染。转染后采用实时荧光定量聚合酶链反应（qRT-PCR）检测3组雪旺细胞的LncRNA MX1mRNA表达情况,采用Transwell小室检测雪旺细胞的迁移能力,采用5-乙炔基-2'-脱氧尿苷（EDU）实验检测雪旺细胞的增殖能力。结果 各组大鼠均造模成功,实验过程中无大鼠死亡。大鼠损伤坐骨神经组织LncRNA差异基因Heatmap聚类分析的热图显示,与T0组比：T1组共3 066个LncRNA基因发生差异性表达,其中1 634个LncRNA基因表达上调,1 432个LncRNA基因表达下调;T2组共2 498个LncRNA基因发生差异性表达,其中1 634个LncRNA基因表达上调,864个LncRNA基因表达下调;T3组3 567个LncRNA基因发生差异性表达,其中有1 643个LncRNA基因表达上调,1 924个 LncRNA基因表达下调。各组差异表达的LncRNA基因中LncRNA MX1的差异倍数数值较大,差异表达显著。大鼠雪旺细胞qRT-PCR结果显示,转染LncRNA MX1 siRNA后,siRNA-MX1组LncRNA MX1的相对表达量为1.0±0.2,低于对照组的2.3±0.2和siRNA组的2.2±0.2,差异有统计学意义（F=78.47,P<0.001）;而对照组和siRNA组组间差异无统计学意义（P>0.05）。迁移、增殖试验结果显示,siRNA-MX1组细胞迁移数量为（24.1±4.2）个、EDU阳性细胞与DAPI阳性细胞的比率为27.5%±2.8%,低于对照组的（50.3±7.8）个、44.1%±7.2%和siRNA组的（49.2±6.2）个、41.8%±7.0%,差异均有统计学意义（F=93.15、121.26,P值均<0.001）;而对照组和siRNA组间差异均无统计学意义（P值均>0.05）。结论 大鼠坐骨神经损伤后LncRNA MX1差异表达显著,下调LncRNA MX1在大鼠雪旺细胞中的表达可显著降低细胞的迁移、增殖能力。     

脑源性神经营养因子对急性高眼压后大鼠视网膜Mueller细胞的影响

目的：检测脑源性神经营养因子（BDNF）干预急性高眼压后大鼠视网膜Mueller细胞谷氨酰胺合成酶（GS）和谷氨酸／天冬氨酸转运体（GLAST）表达的变化，探讨BDNF保护节细胞（RGCs）的可能机制。方法：成年大鼠分为3个BDNF干预组和3个溶媒对照组并进行玻璃体内注射。2d后将预处理动物左眼眼压升高至闪光视网膜电图b波消失的临界眼压且维持缺血60min。实验动物分别存活1、3或7d后通过免疫组织化学检测大鼠视网膜GS和GLAST的表达变化。结果：与正常对照组比较，溶媒对照组GS和GLAST在存活1d和3d时表达上调，7d时下降；BDNF干预组未出现表达明显变化。结论：BDNF可能不是通过Mueller细胞上调GS和GLAST，降低胞外Glu来保护RGCs。     

p53基因对移植心脏冠状动脉内膜增厚的抑制

背景:研究表明野生型p53基因具有抑制血管平滑肌细胞增殖、减轻血管内膜损伤后内膜增生和管腔狭窄的作用。目的:观察野生型p53基因对心脏移植后移植心脏冠状动脉内膜增厚的抑制作用。方法:以Wistar大鼠为供体,SD大鼠为受体建立大鼠腹腔异位心脏移植模型,取出供心后,经供心冠状动脉分别注射携带野生型p53基因的重组腺病毒液(Ad-p53组)、携带β-半乳糖酐酶基因的重组腺病毒液(Ad-LacZ组)和生理盐水(对照组)800μL,4℃静置30min后进行心脏移植。移植后5d,采用RT-PCR扩增法检测各组供心冠状动脉组织外源性p53表达;移植后28d,组织学观察移植心脏,计算各组冠状动脉内膜与中膜厚度比和血管壁厚度与管腔直径比。结果与结论:移植后5d,Ad-p53组供心内可见野生型p53基因的表达。移植后28d,Ad-p53基因组冠状动脉内膜与中膜厚度比和血管壁厚度与管腔直径比均较Ad-LacZ组及对照组明显减小(P0.05),Ad-LacZ组和对照组比较差异无显著性意义(P0.05)。结果证实在心脏移植中,采用冠状动脉注射法可以将目的基因转移至供心内,供心内转染野生型p53基因能显著抑制移植心脏冠状动脉内膜增生,减轻管腔狭窄程度。     

