EPO修饰增强Müller细胞对RCS大鼠视网膜变性的干预效果  

hEPO gene modified Müller cells protects against retinal degeneration of RCS rats

作　　者：李宗义[1] 李鹏[2] 高芙蓉[3] 范氏雪幸 张敬法[2] 王方[1] 吕立夏[2] 徐国彤[2] 

机构地区：[1]同济大学附属第十人民医院眼科,上海200092 [2]同济大学医学院眼科研究所及干细胞研究中心 [3]同济大学生命科学与技术学院

出　　处：《中华细胞与干细胞杂志（电子版）》2014年第1期41-47,共7页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家重点基础研究发展计划(2013CB967501);国家自然科学基金(C120111;31171419);高等学校博士学科点专项科研基金(新教师基金:20100072120051)

摘　　要：目的研究人源促红细胞生成素(hEPO)修饰的Müller(hEPO-Müller)细胞对视网膜退行性病变大鼠的干预作用。方法通过质粒转染法构建hEPO和GFP的Müller细胞稳转株(hEPO-Müller和GFP-Müller);以体外共培养和体内细胞移植为研究体系,利用RT-PCR和冰冻切片及免疫荧光染色的方法检测hEPO-Müller对RCS大鼠视网膜退行性病变的干预作用。内核层与外核层厚度比较采用t检验。结果本实验成功构建了hEPO-Müller和GFP-Müller细胞系。将RCS大鼠的视网膜组织剥离并在体外不同条件下培养两周后测定视网膜各核层厚度发现,与对照细胞裂解液共培养组的内核层(15.94±1.77)μm和外核层(24.81±3.03)μm的厚度相比较,两核层的厚度分别在hEPO组为(23.03±3.29)μm,(33.92±7.59)μm(P<0.05);Müller组为(24.81±2.02)μm,(32.15±3.03)μm(P<0.05);hEPO-Müller组为(32.40±8.35)μm,(40.25±3.29)μm(n=3,P<0.01);以hEPO-Müller组厚度增加最为显著(P<0.05)。提示EPO和Müller细胞对视网膜变性都有干预作用且两者可以叠加。将hEPO-Müller和GFP-Müller分别移植到RCS大鼠的视网膜下腔,四周后取视网膜进行冰冻切片检测,染色结果显示,细胞移植后有更多的外核层细胞存活,且同样也是hEPO-Müller组的外核层细胞更多。此外,Müller移植并不会促进视网膜的胶质化。结论移植Müller细胞可以减缓RCS大鼠视网膜变性,而经hEPO修饰的Müller细胞对视网膜变性有更好的干预作用。因此,Müller细胞可以作为一种供体细胞兼携带hEPO等营养因子的载体用于视网膜变性的治疗。Objective To study the effects of hEPO gene modified Müller cells （hEPO-Müller） in the rat model of retinal degeneration. Methods The hEPO-Müller and GFP modified Müller cells （GFP-Müller） were established by the transfection of Müller cells with hEPO or GFP plasmids, and then selected by Puromycin. To study the effect of hEPO-Müller, retinal explant cultures and subretinal transplantation of hEPO-Müller and GFP-Müller cells were used. The retinas were embedded, sectioned and stained or collected for RT-PCR. Results The stable hEPO-Müller and GFP-Müller were established. The retinal tissue from the 21-day-old RCS rats were dissected and cultured under 5 different conditions for 2 weeks. The thicknesses of the inner nuclear layer （INL） and the outer nuclear layer （ONL） with the Müller lysate were （15.94 ± 1.77）μm and （24.81 ± 3.03）μm, respectively. Compared with these, the thicknesses of INL and ONL were （23.03 ± 3.29）μm,（33.92 ± 7.59）μm in hEPO group （P 〈 0.05）;（24.81 ± 2.02）μm,（32.15 ± 3.03）μm in Müller group （P〈0.05）;（32.40 ± 8.35）μm,（40.25 ± 3.29）μm in hEPO-Müller group （n=3,P〈0.01）. And the thicknesses in hEPO-Müller group were thicker significantly （P 〈 0.05）. The results suggested that both hEPO and Müller can intervene the retinal degeneration and these two can work together. Then, the hEPO-Müller or GFP-Müller was transplanted into the subretinal space of 21-day-old RCS rats. Four weeks later, the expressions of GFAP and Vimentin, the markers of gliosis, were found not increased in these rats, when compared with the control group. DAPI and Recoverin staining showed more photoreceptor cells survived in the rats treated with hEPO-Müller or GFP-Müller transplantation, and there were much more photoreceptors in the hEPO-Müller group. Conclusion Müller cell can slow down the process of retinal degeneration in RCS rats, and the hEPO modified Müller cells can further improve such effects.

关 键 词：细胞移植 视网膜变性 红细胞生成素 MÜLLER细胞 

分 类 号：R774.1[医药卫生—眼科]