BMSCs视网膜下移植对大鼠视网膜光感受器细胞凋亡的影响及机制研究

Effect and mechanism of BMSCs subretinal transplantation on rat retinal photoreceptor cell apoptosis

目的研究骨髓间充质干细胞(BMSCs)视网膜下移植对大鼠视网膜光感受器细胞凋亡的影响及机制。方法取32只6周龄的SPF级SD雄性大鼠,随机分为A组(未进行任何处理)、B组(于视网膜下注入无菌生理盐水)、C组(以BMSCs为溶质,于视网膜下注入磷酸盐溶液)、D组(于视网膜下移植BMSCs2×10~5/g个),每组各8只。其中,C、D组采用光照射1 d,于7 d后进行手术,术后2周摘除眼球。大鼠BMSCs原代和传代培养,切片并行苏木精-伊红染色,观察BMSCs细胞形态学情况,并对视网膜外核层的层数进行计算。根据免疫荧光法,对大鼠眼内BMSCs中的睫状神经营养因子(CNIF)、碱性成纤维细胞生长因子(b FGF)、脑源性神经营养因子(BDNF)的表达水平进行检测,并根据Tunel细胞凋亡检测试剂盒对细胞凋亡情况进行检测。结果 BMSCs细胞刚接种时呈圆形,大小均匀,胞体透亮,胞膜清晰;混有红细胞且呈扁平状、大小不均。BMSCs细胞接种1 d后,贴壁细胞出现,且呈多角形、椭圆形或梭形,而在定期换液中未贴壁的细胞逐渐被清除。BMSCs细胞接种1周后迅速增长,2周后80%以上的贴壁细胞融合,并呈现漩涡状。D组视网膜外核层的层数明显少于A组,而明显多于B、C组(P <0. 01)。D组细胞凋亡率明显高于A组,而明显低于B、C组(P <0. 01)。DAPI阳性的BMSCs于视网膜下腔并不表达CNIF与BDNF,但其表达b FGF。结论于视网膜下移植BMSCs具有抑制视网膜光感受器细胞凋亡的作用,作用机制可能与其于视网膜下腔分泌b FGF有关。

Objective To study the effect of subretinal transplantation of bone marrow mesenchymal stem cells(BMSCs)on rat retinal photoreceptor cell apoptosis and its mechanism.Methods Thirty-two 6-week-old SPF SD male rats were randomly divided into group A(without any treatment),group B(injecting sterile saline into the retina),group C(injecting phosphate solution into the retina with BMSCs as solute),and group D(transplanting BMSCs into the retina with 2×105/g),with 8 rats in each group.Group C and D were treated with light for 1 day,followed by surgery 7 days later,and eyeballs were removed 2 weeks after operation.The layers of outer retinal nucleus were calculated by hematoxylin-eosin staining in primary and passage culture of rat BMSCs.Morphology of BMSCs cells was observed and the layers of the outer retinal nucleus were calculated.The expression of ciliary neurotrophic factor(CNIF),basic fibroblast growth factor(bFGF)and brain-derived neurotrophic factor(BDNF)in rat BMSCs was determined by immunofluorescence method.The level of apoptotic cells was detected,and the apoptotic status was detected according to the Tunel cell apoptosis detection kit.Results When BMSCs cells were inoculated,showing round,uniform in size,transparent in body and clear in membrane,and mixed with red blood cells,which were flat and uneven in size.One day after BMSCs cells were inoculated,adherent cells appeared in polygonal,elliptical or spindle shape,while the non-adherent cells were gradually removed by regular fluid exchange.BMSCs cells grew rapidly after 1 week of inoculation,and more than 80%adherent cells fused after 2 weeks,showing a whirlpool shape.The number of layers of the outer retinal nucleus in group D was significantly less than that in group A,but significantly more than that in group B and C(P<0.01).The apoptotic rate in group D was significantly higher than that in group A,but significantly lower than that in group B and C(P<0.01).DAPI-positive BMSCs did not express CNIF and BDNF in the subretinal space,but expressed bFGF.Conclusion Subretinal transplantation of BMSCs can inhibit the apoptosis of photoreceptor cells,and the mechanism may be related to the secretion of bFGF in the subretinal space.