血管内皮细胞生长因子基因转染骨髓基质细胞的瞬时表达与稳定表达

目的探讨血管内皮细胞生长因子（VEGF）基因修饰的大鼠骨髓基质细胞（BMCS）的表达能力及表达活性。方法取6周龄SD大鼠BMCS传代培养后以3μl阳离子脂质体（Lipofectamine）：1μg pc DNA3．1-VEGF165的比例转染，通过RT—PCR技术、免疫组织化学S—P法检测转染细胞中外源性VEGF165基因的转录及瞬时表达和稳定表达，用血管内皮细胞（VEC）增殖试验测定转染细胞培养上清中VEGF的生物活性。结果VEGF基因转染的大鼠骨髓基质细胞可有效转录VEGF165，其分泌培养上清液中的表达产物可促进血管内皮细胞增殖，具有很强的生物活性。结论采用基因转染技术可将VEGF转染至BMCS中，并可有效表达具有生物活性的VEGF。     

Long-term preservation of cortically dependent visual function in RCS rats by transplantation.

Cell transplantation is one way of limiting the progress of retinal degeneration in animal models of blinding diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Here we transplanted a human retinal pigment epithelial (RPE) cell line into the subretinal space of one such model, the Royal College of Surgeons (RCS) rat, and showed, using head tracking to moving stripes and pattern discrimination in conjunction with single-unit cortical physiology, that cortically mediated vision can be preserved with this treatment.     

Human cord blood cells can differentiate into retinal nerve cells

Retinal degeneration and dystrophy are the major causes of blindness in the developed world. It has been reported that human cord blood cells (HCBCs) can differentiate into neuron-like cells in vitro. We have recently demonstrated that bone marrow cells (BMCs) of both mice and rats can differentiate into retinal nerve cells (RNCs). In the present study, we show the differentiation capacity of HCBCs into RNCs in vivo. We transplanted lineage-negative HCBCs into the subretinal space of severe combined immunodeficiency (SCID) mice. Two weeks after the transplantation, some of the transplanted cells expressed human nestin, human MAP2, human neuron specific enolase (NSE), beta-III tubulin and also rhodopsin. These results indicate that HCBCs can differentiate into RNCs and suggest that our new strategy could be used for the regeneration of retinal nerve cells in degenerative or dystrophic diseases.     

Behavior tests and immunohistochemical retinal response analyses in RCS rats with subretinal implantation of Okayama-University-type retinal prosthesis

We have developed a photoelectric dye-coupled polyethylene film as a prototype of retinal prosthesis, which we named Okayama University-type retinal prosthesis. The purposes of this study are to conduct behavior tests to assess vision in Royal College of Surgeons (RCS) rats that underwent subretinal implantation of the dye-coupled film and to reveal retinal response to the dye-coupled film by immunohistochemistry. Polyethylene films were made of polyethylene powder at refined purity, and photoelectric dyes were coupled to the film surface at higher density compared with the prototype. Either dye-coupled film or dye-uncoupled plain film used as a control was implanted subretinally from a scleral incision in both eyes of an RCS rat at 6 weeks of the age. Behavior tests 2, 4, 6, and 8 weeks after implantation were conducted by observing head turning or body turning in the direction consistent with clockwise or counterclockwise rotation of a black-and-white-striped drum around a transparent cage housed with the rat. After the behavior tests at 8 weeks, rats’ eyes were enucleated to confirm subretinal implantation of the films and processed for immunohistochemistry. In the behavior tests, the number of head turnings consistent with the direction of the drum rotation was significantly larger in RCS rats with dye-coupled- compared with plain-film implantation [P < 0.05, repeated-measure analysis of variance (ANOVA), n = 7]. The number of apoptotic neurons was significantly smaller in eyes with dye-coupled- compared with plain-film implantation (P < 0.05, Mann–Whitney U test, n = 6). In conclusion, subretinal implantation of photoelectric dye-coupled films restored vision in RCS rats and prevented the remaining retinal neurons from apoptosis.     

辣根过氧化物酶标记的纯视细胞层移植

目的　利用HRP标记Wistar鼠的视细胞 ,观察移植的纯视细胞在受体的分布。　方法　 30 %的HRP标记Wistar鼠视细胞 ,经外路途径移入皇家外科学院鼠 (RoyalCollegeofSurgeonRat,RCS)的视网膜下腔 ,术后 2周取RCS鼠眼做冰冻切片 ,组织化学染色法染色 ,普通光学显微镜下观察。　结果　在视细胞移植术后 2周 ,移植视细胞存活 ,HRP标记的Wistar鼠纯视细胞层在RCS鼠视网膜内呈褐色 ,排列平覆。　结论　在RCS鼠视网膜内可以观察到移植的HRP标记的Wistar鼠纯视细胞     

