角膜缘损伤模型静脉移植兔骨髓间充质干细胞体内动态分布特点的初步研究

Research on dynamic distribution of the bone marrow mesenchymal stem cells after intravenous transplantation in the corneal injury model

目的骨髓间充质干细胞(bone marrow mesenchymal stem cells,MMSCs)取材方便,可使器官、组织的修复和重建成为可能,但MMSCs在体内的分布、归巢等问题一直是研究热点和难点。文中探讨角膜缘损伤模型静脉移植兔MMSCs的体内动态分布特点。方法选用2.5 kg,月龄2个月的新西兰大白兔60只,入笼养至7 d。随机分为2组,每组30只,正常兔作为对照组,角膜缘碱烧伤兔(制备右眼)作为实验组。分别进行静脉移植MMSCs,观察动物进食、休息状态、行为、排便,是否有腹泻、毛色变化、脱毛等;观察眼部是否有睑球粘连、感染。分别于细胞移植后第3、21、60天耳缘静脉抽取2ml外周血后,空气栓塞处死兔(每组每次处死6只),将眼、肺、心、肝、脾、肾、肌肉和脑组织作冰冻切片,片厚4μm。结果对照组移植术后不同天数MMSCs细胞数目比较的差异无统计学意义(P>0.05);实验组移植术后第3天,外周血中的MMSCs数量较高(2.98±0.11)个,与第21、60天比较,差异有统计学意义(P<0.05),且移植MMSCs的起始数量与外周血中第3天的流式细胞学检测结果呈正相关(r=0.567,P<0.05)。2组间移植术后第3、21、60天细胞数目比较的差异均有统计学意义(P<0.05)。激光共聚焦显像镜下可观察到外源性MMSCs迁移至角膜。对照组在角膜、虹膜、视网膜、脉络膜、虹膜可偶见MMSCs分布。实验组MMSCs数量由多至少依次为角膜缘、角膜中央、结膜、视网膜、脉络膜、虹膜;且MMSCs在角膜缘的分布有"聚集现象";MMSCs经静脉移植后,心、肝、脾肺、肾、肌肉、脑均可见到分布。移植术后不同时间,肺MMSCs数量变化差异有统计学意义(P<0.05),而脾组织中MMSCs数量变化差异无统计学意义(P>0.05)。结论在角膜损伤修复过程中,静脉移植MMSCs可归巢至角膜损伤部位,在全身实质器官中均有分布,肺组织是主要截流场所,脾是MMSCs破坏与溶解场所。

Objective It is easy to obtain bone marrow mesenchymal stem ceils （MMSCs）, which makes repair and recon- struction of tissue and organs possible, but their distribution and homing in vivo are research difficulty and hot spot. The objective of this study was to to investigate the dynamic in vivo distribution characteristics of MMSCs after intravenous transplantation into the rabbitmodel of corneal injury. Methods Sixty two-month-old New Zealand rabbits with body weight of 2.5 kg each were selected and fed in a cage for 7 days. They were randomly divided into two groups with 30 rabbits in each group. Normal rabbits were used as normal control group, and rabbits suffering from limbal alkali bum （preparation ofthe right eye） as experimental group. MMSCs were implanted intravenously and rabbits＇ feeding, resting state, incontinent, any diar- rhea, hair color, hair removal and other changes were observed. All of them were observed whether symblepharon or infection in the eyes or not. After transplantation for 3, 21,60 days, 2 ml of peripheral blood was extracted through ear vein. Rabbits were sacrificed through air embolism （ n = 6 for each time point in each group）, frozen sections with thick slices of 4 μm of the eye, heart, lung, liver, spleen, kidney and muscle, brain were made. Results In the normal control group, there was no difference for the number of MMSCs among different time points after transplantation （ P 〉 0.05 ）. Numbers of MMSCs of peripheral blood （2.98 ＋ 0. 11 ） were higher after transplantation for 3 days that those after transplantation for 21, 60 days （P 〈 0.05）, and initial numbers of transplanted MMSCs positively correlated with flow cytometry assay results of peripheral blood 3 days after transplantation （ r = 0. 567, P 〈 0.05 ）. There was difference for numbers of MMSCs for both groups 3, 21, 60 days after transplantation （P 〈 0.05）. The exogenous MMSCs migrating to cornea were observed under confocal laser microscopy. The labeled MMSCs can be occasionally observed in the corneal limbus, cornea central, conjunctiva, retina, choroid, iris in the normal groups, while the labeled MMSCs were from more to less in order in the corneal limbus, central cornea, conjunctiva, retina, choroid, and iris, and MMSCs tended to gather in the corneal limbus. MMSCs can be observed in the heart, liver, spleen, lungs, muscle, and brain after intravenous injection. There was statistical differ- ence for the numbers of MMSCs in the lungs at the different time points after transplantation （ P 〈 0.05 ）, while there was no difference for the numbers of MMSCs in the spleen （ P 〉 0.05 ）. Conclusion Intravenously injected MMSCs can home to injury location of cornea during healing of cornea injury. These MMSCs can distributed in all parenchymal organs, lung is the main closure organ, and spleen is the organ of destruction and dissolution.