摘要
目的以RCS视网膜色素变性大鼠为模型,研究经诱导产生神经干细胞特性的Müller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和胰岛素)刺激视网膜Müller细胞。1周后取材分离Müller细胞体外原代培养。将细胞标记后,以每眼1×105个细胞密度移植到6周龄RCS大鼠右眼视网膜下腔,左眼分组注射正常Müller细胞和PBS作为同型对照及阴性对照。术后分别于第1、3、5、7周,行视网膜铺片、组织病理学及视觉电生理检查。结果培养的Müller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。组织病理学观察显示,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)检查结果与病理学结果相符。结论视网膜下腔移植经诱导产生神经干细胞特性的Müller细胞可以有效延缓RCS大鼠视网膜光感受器变性,为治疗视网膜变性疾病提供了新的途径。
Objective The purpose of this study was to investigate whether subretinal transplantation of Müller 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 6-week-old VC rats （ten rats） to activate Müller cells and the physical salt solution was injected in the left eyes as control. One week later, the activated Müller cells were gathered, cultured and purified. After being labeled with DAPI, the activated Müller cells were transplanted into subretinal space of the right eyes of 6-week-old RCS rats （ eight rats） , and the left eyes received unactivated Müller cells as homotype control in three RCS rats and PBS as negtive control eyes in three RCS rats. The histologic evaluation 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 purified Müller 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 Müller cells were seen in the retina of transplantation zone and only one layer in non-transplantation zone under the light microscope. The number of survival photoreceptors in transplantation eyes of Müller cells was significantly increased than that of control eyes （P 〈 0. 01 ）. The a-wave amplitudes of electrophysiology showed a statistically significant difference among the active cells injection group,inactive 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, inactive cells injection group and negative control group at 1,3,5 and 7 weeks after injection （ P 〈 0. O1 ）. Conclusion Subretinal transplantation of Müller glia with neural stem cell properties can effectively delay photoreceptor degeneration and functional deterioration time in RCS rats, which offer a new method of theraputic way of the retinal degenerative disease.
出处
《眼科研究》
CSCD
北大核心
2009年第4期279-284,共6页
Chinese Ophthalmic Research
基金
国家自然科学基金资助(30271391)
