[1]常鲁,许丽,韩新红,等.色素上皮衍生因子基因修饰的人脐带间充质干细胞对大鼠视网膜缺血-再灌注损伤的保护作用[J].眼科新进展,2018,38(4):324-328.[doi:10.13389/j.cnki.rao.2018.0076]
　CHANG Lu,XU Li,HAN Xin-Hong,et al.Protective effects of PEDF gene modified human umbilical cord mesenchymal stem cells in rat with retinal ischemia-reperfusion injury[J].Recent Advances in Ophthalmology,2018,38(4):324-328.[doi:10.13389/j.cnki.rao.2018.0076]
点击复制

色素上皮衍生因子基因修饰的人脐带间充质干细胞对大鼠视网膜缺血-再灌注损伤的保护作用/HTML
分享到：

《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
38卷
期数:
2018年4期
页码:
324-328
栏目:
实验研究
出版日期:
2018-04-05

文章信息/Info

Title:
Protective effects of PEDF gene modified human umbilical cord mesenchymal stem cells in rat with retinal ischemia-reperfusion injury
作者:
常鲁; 许丽; 韩新红; 王慧凤; 李艳
261000　山东省潍坊市，潍坊医学院（常鲁，许丽，韩新红，王慧凤）；261000　山东省潍坊市，潍坊医学院附属医院眼科（李艳）
Author(s):
CHANG Lu; XU Li; HAN Xin-Hong; WANG Hui-Feng; LI Yan
Weifang Medical University （CHANG Lu，XU Li，HAN Xin-Hong，WANG Hui-Feng），Weifang 261000，Shangdong Province，China；Department of Ophthalmology，Affiliated Hospital，Weifang Medical University （LI Yan），Weifang 261000，Shangdong Province，China
关键词:
色素上皮衍生因子; 血管内皮生长因子; 基因转染; 视网膜缺血-再灌注损伤
Keywords:
PEDF; VEGF; gene transfection; RIRI
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2018.0076
文献标志码:
A
摘要:
目的　观察色素上皮衍生因子色素上皮衍生因子（pigment epithelial-derived factor，PEDF）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）对大鼠视网膜缺血-再灌注损伤（retinal ischemia-reperfusion injury，RIRI）模型中PEDF、血管内皮生长因子（vascular endothelial growth factor，VEGF）表达的影响及对视网膜神经节细胞的保护作用。方法　绿色荧光蛋白（green fluorescent protein，GFP）标记的慢病毒（lentivirus，LV）为载体，将PEDF基因以感染复数（multiplicity of infection，MOI）为1、10、20、50体外转染至人脐带间充质干细胞（human umbilical cord mesenchymal stem cells，hUCMSCs）中，应用RT-PCR及ELISA法分别检测以最佳MOI值转染细胞中PEDF的表达情况。选取健康SD雄性大鼠，采用高眼压的方法制成RIRI模型，随机分为正常对照组（N组）、磷酸盐缓冲液（phosphate buffered saline，PBS）治疗组（PBS组）、hUCMSCs治疗组（M组）及 PEDF基因转染的hUCMSLs治疗组（P组）；N组不予特殊处理，其余三组大鼠造模成功后立即进行干预治疗，PBS组给予PBS治疗，P组与M组分别给予 PEDF-MSCs、hUCMSCs治疗。5 d后处死大鼠，尼氏染色计数视网膜神经节细胞数目，计算视网膜内层厚度，RT-PCR检测大鼠视网膜 PEDF和VEGF的mRNA表达情况。结果　荧光显微镜下观察到转染率高达75.8％。RT-PCR结果显示，转染后PEDF-MSCs的上清液中PEDF mRNA相对表达量（4.34±0.29)明显高于hUCMSCs(1.08±0.15)，差异有统计学意义（P<0.05）。ELISA检测结果显示，转染后 PEDF-MSCs上清液中PEDF蛋白表达量［（83.09±7.58）μg·L-1］明显高于hUCMSCs［（12.30±1.24）μg·L-1］，差异有统计学意义（P<0.05）。尼氏染色结果显示造模后PBS组大鼠神经节细胞数量变少，治疗5 d后，P组、M组大鼠神经节细胞数量均高于PBS组，差异均有统计学意义（均为P<0.05）；P组高于M组，差异有统计学意义（P<0.05）；治疗5 d后，P组、M组大鼠视网膜厚度均厚于PBS组，差异均有统计学意义（均为P<0.05）；P组厚于M组。RT-PCR检测结果显示，P组PEDF mRNA表达水平显著升高，VEGF mRNA 表达水平下降，与PBS组、M组相比差异均有统计学意义（均为P<0.05）。结论　玻璃体内注射PEDF-MSCs能上调RIRI模型大鼠视网膜PEDF表达，并下调VEGF 表达，对RIRI大鼠视网膜神经节细胞有保护作用。
Abstract:
