Growth-associated protein-43 gene modified adipose-derived mesenchymal stem cells protect against retinitis pigmentosa

doi:10.3969/j.issn.2095-4344.2017.25.007

Abstract

Abstract:

BACKGROUND: Gene therapy and cell transplantation therapy, especially their combined use, have certain therapeutic effects on retinitis pigmentosa. However, little attention has been paid to the combination of gene therapy and cell transplantation in the treatment of retinitis pigmentosa.
OBJECTIVE: To investigate the protective effect of adipose-derived mesenchymal stem cells (ADMSCs) modified by growth-associated protein-43 (GAP-43) gene on the retina of rats with retinitis pigmentosa.
METHODS: ADMSCs from rats with retinitis pigmentosa were isolated and cultured, and then transfected with GAP-43 lentiviral vector. Sixty retinitis pigmentosa rats were randomized into experimental group (subretinal injection of GAP-43-transfected ADMSCs), control group (subretinal injection of ADMSCs) and sham operation group (PBS injection). The expressions of GAP-43 protein, Rho protein and GS protein in the retina of rats were determined by western blot. The outer nuclear layer thickness of the retina was determined by hematoxylin-eosin staining.
RESULTS AND CONCLUSION: After transfected cell transplantation, the expression of GAP-43 protein in the retina was gradually increased with time and showed significant differences at different time post-transplantation (P < 0.05). The expression levels of Rho protein and GS protein in the retina of experimental and control groups were higher than that in the sham operation group (P < 0.05). The expression of Rho protein in the retina of the experimental group was higher than that in the control group (P < 0.05). The expression of GS protein in the retina of the experimental group was lower than that in the control group (P < 0.05). The thickness of retinal outer nuclear layer was ranked as follows: the experimental group > the control group > the sham group, and there was significant difference between groups (P < 0.05). These results show that the ADMSCs modified by GAP-43 gene have protective effect on the retina of rats with retinitis pigmentosa.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Mesenchymal Stem Cells, GAP-43 Protein, Retinal Degeneration, Tissue Engineering

CLC Number:

R394.2

Gao Jun-yan, Zhang Yun, Li Ming, Liu Xue-min, Wu Zhi-bing, Cao Yan-fei. Growth-associated protein-43 gene modified adipose-derived mesenchymal stem cells protect against retinitis pigmentosa[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 3977-3982.

Figures/Tables 2

2.1 脂肪间充质干细胞的培养及鉴定脂肪间充质干细胞原代培养过程中逐渐贴壁，48 h后换液去掉未贴壁生长细胞，贴壁脂肪间充质干细胞呈梭形；当脂肪间充质干细胞培养至第3代时，细胞呈规则的梭形生长，见图1。脂肪间充质干细胞CD29、CD90、CD49d呈阳性表达，阳性表达率分别为98.34%、99.47%、87.02%；CD106呈阴性表达(0.82%)。表明培养的为脂肪间充质干细胞。 2.2 Western Blot检测脂肪间充质干细胞GAP-43蛋白表达情况感染LV5-GAP病毒的脂肪间充质干细胞可表达GAP-43蛋白，表达量为(96.26±5.24)%，阴性对照组和空白对照组脂肪间充质干细胞不表达GAP-43蛋白，均为(0.01±0.01)%，见图2。 2.3 大鼠视网膜内脂肪间充质干细胞的变化情况脂肪间充质干细胞移植后，在大鼠变性视网膜内逐渐消失，移植后3 d见荧光细胞，移植后7 d荧光细胞明显减少，移植后21 d基本无荧光细胞，见图3。 2.4 大鼠视网膜内GAP-43蛋白表达情况实验组脂肪间充质干细胞移植后，随着时间的延长，GAP-43的表达量逐渐升高，脂肪间充质干细胞移植后3，7，14，21 d的GAP-43蛋白相对表达量分别为0.12±0.03、0.37±0.05、0.89±0.12及1.38±0.23，不同时间点间两两比较差异均有显著性意义(P < 0.05)。 2.5 大鼠视网膜Rho蛋白和GS蛋白表达情况移植后 28 d，3组之间视网膜Rho蛋白和GS蛋白表达量比较差异有显著性意义，其中实验组和对照组大鼠视网膜Rho蛋白和GS蛋白表达量高于假手术组(P < 0.05)；实验组大鼠视网膜Rho蛋白表达量高于对照组(P < 0.05)，GS蛋白表达量低于对照组(P < 0.05)，见表1。 2.6 大鼠视网膜外核层厚度移植后28 d，实验组、对照组、假手术组大鼠视网膜外核层厚度分别为(76.35± 14.65)，(53.24±12.13)，(25.46±7.48) μm，组间两两比较差异均有显著性意义(P < 0.05)。"

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