Transplantation of tissue-engineered retinal pigment epithelial cell sheets in a rabbit model

The retinal pigment epithelium (RPE) plays an important role in maintaining a healthy neural retina. With changes due to age, morbidity or removal of choroidal neovascularis developed as a means ofation, damage or defects of the RPE occur. Accordingly, RPE transplantation techniques have been repairing the damaged RPE. We conducted a study to transplant tissue-engineered RPE cell sheets in a rabbit model. RPE cells were isolated from pigmented rabbit eyes and seeded on temperature-responsive culture surfaces. Cultured RPE cells were arranged as a monolayer with a cobblestone cell shape that is characteristic of native RPE. The pigmented RPE cell sheets were non-invasively harvested without enzymatic treatment simply by reducing the culture temperature. Using 3-port vitrectomy, RPE cell sheets were transplanted into the subretinal space of albino rabbits. Seven days after surgery, the rabbits were sacrificed, and the eyes were enucleated and examined under both light and electron microscopy. After transplantation, our results show that the RPE cell sheets attached to the host tissues in the subretinal space more effectively than with the injection of isolated cell suspensions. Although the cell sheets maintained a monolayer structure in most areas, they were slightly folded or wrinkled in some regions. We conclude that tissue-engineered RPE cell sheets harvested from temperature-responsive culture dishes can be effectively transplanted beneath the neural retina.     

胚胎干细胞C3B鼠视网膜下腔移植分化研究

目的：探讨胚胎干细胞在具有正常视网膜结构的C3B鼠视网膜下腔中的诱导分化情况。 方法:胚胎干细胞传1代后进行拟胚体培养。拟胚体消化成单细胞后联合视黄酸注入C3B鼠视网膜下腔，注射后1周、3周、2月处死小鼠取材进行病理切片、电镜检查和免疫组化检测。 结果:1周时，可见到注射部位视网膜层水肿增厚。3周和2个月时，3只移植眼内出现畸胎瘤，占所有移植眼的25%，其余眼球注射部位仅见瘢痕组织或眼球萎缩。电镜发现增殖的细胞核异型性明显，具肿瘤细胞特征。免疫组化显示：畸胎瘤部分区域MAP-2强阳性反应；可见团状或集落状细胞GFAP强阳性反应；个别细胞Nestin阳性反应。 结论:胚胎干细胞移植入具有正常视网膜结构的C3B鼠视网膜下腔，未能出现ESC向视网膜组织分化或嵌入视网膜组织，相当部分的鼠眼出现了畸胎瘤，其临床安全性和致瘤性是非常值得关注的问题。     

Chondroitin sulfate proteoglycans and microglia prevent migration and integration of grafted Müller stem cells into degenerating retina

At present, there are severe limitations to the successful migration and integration of stem cells transplanted into the degenerated retina to restore visual function. This study investigated the potential role of chondroitin sulfate proteoglycans (CSPGs) and microglia in the migration of human Müller glia with neural stem cell characteristics following subretinal injection into the Lister hooded (LH) and Royal College of Surgeons (RCS) rat retinae. Neonate LH rat retina showed minimal baseline microglial accumulation (CD68-positive cells) that increased significantly 2 weeks after transplantation (p < .001), particularly in the ganglion cell layer (GCL) and inner plexiform layer. In contrast, nontransplanted 5-week-old RCS rat retina showed considerable baseline microglial accumulation in the outer nuclear layer (ONL) and photoreceptor outer segment debris zone (DZ) that further increased (p < .05) throughout the retina 2 weeks after transplantation. Marked deposition of the N-terminal fragment of CSPGs, as well as neurocan and versican, was observed in the DZ of 5-week-old RCS rat retinae, which contrasted with the limited expression of these proteins in the GCL of the adult and neonate LH rat retinae. Staining for CSPGs and CD68 revealed colocalization of these two molecules in cells infiltrating the ONL and DZ of the degenerating RCS rat retina. Enhanced immune suppression with oral prednisolone and intraperitoneal injections of indomethacin caused a reduction in the number of microglia but did not facilitate Müller stem cell migration. However, injection of cells with chondroitinase ABC combined with enhanced immune suppression caused a dramatic increase in the migration of Müller stem cells into all the retinal cell layers. These observations suggest that both microglia and CSPGs constitute a barrier for stem cell migration following transplantation into experimental models of retinal degeneration and that control of matrix deposition and the innate microglial response to neural retina degeneration may need to be addressed when translating cell-based therapies to treat human retinal disease.     

