嗅鞘细胞移植后通过分泌MMP-3在RCS-P^+大鼠视网膜中迁移  

Transplanted olfactory ensheathing cells migrate in RCS-P^+ rat retina through secreting MMP-3

作　　者：谢晶[1] 李瑶琛[1] 阴正勤[1] 

机构地区：[1]第三军医大学西南医院全军眼科中心,重庆400038

出　　处：《第三军医大学学报》2012年第9期843-847,共5页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划(973计划;2007CB512200)~~

摘　　要：目的初步研究将嗅鞘细胞(olfactory ensheathing cells,OECs)及嗅球成纤维细胞(olfactory nerve fibroblasts,ONF)混合细胞移植到皇家外科学院大鼠(Royal College of Surgeon rat,RCS-P+rat)的视网膜下腔后,OECs/ONF迁移进入视网膜的机制。方法离体实验中,取成年RCS-rdy+-P+大鼠的嗅球培养OECs/ONF至14 d行OECs/ONF的基质金属蛋白酶-3(matrix-metalloproteinase-3,MMP-3)细胞免疫荧光染色。收集5、8、11、14 d OECs/ONF培养液上清,与普通培养液超滤后进行酶联免疫吸附实验(ELISA),检测MMP-3的含量变化。在体实验中,制作40只RCS-P+大鼠单眼视网膜下腔细胞移植,并以对侧眼作为伪手术组以及相同天龄未处理大鼠作为对照组。术后7、14、21、28 d用ELISA法检测细胞移植组、伪手术组及未处理组大鼠视网膜中MMP-3含量的变化。将携带绿色荧光的慢病毒感染后的OECs/ONF移植到4只RCS-P+大鼠视网膜下腔,激光共聚焦显微镜观察移植后7、14、21 d及28 d OECs/ONF在视网膜中迁移情况。结果离体实验中,培养14 d时OECs/ONF的MMP-3免疫细胞化学染色阳性。OECs/ONF培养液上清MMP-3含量分别为5 d(2.83±0.80)、8 d(6.34±1.12)、11 d(11.65±1.35)、14 d(19.11±2.11),明显高于普通D/F12+10%FBS培养液(1.65±0.44)(P<0.01);在体实验中,移植后21 d及28 d,OECs/ONF移植组视网膜MMP-3的含量[(1.80±0.29)、(3.96±0.51)]明显高于伪手术组[(1.17±0.20)、(1.83±0.26)]和未处理组[(1.19±0.17)、(1.92±0.25)](P<0.01),伪手术组与未处理组之间无明显统计学差异(P>0.05)。激光共聚焦显微镜观察可见移植后7～28 d,OECs/ONF在视网膜中迁移,最远能够达到神经节细胞层。结论 OECs/ONF移植到RCS视网膜下腔后,可能通过分泌MMP-3在RCS-P+大鼠视网膜中迁移。Objective To investigate how olfactory ensheathing cells（OECs） migrate in the retina of pigmented Royal College of Surgeon rats（RCS-P＋ rats） after being transplanted into the subretinal space with olfactory nerve fibroblasts（ONFs） together.Methods OECs/ONFs were primarily cultured from RCS-rdy＋-P＋ rat olfactory bulbs and identified by immunofluorescence staining.The expression of anti-matrix metalloproteinase-3（MMP-3） in the OECs were detected by cytological immunological fluorescence staining.After GFP lentivirus infection,the secretion of MMP-3 in the supernatants of OECs collected at 5,8,11,and 14 d were detected by enzyme-linked immunosorbent assay（ELISA）.For the experiment in vivo,the suspension of OECs/ONFs was injected into the subretinal space of one eye of 40 RCS-P＋ rats.PBS was injected into the subretinal space of the other eye.For the untreated group,no operation was done.In 7,14,21 and 28 d after surgery,their retina tissues were collected.ELISA was used to detect the changes of MMP-3 in the retina.After EGFP labeled OECs/ONF were injected into the subretinal space of 4 RCS-P＋ rats,laser confocal microscopy was used to observe the migration of OECs/ONF in 7,14,21 and 28 d after transplantation.Results In 14 d after culture,OECs/ONF secreted MMP-3.The MMP-3 level in the OECs/ONF supernatant was 2.83±0.80,6.34±1.12,11.65±1.35,19.11±2.11 at the 5th,8th,11th,and 14th days after culture respectively,which was significantly higher than that of control supernatants（1.65±0.44,P0.01）.For the experiment in vivo,the MMP-3 levels were 1.80±0.29 and 3.96±0.51 respectively in RCS-P＋ in 21 and 28 after OECs/ONF transplantation,which were significantly higher than those of the PBS group（1.17±0.20 and 1.83±0.26） and untreated group（1.19±0.17 and 1.92±0.25）（P0.01）.There was no significant difference between the 2 latter groups（P0.05）.From the 7th d to 28th day,laser confocal microscopy indicated that OECs/ONF migrated to the retina and most of them moved t

关 键 词：嗅鞘细胞 皇家外科学院大鼠 视网膜 移植 迁移 基质金属蛋白酶-3 

分 类 号：R322.91[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]