压力诱导兔视网膜干细胞向视网膜神经节细胞分化研究  

Pressure-induced differentiation of rabbit retinal stem cells into retinal ganglion cells

作　　者：戴敏[1] 亢泽峰[2] 胡竹林 李妍[1] Dai Min;Kang Zefeng;Hu Zhulin;Li Yan(Department of Ophthalmology,Yunnan University Affiliated Hospital,Yunnan Eye Hospital,Yunnan Eye Institute,Key Laboratory of Yunnan Province for the Prevention and Treatment of Ophthalmology,Provincial Innovation Team for Cataract and Ocular Fundus Disease,The Second People's Hospital of Yunnan Province,Expert Workstation of Yao Ke,Eye Disease Clinical Medical Research Center of Yunnan Province,Eye Disease Clinical Medical Center of Yunnan Province,Kunming 650021,China;Eye Hospital of China Academy of Traditional Chinese Medicine,Beijing 100040,China)

机构地区：[1]云南大学附属医院眼科、云南省眼科医院、云南省眼科研究所、云南省眼科疾病防治研究重点实验室、云南省第二人民医院白内障与眼底疾病防治省创新团队、云南省姚克专家工作站、云南省眼部疾病临床医学研究中心云南省眼科疾病临床医学中心,昆明650021 [2]中国中医科学院眼科医院,北京100040

出　　处：《中华实验眼科杂志》2022年第12期1134-1140,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目(81660160);云南省自然科学基金项目(2017FB124);云南省卫生健康委员会医学后备人才培养计划项目(H-2018023);云南省高层次人才培养支持计划-青年拔尖人才专项项目(YNWR-QNBJ-2020-237);云南省眼科疾病防治研究重点实验室项目(2017DG008);院士和领军人才培养项目(2017HC010);北京市石景山区亢泽峰名医传承工作室项目;中医药传承与创新“百千万”人才工程(岐黄工程)岐黄学者项目。

摘　　要：目的探讨压力对与兔视网膜神经节细胞(RGCs)共培养的视网膜干细胞(RSCs)分化的影响。方法选取SPF级新西兰孕22 d兔,取出胚胎,获取视网膜睫状缘色素上皮组织,培养原代RSCs。选取SPF级新生新西兰大白兔6只,分离视网膜神经上皮层组织,培养原代RGCs。采用巢蛋白(Nestin)抗体免疫荧光染色法、溴脱氧核苷尿嘧啶(BrdU)流式细胞术分析、RSCs自发分化细胞免疫荧光检测及流式细胞术分析鉴定体外培养的兔RSCs,采用免疫荧光染色法以Brn3b抗体及Thy1.1抗体鉴定RGCs;将RGCs和RSCs分别放在Transwell的上下层培养,构建共培养体系。分别采用实时荧光定量PCR法和Western blot法检测0、20、40、60、80 mmHg(1 mmHg=0.133 kPa)压力条件下室RSCs及分化所得细胞的Nestin和Thy1.1 mRNA和蛋白表达变化。结果体外培养的兔RSCs特异性标志物Nestin抗体染色阳性;BrdU流式细胞术分析结果显示,BrdU阳性细胞占分离RSCs细胞的(92.26±3.28)%;免疫荧光检测结果显示,RSCs分化细胞中,部分细胞Brn3b抗体表达阳性,部分细胞GS抗体表达阳性。流式细胞术双色分析结果显示,Brn3b和GS阳性细胞率分别为(13.00±3.06)%和(31.60±3.67)%。免疫荧光染色结果显示,体外培养的兔RGCs Brn3b抗体及Thy1.1抗体染色阳性。不同压力条件下RSCs及分化所得细胞的Nestin、Thy1.1 mRNA和蛋白相对表达量比较差异均有统计学意义(mRNA:F=127.600、137.400,均P<0.01;蛋白:F=82.480、158.700,均P<0.001),其中与0 mmHg相比,20、40、60、80 mmHg条件下,Nestin mRNA及蛋白相对表达量降低,Thy1.1 mRNA及蛋白相对表达量增加,差异均有统计学意义(均P<0.05)。40 mmHg时,Nestin mRNA和蛋白相对表达量最低,Thy1.1 mRNA和蛋白相对表达量最高。结论在一定范围内,压力能够促进与RGCs共培养的RSCs分化成视网膜神经节样细胞,压力过大会抑制RSCs的分化。Objective To investigate the effect of pressure on the differentiation of rabbit retinal stem cells(RSCs)co-cultured with retinal ganglion cells(RGCs).Methods SPF grade New Zealand rabbits on the day 22 of gestation were selected,and embryos were removed to obtain retinal ciliary margin pigment epithelial tissue and culture primary RSCs.Six SPF grade newborn New Zealand rabbits were selected,and retinal neuroepithelial layer tissues were isolated to culture primary RGCs.Rabbit RSCs cultured in vitro were identified by immunofluorescence staining of nestin antibody,bromodeoxyuridine(BrdU)cell proliferation assay kit,RSCs spontaneously differentiated cells immunofluorescence detection and flow cytometry.RGCs were identified through immunofluorescence staining of Brn3b antibody and Thy1.1 antibody.A co-culture system of RGCs and RSCs cultured in the upper and lower layers of a transwelll plate respectively was constructed.The mRNA and protein expression levels of nestin and Thy1.1 in RSCs and differentiated cells under pressures of 0,20,40,60,80 mmHg(1 mmHg=0.133 kPa)were detected by real-time fluorescence quantitative PCR and Western blot.The feeding and use of laboratory animals were in accordance with the Regulations on the Administration of Laboratory Animals promulgated by the State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Yunnan University Affiliated Hospital(No.KPRC-IACUC17008).Results RSCs cultured in vitro were nestin-positive.The percentage of BrdU-positive isolated RSCs was(92.26±3.28)%.Some cells differentiated from RSCs were Brn3b-positive,accounting for(13.00±3.06)%,and some were GS-positive,accounting for(31.60±3.67)%.RGCs cultured in vitro were Brn3b-and Thy1.1-positive.There were statistically significant differences in the relative mRNA and protein expressions of nestin and Thy1.1 between RSCs and differentiated cells under different pressures(mRNA:F=127.600,137.400;both at P<0.01;protein:F=82.480,158.700;both at P<0.001).The relative mRNA and

关 键 词：视网膜 干细胞 视网膜神经节细胞 压力 细胞分化 青光眼 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]