Objective　To observe the effects of pigment epithelial-derived factor gene-modified human umbilical cord mesenchymal stem cells (PEDF-MSCs) on the expression of pigment epithelial-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in a rat model of retinal ischemia-reperfusion injury (RIRI) and its protection on retinal ganglion cells.Methods　Lentivirus (LV) labeled with green fluorescent protein (GFP) was used as a vector to transfect the PEDF gene into human umbilical cord mesenchymal stem cells (hUCMSCs) at a multiplicity of infection (MOI) of 1，10，20，and 50 in vitro，and then the expression of PEDF gene and protein in cells transfected with the best MOI value was detected by RT-PCR and ELISA.The healthy male SD rats were randomly divided into normal group (N group) and experimental group.The RIRI model was made by high intraocular pressure in the experimental group，and the RIRI rats with PBS treatment were allocated as the PBS group，with hUCMSCs treatment as M group and LV-PEDF-MSCs treatment as P group，and the N group was left untreated.All rats were sacrificed on day 5，and the number of retinal ganglion cells were counted by Nissl staining，the thickness of the retina was calculated，as well as the expression of PEDF and VEGF mRNA in rat retina was detected by RT-PCR.Results　The transfection efficiency was as high as 75.8% under fluorescence microscope.The results of RT-PCR showed that the relative expression of PEDF mRNA in PEDF-MSCs supernatant (4.34±0.29) was significantly higher than that in hUCMSCs (1.08±0.15)，and the difference was statistically significant (P<0.05)；moreover，the results of ELISA showed that PEDF protein expression in PEDF-MSCs supernatant ［(83.09±7.58)μg·L-1］ was significantly higher than that in hUCMSCs ［(12.30±1.24)μg·L-1］，and the difference was statistically significant (P<0.05).Nissl staining results showed that the number of ganglion cells in group PBS decreased after model establishment.After 5 days of treatment，the number of ganglion cells in P group and M group was higher than that in PBS group，and the difference was statistically significant (both P<0.05)；and P group was higher than M group，with the significant difference (P<0.05).And this was true of the thickness of the retina.RT-PCR showed that the expression of PEDF mRNA in P group was significantly up-regulated，but VEGF mRNA expression was significantly down-regulated，and the differences were statistically significant when compared with PBS and M group (both P<0.05).Conclusion　Intravitreal injection of PEDF-MSCs can up-regulate the expression of PEDF but down-regulate the expression of VEGF in the retina of RIRI rats，which can protect the retinal ganglion cells against RIRI.

