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  

Effect of human umbilical cord derived mesenchymal stem cells on apoptosis of rat retinal ganglion cells under high glucose and on the regulation of p38MAPK pathway

作　　者：刘曦[1] 孙晓萍[1] 杨建伟[1] 朱冬梅[1] 王媛[1] Liu Xi;Sun Xiaoping;Yang Jianwei;Zhu Dongmei;Wang Yuan(Department of Ophthalmology,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]郑州大学附属郑州市中心医院眼科,450000

出　　处：《中华实验眼科杂志》2022年第1期21-28,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：河南省医学科技攻关计划项目(2018020799)。

摘　　要：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)和体质量,采用眼底血管造影观察视网膜血管走行和渗漏情况。将40只建模成功大鼠按照随机数字表法随机分为模型组和hUC-MSC注射组,每组20只;另选取20只正常大鼠腹腔注射等量柠檬酸缓冲液并常规饲养,作为对照组。hUC-MSC注射组玻璃体内注射hUC-MSC,对照组和模型组玻璃体内注射等量磷酸盐缓冲液(PBS)。采用荧光金(FG)逆行示踪标记RGCs观察存活RGCs数目;采用苏木精-伊红染色观察视网膜病理学变化;采用TUNEL法观察RGCs凋亡情况;采用Western blot法检测视网膜组织中B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、p38MAPK和磷酸化p38MAPK(p-p38MAPK)蛋白表达量。结果未造模大鼠FBG维持在正常水平,体质量随时间逐步增长,视网膜血管走行正常,无荧光素渗漏;造模大鼠FBG维持在较高水平,体质量增长缓慢,且于造模2周开始随着病程延长,体质量逐渐下降,视网膜远端血管发生扭曲,可见毛细血管荧光素渗漏,渗漏面积较大。对照组、模型组和hUC-MSC注射组RGCs密度分别为(2136.10±215.17)、(849.40±167.82)和(1549.20±183.26)个/mm^(2),总体比较差异有统计学意义(F=115.218,P<0.01);模型组和hUC-MSC注射组RGCs密度明显低于对照组,hUC-MSC注射组RGCs密度明显高于模型组,差异均有统计学意义(均P<0.05)。对照组视网膜结构清晰,层次分明,RGCs形态完整,数目较多;模型组RGCs数量明显减少,出现核固缩,RGC层变薄萎缩;hUC-MSC注射组视网膜结构较完整,RGCs数较模型组多。对照组、模型组和hUC-MSC注射组RGCs凋亡率分别为(2.16±1.11)%、(43.47±2Objective To investigate the effect of human umbilical cord mesenchymal stem cells(hUC-MSC)on the apoptosis of retinal ganglion cells(RGCs)in diabetic retinopathy(DR)model rats and on the regulation of p38 mitogen-activated protein kinase(p38MAPK)pathway.Methods Forty-five SPF male 8-week old SD rats were selected.The DR rat model was established by intraperitoneal injection of streptozotocin(STZ)combined with a high-sugar and high-fat diet.The fasting blood glucose(FBG)and body weight of the rats were measured every week during the high-sugar and high-fat diet,and fundus angiography was used to observe the circulation and leakage of retinal blood vessels.Forty rats with successful modeling were randomly divided into model group and hUC-MSC injection group according to the random number table method,with 20 rats in each group.Another 20 normal rats fed routinely were served as control group,and intraperitoneally injected with the same amount of citric acid buffer.The hUC-MSC injection group was injected intravitreously with hUC-MSC,and the control group and model group were injected intravitreously with the same amount of phosphate buffer saline(PBS).Fluoro gold(FG)retrograde tracer labeling RGCs was used to observe the number of survived RGCs.Hematoxylin-eosin staining was used to observe the pathological changes of retina.TUNEL method was used to observe the apoptosis of RGCs.Western blot was used to detect B cell lymphoma/leukemia-2(Bcl-2),Bcl-2 associated X protein(Bax),p38MAPK and phosphorylated(p-)p38MAPK protein expression in retinal tissues.The use and care of the rats complied with the ARVO statement.The study protocol was approved by an Animal Ethics Committee of Zhengzhou central Hospital Affiliated to Zhengzhou University(NO.2980316).Results The FBG of control rats was maintained at a normal level,and the body weight gradually increased over time,and was gradually decreased as the course of disease prolonged.The retinal blood vessels ran normally without fluorescein leakage in the control group.In the

关 键 词：糖尿病视网膜病变 人脐带间充质干细胞 视网膜神经节细胞 凋亡 

分 类 号：R774.1[医药卫生—眼科] R587.2[医药卫生—临床医学]