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。
ObjectiveTo investigate the effect of pressure on the differentiation of rabbit retinal stem cells (RSCs) co-cultured with retinal ganglion cells (RGCs).
MethodsSPF 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).
ResultsRSCs 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 protein expressions of nestin were significantly reduced in RSCs, and relative mRNA and protein expressions of Thy1.1 were significantly increased in differentiated cells at 20, 40, 60 and 80 mmHg in comparison with 0 mmHg (all at P<0.05). When the pressure was 40 mmHg, the relative mRNA and protein expressions of nestin were lowest in RSCs, and the relative mRNA and protein expressions of Thy1.1 in differentiated cells were highest.
ConclusionsWithin a certain range, pressure can promote the differentiation of RSCs co-cultured with RGCs into ganglion-like cells, and excessive pressure can inhibit the differentiation of RSCs.
戴敏,亢泽峰,胡竹林,等. 压力诱导兔视网膜干细胞向视网膜神经节细胞分化研究[J]. 中华实验眼科杂志,2022,40(12)：1134-1140.
DOI:10.3760/cma.j.cn115989-20200519-00361版权归中华医学会所有。
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戴敏：实验操作、论文撰写、数据整理、统计分析；亢泽峰：实验设计、论文修改及定稿；胡竹林：论文修改；李妍：部分实验操作
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