参考文献/References:

［1］　HE X，CHENG R，BENYAJATI S，MA JX.PEDF and its roles in physiological and pathological conditions：implication in diabetic and hypoxia-induced angiogenic diseases［J］.Clin Sci （Lond），2015，128 （11）；805-823.
［2］　CRAWORD SE，FITCHEV P，VELICEASA D，VOLPERT OV.The many facets of PEDF in drug discovery and disease：a diamond in the rough or split personality disorder［J］？Expert Opin Drug Discov，2013，8 （7）；769-792.
［3］　KOZULIN P，NATOLI R，BUMSTED OK，Madigan MC，Provis JM.The cellular expression of antiangiogenic factors in fetal primate macula［J］.Invest Ophthalmol Vis Sci，2010，51（8）：4298-4306.
［4］　YOKOI M，YAMAGISHI S，SAITO A，YOSHIDA Y，MATSUI T，SAITO W，et al.Positive association of pigment epithelium-derived factor with total antioxidant capacity in the vitreous fluid of patients with proliferative diabetic retinopathy［J］.Br J Ophthalmol，2007，91（7）：885-887.
［5］　TAN ML，CHOONG PF，DASS CR.Anti-chondrosarcoma effects of PEDF mediated via molecules important to apoptosis，cell cycling，adhesion and invasion［J］.Biochem Biophys Res Commun，2010，398（4）：613-618.
［6］　AGARDH CD，GUSTAVSSON C，HAGERT P，NILSSON M，AGARDH E.Expression of antioxidant enzymes in ratretinal ischemia followed by reperfusion［J］.Metabolism，2006，55（7）：892-898.
［7］　NISHIWAKI A，UEDA T，UGAWA S，SHIMADA S，OGURA Y.Upregulation of P- selectin and intercellular adhesion molecule-1 after retinal ischemia-reperfusion injury［J］.Invest Ophthalmol Vis Sci，2003，44（11）：4931-4935.
［8］　HIROOKA K，MIYAMOTO O，JINMING P，DU Y，BABA T，TOKUDA M，et al.Neuroprotective effects of D-allose against retinal ischemia- reperfusion injury［J］.Invest Ophthalmol Vis Sci，2006，47（4）：1653-1657.
［9］　PARK K，JIN J，HU Y，ZHOU K，MA JX.Overexpression of pigment epithelium-derived factor inhibits retinal inflammation and neovascularization［J］.Am J Pathol，2011，178（2）：688-698.
［10］　LI WW，YAO XY，YANG LM，ZHAO PW，ZHANG GH.Induced differentiation of human umbilical cord mesenchymal stem cells into neural stem cells in vitro［J］.Chin J Tissue Engineering Res，2014，18（1）：75-80.
李伟伟，姚星宇，杨丽敏，赵鹏伟，张国华.体外诱导人脐带间充质干细胞向神经干细胞的分化［J］.中国组织工程研究，2014，18（1）：75-80.
［11］　DUYA P，BIAN Y，CHU X，ZHANG Y.Stem cells for reprogramming：could h UMSCs be a betterchoice［J］?Cytotechnology，2013，65（3）：335-345.
［12］　YANG H，XIE Z，WEI L，YANG H，YANG S，ZHU Z，et al.Human umbilical cord mesenchymal stem cell-derived neuron-like cells rescue memory deficits and reduce amyloid-beta deposition in an AβPP/PS1 transgenic mouse model［J］.Stem Cell Res Ther，2013，4（4）：76.
［13］　ZHANG ZP，KAI-JIN MA，DENG YG，et al.Experimental study on effect of citicoline in inducing differentiation of human umbilical cord-derived mesenchymal stem cells to neuron-like cells［J］.Pharm Today，2013，23（3）：129-131.
张紫萍，马凯进，邓英光，余卫，胡志兵.胞磷胆碱体外诱导人脐带间充质干细胞分化为神经元样细胞的实验研究［J］.今日药学，2013，23（3）：129-131.
［14］　TOMBRAN-TINK J，APARICIO S，XU X，TINK AR，LARA N，SAWANT S，et al.PEDF and the serpins：phylogeny，sequence conservation，and functional domains［J］.J Struct Biol，2005，151（2）：130-150.
［15］　OGATA N，WADA M，OTSUJI T，JO N，TOMBRAN-TINK J，MATSUMURA M.Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization［J］.Invest Ophthalmol Vis Sci，2002，43（4）：1168-1175.
［16］　ORTEGO J，ESCRIBANO J，BECERRA SP，COCA-PRADOS M.Gene expression of the neurotrophic pigment epithelium-derived factor in the human ciliaryepithelium.Synthesis and secretion into the aqueous humor［J］.Invest Ophthalmol Vis Sci，1996，37（13）：2759-2767.
［17］　KOZULIN P，NATOLI R，BUMSTED O K，MADIGAN MC，PROVIS JM.The cellular expression of antiangiogenic factors in fetal primate macula［J］.Invest Ophthalmol Vis Sci，2010，51（8）：4298-4306.
［18］　DAWSON DW，VOLPERT OV，GILLIS P，CRAWFORD SE，XU H，BENEDICT W，et al.Pigment epithelium-derived factor：a potent inhibitor of angiogenesis［J］.Science，1999，285（5425）：245-248.
［19］　HE SS，SHI H S，YIN T，LI YX，LUO ST，WU QJ，et al.Aav-mediated gene transfer of human pigment epithelium-derived factor inhibits Lewis lung carcinoma growth in mice［J］.Oncol Rep，2012，27（4）；1142-1148.
［20］　CLARA CA，MARIE SK，DE ALMEIDA JR，WAKAMATSU A，OBA-SHINJO SM，UNO M，et al.Angiogenesis and expression of PDGF-C，VEGF，CD105 and HIF-1alpha in human glioblastoma［J］.Neuropathology，2014，34（4）；343-352.
［21］　DUH EJ，YANG HS，SUZUMA I，MIYAGI M，YOUNGMAN E，MORI K，et al.Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth［J］.Invest Ophthalmol Vis Sci，2002，43（3）：821-829.
［22］　ZHANG Y，HAN J，YANG X，SHAO C，XU Z，CHENG R，et al.Pigment epithelium-derived factor inhibits angiogenesis and growth of gastric carcinoma by down-regulation of VEGF［J］.Oncol Rep，2011，26（3）：681-686.
［23］　YOSHIDA Y，YAMAGISHI S，MATSUI T，JINNOUCHI Y，FUKAMI K，IMAIZUMI T，et al.Protective role of pigment epithelium-derived factor（PEDF） in early phase of experimental diabetic retinopathy［J］.Diabetes Metab Res Rev，2009，25（7）：678-686.
［24］　TAKITA H，YONEYA S，GEHLBACH PL，DUH EJ，WEI LL，MORI K.Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor［J］.Invest Ophthalmol Vis Sci，2003，44（10）：4497-4504.