The in vitro and in vivo behaviour of retinal pigment epithelial cells cultured on ultrathin collagen membranes

The transplantation of pigment epithelial cells as a therapeutic modality for retinal degeneration requires that the transplanted cells form a monolayer in the subretinal space that will establish communication with photoreceptors. Since previous studies have shown that transplanted cells in suspension do not form a monolayer, it will be necessary to transplant preformed pigment epithelial cell monolayers at the location of the exposed photoreceptors. To establish cell monolayers, retinal pigment epithelial (RPE) cells were cultured on ultrathin collagen membranes. Cells were examined for morphology, for characteristics of differentiation and viability. Membrane degradation and long-term biocompatibility in vivo were assessed following subconjunctival and subretinal implantation in rabbits. These studies have shown that RPE cells adhere, proliferate, form monolayers, and acquire differentiated properties on a collagen membrane that has features similar to Bruch's membrane. Membranes transplanted subconjunctivally and subretinally exhibit excellent biocompatibility without any evidence of inflammation or rejection. RPE cells cultured on collagen membranes acquire differentiated characteristics similar to those of RPE cells in vivo and form complete monolayers that are amenable to be transplanted to the subretinal space. The collagen membranes are non-toxic and do not elicit any rejection or inflammatory response when implanted subconjunctivally or subretinally in rabbits.     

Safety, efficacy, and quality control of a photoelectric dye-based retinal prosthesis (Okayama University-type retinal prosthesis) as a medical device

Patients with retinitis pigmentosa lose photoreceptor cells as a result of genetic abnormalities and hence become blind. Neurons such as bipolar cells and ganglion cells remain alive even in the retina of these patients, and ganglion cells send axons to the brain as the optic nerve. The basic concept of retinal prostheses is to replace dead photoreceptor cells with artificial devices to stimulate the remaining neurons with electric currents or potentials. Photodiode arrays and digital camera-type electrode arrays are the two main approaches for retinal prostheses to stimulate retinal neurons, but these arrays have the problems of poor biocompatibility, low sensitivity, and low output of electric currents, and hence have a requirement for external electric sources (batteries). To overcome these problems, we are developing photoelectric dye-based retinal prostheses that absorb light and convert photon energy to generate electric potentials. The prototype, using a photoelectric dye-coupled polyethylene film, could induce intracellular calcium elevation in photoreceptor-lacking embryonic retinal tissues and cultured retinal neurons. The subretinal implantation of the prototype in the eyes of Royal College of Surgeons (RCS) rats led to vision recovery as proved by a behavior test. The photoelectric dye that was chosen for the prototype did not exhibit any cytotoxicity. The surface potentials of the photoelectric dye-coupled film showed a rapid on-and-off response to illumination with a threshold for light intensity as measured by a Kelvin probe system. Photoelectric dye-based retinal prostheses are thin and soft, and therefore, a sheet of the film of large size, corresponding to a large visual field, could be inserted into the vitreous and then to the subretinal space through a small opening by rolling up the film. Clinical studies of photoelectric dye-based retinal prostheses in patients with retinitis pigmentosa who lose sight will be planned after the manufacturing control and the quality control had been established for the medical device.     

Embryonic stem cells-derived exosomes enhance retrodifferentiation of retinal Müller cells by delivering BDNF protein to activate Wnt pathway

ObjectiveThe present study was intended to investigate the role of embryonic stem cell-derived exosomes (ESC-Exos) in Müller cell retrodifferentiation and their specific mechanism.MethodsFollowing co-incubation of the extracted ESC-Exos and Müller cells, their effects on the retrodifferentiation and proliferation of Müller cells were measured by EdU staining, immunofluorescence, and western blot. ESCs transfected with small interfering RNA of BDNF were co-incubated with Müller cells to determine Müller cell proliferation and retrodifferentiation. β-catenin expression in the nucleus and GSK-3β phosphorylation were measured to determine the role of the Wnt pathway in Müller cells. The function of the retina in RCS rats was observed using flash electroretinogram.ResultsCo-incubation of ESCs with Müller cells or overexpression of BDNF contributed to Müller cell retrodifferentiation and proliferation, as evidenced by increased cell proliferation, fluorescence intensities of proliferation markers and retinal stem cell markers, and expression of BDNF and β-catenin, and suppressed GSK-3β phosphorylation. However, co-incubation with ESCs silencing BDNF or treatment with GW4869 inhibited the proliferation and retrodifferentiation of retinal Müller cells. In addition, exosome injection increased BDNF, BrdU, PH3, SOX2, and Pax6 expression, enhanced β-catenin expression in the nucleus, diminished GSK-3β, and improved retinal degeneration in RCS rats.ConclusionESC-Exos accelerated Müller cell retrodifferentiation and proliferation through Wnt pathway activation by delivering BDNF protein to Müller cells.