目的 体外观察脐带间充质干细胞(UCMSCs)对光诱导视网膜色素上皮(RPE)细胞损伤是否具有保护作用.方法 培养扩增人UCMSCs,对UCMSCs的免疫表型进行流式检测鉴定.原代分离培养人眼RPE细胞,制备蓝光损伤的RPE细胞模型.利用Transwell小室建立光损伤RPE细胞与UCMSCs的非接触共培养体系.RPE细胞分为正常对照组、模型对照组和UCMSCs共培养组.正常对照组不予处理,模型对照组采用蓝光照射诱导RPE细胞损伤,UCMSCs共培养组为光损伤RPE细胞+UCMSCs共培养.光损伤RPE细胞与UCMSCs共培养后24 h和48 h,采用四甲基偶氮唑盐(MTT)法检测各组RPE细胞增生活力;共培养48 h,收集各组细胞培养上清液,酶联免疫吸附测定(ELISA)法定量检测色素上皮衍生因子(PEDF)、碱性成纤维细胞生长因子(bFGF)质量浓度,并进行RPE细胞吞噬感光细胞外节段膜盘(POS)试验.结果 UCMSCs形态呈梭形,细胞表面抗原CD29、CD44、CD90和CD105呈阳性表达,同时细胞表面抗原CD34和CD45呈阴性表达.RPE细胞呈多边形,阳性表达特异性标志物RPE65蛋白.各组共培养后不同时间点RPE细胞增生能力(A值)总体比较,差异均有统计学意义(F组别=132.388,P=0.000;F时间=231.440,P=0.000),其中培养后24 h、48 h,模型对照组和UCMSCs共培养组的细胞A值均较正常对照组明显下降,差异均有统计学意义(均P<0.01);UCMSCs共培养组细胞在相应时间点的细胞A值显著高于模型对照组,差异均有统计学意义(均P<0.01).RPE细胞POS吞噬试验的量化计算结果显示,3个组RPE细胞平均吞噬POS颗粒数总体比较,差异有统计学意义(F=28.087,P=0.000),其中模型对照组RPE细胞平均吞噬POS颗粒数较正常对照组明显减少,UCMSCs共培养组RPE细胞平均吞噬POS颗粒数较模型对照组明显增加,差异均有统计学意义(均P<0.01).ELISA法检测结果显示,正常对照组、模型对照组和UCMSCs共培养组RPE细胞上清液中PEDF质量浓度分别为(18.8±1.9)、(10.0±1.7)和(20.2±6.0)ng/ml,bFGF质量浓度分别为(25.2±1.5)、(26.3±3.6)和(61.9±14.3)pg/ml,总体比较差异均有统计学意义(F=8.654,P=0.008;F=23.698,P=0.000).模型对照组PEDF质量浓度较正常对照组明显下降,UCMSCs共培养组PEDF质量浓度较模型对照组明显升高,差异均有统计学意义(均P<0.01);UCMSCs共培养组bFGF质量浓度较正常对照组和模型对照组均明显升高,差异均有统计学意义(均P<0.01).结论 与UCMSCs非接触共培养可以改善光损伤RPE细胞的增生能力和吞噬功能,促进RPE细胞分泌PEDF.UCMSCs可能通过旁分泌的方式分泌bFGF,减轻RPE细胞的光损伤.
Objective To investigate the protective effect of human umbilical cord mesenchymal stem cells ( UCMSCs) on light-damaged retinal pigment epithelial ( RPE) cells in vitro. Methods Human UCMSCs were cultured,and then identified by flow cytometry. Human RPE cells were isolated and cultured,and then the model of light-damaged RPE cells was prepared. The noncontact co-culture system of light-damaged RPE cells and UCMSCs was established by Transwell chamber. RPE cells were divided into normal control group, model control group and UCMSCs co-culture group. RPE cells in the normal control group were not treated. RPE cells in the model control group were treated with blue light to induce RPE cell damage. Co-culture system of light-damaged RPE cells and UCMSCs was established in the UCMSCs co-culture group. The proliferative ability of RPE cells was measured by methyl thiazolyl tetrazolium ( MTT ) assay at 24 hours and 48 hours after co-culture. ELISA kits were used to quantitatively measure the levels of pigment epithelium-derived factor ( PEDF) and basic fibroblast growth factor ( bFGF) in the culture supernatant at 48 hours after co-culture. And the photoreceptor outer segments ( POS ) phagocytosis assay of RPE cells was also conducted. Results UCMSCs displayed spindle-shaped morphology, positively expressed CD29, CD44, CD90 and CD105, while negatively expressed CD34 and CD45. RPE cells were polygonal in morphology and positive for the specific marker RPE65 protein. The proliferative ability( A value) of RPE cells in the three groups at different timepoints were significantly different ( Fgroup =132. 388, P=0. 000;Ftime =231. 440,P= 0. 000 ) , the A values of RPE cells in model control group and UCMSCs co-culture group were significantly lower than that in the normal control group,the A value of RPE cells in UCMSCs co-culture group was significantly higher than that in the model control group, and the differences were statistically significant both at 24 hours and 48 hours after co-culture (all at P<0. 01). POS phagocytosis test showed that there was a significant difference in the average number of POS particles phagocytized by RPE cells among the three groups( F=28. 087,P=0. 000) . The average number of POS particles phagocytized by RPE cells in model control group was significantly lower than that in normal control group, and the average number of POS particles phagocytized by RPE cells in UCMSCs co-culture group was significantly more than that in model control group (all at P<0. 01). ELISA showed that the concentrations of PEDF in RPE cell supernatant of normal control group,model control group and UCMSCs co-culture group were (18. 8±1. 9),(10. 0±1. 7) and (20. 2±6. 0)ng/ml,respectively. The concentrations of bFGF in RPE cell supernatant were (25.2±1.5),(26.3±3.6) and (61.9±14.3)pg/ml,respectively. There were significant differences in PEDF and bFGF concentrations among the three groups ( F=8. 654, P=0. 008;F=23. 698, P=0. 000) . The concentration of PEDF in the model control group was significantly lower than that in the normal control group,and the concentration of PEDF in the UCMSCs co-culture group was significantly higher than that in the model control group ( all at P<0. 01) . The concentration of bFGF in UCMSCs co-culture group was significantly higher than that in the normal control group and model control group ( all at P<0. 01 ) . Conclusions Cocultivation with UCMSCs can improve the proliferation ability and phagocytosis function of light-damaged RPE cells,and promote the secretion of PEDF by RPE cells. UCMSCs may protect RPE cells from light damage through paracrine secretion of bFGF.