尾静脉注射骨髓间充质干细胞对小鼠视网膜激光损伤后细胞凋亡的影响

Retinal apoptosis of laser-induced retinal injury in mice after bone marrow mesenchymal stem cellstransplantation

目的观察尾静脉注射骨髓间充质干细胞（MSCs）对小鼠视网膜激光损伤后细胞凋亡的影响。方法体外培养绿色荧光蛋白（GFP）标记的C57BL／6小鼠骨髓间充质干细胞（GFP-MSCs）。135只C57BL／6小鼠随机分为正常对照组、损伤对照组和MSCs治疗组，分别为15、60、60只。采用激光光凝方式建立视网膜激光损伤模型。激光光凝后1d，MSCs治疗组小鼠尾静脉注射0．2mlGFP-MSCs细胞悬液（含MSCs1×106个），损伤对照组小鼠尾静脉注射等体积磷酸盐缓冲液。分别于激光光凝后3、7、14、21d，处死各组小鼠并摘除眼球。采用苏木精一伊红（HE）染色观察视网膜组织形态，并用图像处理软件测量激光斑直径和外核层光感受器细胞核完全缺损区域直径；原位末端标记（TUNEL）法检测细胞凋亡情况；荧光显微镜观察MSCs治疗组GFP-MSCs的迁移情况。结果光学显微镜观察发现，正常对照组视网膜组织结构完整；损伤对照组及MsCs治疗组均可见视网膜组织结构损伤，但MSCs治疗组较损伤对照组损伤程度减轻。激光光凝后3d，损伤对照组与MSCs治疗组激光斑直径比较，差异无统计学意义（t=0．964，P〉0．05）；MSCs治疗组外核层细胞核完全缺损区域直径小于损伤对照组，差异有统计学意义（t=5．769，P〈0．05）。激光光凝后7、14、21d，MSCs治疗组激光斑直径（t=5．180、5．417、2．381）和外核层细胞核完全缺损区域直径（t=3．530、3．224、3．162）均小于损伤对照组，差异均有统计学意义（P〈O．05）。激光光凝后3、7、14、21d，正常对照组小鼠视网膜内均未见凋亡细胞；损伤对照组及MSCs治疗组小鼠损伤部位视网膜内可见凋亡细胞；MSCs治疗组平均细胞凋亡数均小于损伤对照组，差异均有统计学意义（t=11．142、7．479、6．678、3．953，P〈0．05）。荧光显微镜观察发现，激光光凝后3、7、14、21d均未见MSCs治疗组GFP-MSCs大量向视网膜内迁移；仅于激光光凝后7、14d时在视网膜下及脉络膜新生血管处有少量迁移。结论尾静脉注射MSCs能有效限制小鼠视网膜激光损伤范围及抑制细胞凋亡。

Objective To observe the retinal apoptosis of laser-induced retinal injury in mice after bone marrow mesenehymal stem cells transplantation. Methods Green fluorescent protein （GFP） labeled MSCs from C57BL/6 mice were cultured in vitro. A total of 135 C57BL/6 mice weredivided into three groups including normal control group （15 mice）, injured control group （60 mice） and MSCs treatment group （60 mice）. Laser retinal injuries were induced by laser photocoagulation. One day after photocoagulation, 0.2 ml cell suspension, which contained 1× 106 GFP-MSCs, were injected into the mice in treatment group via tail vein, and the mice in injured control group were given equal volume of phosphate buffer solution. Animal were execute on three, seven, 14 and 21 clays following laser damage. Hematoxylin and eosin （HE） staining was performed to assess the changes of injured retinas. The diameters of laser spots and areas with total loss of cells in outer nuclear layer （ONL） were analyzed by image processing software. The apoptosis of retinal cells was examined by terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） staining. The migration of GFP-MSCs into the retina was observed by fluorescence microscope. Results HE staining showed that the retinal structures were integrated in normal controlgroup. Retinal damages were observed both in injured control group and MSCs treatment group, but milder in the latter. Though the average diameter of area with total loss of cells in ONL of MSCs treatment group was less than the injured control group （t =5. 769, P〈 0.05）, the diameters of laser spots show no difference （t=0. 964, P〈0.05） on day three. Both the average diameter of laser spots （t=5. 180, 5. 417, 2. 381） and area with total loss of cells in ONL （t=3. 530, 3. 224, 3. 162） were less in the MSCs treatment group on day seven, 14 and 21 （P〈0.05）. TUNEL staining shows that the apoptosis were decreased after MSCs transplantation on day three, seven, 14 and 21 （t= 11. 142, 7. 479, 6. 678, 3. 953, P〈0.05）. No apoptosis was observed in normal control group. Very few GFP-MSCs were observed in the retina at all time-points. They were only seen in the subretinal and choroidal neovascularization occasionally on day seven and 14. Conclusion MSCs transplantation can effectively limit the range of retinal laser damage and inhibit cell apoptosis.