相似文献/References:

[1]程文武 江萍 席祖莲 张汉武 訾世莉 聂军 董彩虹.蛹虫草提取物抑制大鼠角膜新生血管的实验研究[J].眼科新进展,2012,32(5):000.
[2]于静 张明昌.苦参碱对大鼠角膜新生血管的抑制作用[J].眼科新进展,2012,32(6):000.
[3]吴正正 严京 接传红 高健生 陈皆春 宋剑涛.密蒙花方对缺氧状态下脐静脉血管内皮细胞 VEGF-VEGFR 信号转导通路的影响[J].眼科新进展,2012,32(7):000.
[4]庞霞 曲蕴慧 马跃伟 张效房 董敬民 高冬玲.去整合素金属蛋白酶9和血管内皮生长因子蛋白在眼部恶性黑色素瘤中的表达[J].眼科新进展,2012,32(7):000.
[5]李艳 李筱荣 袁佳琴 潘斌.糖尿病大鼠视网膜中VEGF、PEDF的表达与血-视网膜屏障损伤[J].眼科新进展,2013,33(1):000.
[6]毕春潮 王睿 王建洲 雷春灵 董晓娟 王小莉 薛晓辉.Ad-PEDF对糖尿病视网膜病变大鼠视网膜新生血管的抑制作用[J].眼科新进展,2013,33(8):000.
[7]许琴 黄亮 林素香 郑水华 肖煜晨 王雅丽.激光诱导小鼠脉络膜新生血管中膜攻击物与血管生长因子的表达[J].眼科新进展,2013,33(8):000.
[8]刘宁宁 才娜 陈蕾.色素上皮衍生因子的表达与早期糖尿病大鼠视网膜血管形态学的改变[J].眼科新进展,2012,32(2):000.
[9]曾莉 常以力 邵毅.血管内皮生长因子反义寡脱氧核苷酸联合血管生成素-1对糖尿病视网膜病变大鼠视网膜血管渗漏及新生血管生成的影响[J].眼科新进展,2012,32(10):000.
[10]兰兰 惠延年 曾光伟.血管生成素-1对糖尿病大鼠视网膜微血管病变、基质金属蛋白酶和血管内皮生长因子的抑制作用[J].眼科新进展,2012,32(10):000.
[11]李小璐 马雅玲.糖尿病视网膜病变大鼠视网膜VEGF和PEDF的动态表达[J].眼科新进展,2013,33(9):000.
[12]王清 郭乔茜 李琴 谢婷玉 陈雪艺. 臭氧治疗对糖尿病大鼠视网膜细胞凋亡和VEGF、PEDF因子表达的影响[J].眼科新进展,2014,34(3):232.
[13]田楠楠,亢泽峰,张庆.加减驻景方对病理性近视脉络膜新生血管动物模型血管内皮生长因子及色素上皮衍生因子表达的影响[J].眼科新进展,2015,35(10):906.[doi:10.13389/j.cnki.rao.2015.0247]
　TIAN Nan-Nan,KANG Ze-Feng,ZHANG Qing.Effects of modified Zhu Jing prescriptions on expression of VEGF and PEDF in pathologic myopia with choroidal neovascularization animal model[J].Recent Advances in Ophthalmology,2015,35(4):906.[doi:10.13389/j.cnki.rao.2015.0247]
[14]徐硕,张红.角膜新生血管药物治疗的最新进展[J].眼科新进展,2015,35(10):989.[doi:10.13389/j.cnki.rao.2015.0271]
　XU Shuo,ZHANG Hong.Recent advances in drug therapy of corneal neovascularization[J].Recent Advances in Ophthalmology,2015,35(4):989.[doi:10.13389/j.cnki.rao.2015.0271]

备注/Memo

备注/Memo:
山东省高等学校科技计划项目（编号：J15LL08E）
更新日期/Last Update: 2018-04-02