摘要
目的以RCS大鼠为模型,研究经诱导产生神经干细胞特性的Muller细胞视网膜下腔移植对光感受器变性的保护作用。方法向6周龄VC大鼠玻璃体腔内注射神经毒素混合液(包含NMDA、Kainate、FGF2和insulin)刺激视网膜M(u|¨)ller细胞。1周后,取材分离M(u|¨)ller细胞体外原代培养纯化。将细胞标记后,以每眼1×10~5个细胞的密度移植到6周龄RCS大鼠右眼视网膜下腔,左眼分组注射正常M(u|¨)ller细胞和PBS作为同型对照以及阴性对照。术后分别于第1、3、5、7周,行视网膜铺片、病理切片以及视觉电生理检查。结果培养的M(u|¨)ller细胞纯度可达96%以上,其中表达神经干细胞标志物的细胞占总细胞量的53%以上。光镜病理切片观察显示,在相同时间点,移植眼保留的光感受器细胞数量明显较同型对照眼多,同型对照移植眼保留的光感受器细胞数量明显较阴性对照眼多。视网膜电图(ERG)检查结果同病理结果相符。结论视网膜下腔移植经诱导产生神经干细胞特性的M(u|¨)ller细胞可以有效延缓RCS大鼠视网膜光感受器变性,为治疗视网膜变性性疾病提供了新的途径。
Objective The purpose of this study was to investigate whether subretinal transplantation of M(u|¨)ller cells with neural stem cell properties can rescue photoreceptor degeneration in RCS rat.Methods The mixed neurotoxins solution containing N-methyl-D-aspartate(NMDA),kainate,fibroblast growth factor-2(FGF2) and insulin was intravitreally injected in the right eyes of 10 6-week-old Vneversity College(VC) rats to activate M(u|¨)ller cells and the physical salt solution was injected in the left eyes as control. One week later,the activated M(u|¨)ller cells were gathered,cultured and purified.After being labeled with DAPI,the activated M(u|¨)ller cells were transplanted into subretinal space of the right eyes of 8 6-week-old RCS rats,and the left eyes received unactivated M(u|¨)ller cells as homotype control in 3 RCS rats and PBS as negtive control eyes in 3 RCS rats.The histologic assessment and electroretinogram(ERG) were carried out at the 1st,3rd,5th and 7th week after injection.Results Fluorescence-activated cell sorting(FACS) showed that the puritied M(u|¨)ller cells labeled by glutamine synthetase was above 96.07%,and 53.47% of cells presented positive fluorescene staining for nestin.Four to five layer of survival M(u|¨)ller cells were seen in the retina of transplantation zone and only one layer in non-transplantation zone under the light microscope.The numbers of survival photoreceptors in transplantation eyes of M(u|¨)ller cells were significantly increased than those of control eyes(P<0.01).The a-wave amplitudes of electrophysiology showed a statistically significant difference among the active cells injection group,unactive cells injection group and negative control group at 1,5 and 7 weeks after injection(P<0.01),and those of b wave were significantly different among the active cells injection group, unactive cells injection group and negative control group at 1,3,5 and 7 weeks after injection(P<0.01).Conclusion Subretinal transplantation of M(u|¨)ller glia with neural stem cell properties can effectivelly delay photoreceptor degeneration and functional deterioration time in RCS rats,which offer a new method of treating the retinal degenerative disease.
引文
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