人脂肪干细胞移植对兔眼视网膜裂孔修复作用的研究
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摘要
目的:观察人脂肪源性干细胞(adipose-derived stem cells,ADSCs)移植入兔眼视网膜裂孔模型眼内,对裂孔修复的影响及眼内干细胞分化情况。
方法:新西兰白兔20只,取左眼,行玻璃体切除,制作直径约1.5mm的视网膜裂孔模型。一组动物于玻璃体腔内裂孔表面移植ADSCs 20μl或10μl;另一组注入等量生理盐水作为对照。分别于术后第2、4、8、12、20、32日行三维OCT检查,观察裂孔修复情况。术后第12日、32日取出兔眼,行组织学及人细胞核、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、视紫红质N端残基表位Opsin、蛋白激酶C(Protein kinase C,PKC)抗原的免疫荧光检测。
结果:三维OCT及眼底检查示两组视网膜裂孔在术后第4日有修复组织出现,对照组修复组织随时间延长逐渐增多,至20日后不再变化,裂孔中心组织最终厚度薄于正常视网膜。免疫荧光染色显示裂孔处细胞主要为GFAP阳性细胞,Opsin,PKC抗原阴性。ADSCs移植组裂孔修复组织生长迅速,修复组织量大,至术后12日不再变化,20μl ADSCs移植组裂孔组织明显隆起,远高出正常视网膜,其中一眼有增殖膜生成。10μl ADSCs移植组,修复组织稍隆起,无增殖膜出现。免疫荧光染色显示裂孔处修复细胞主要为GFAP抗原阳性细胞,尚有少量细胞Opsin或PKC抗原阳性。受体眼内有抗人细胞核单克隆抗体(anti-human nuclei monoclonalantibody,HuNu)染色阳性细胞存在,并可表达GFAP或Opsin或PKC抗原。
结论:玻璃体切除兔眼视网膜裂孔模型,术后短期裂孔能够自我修复,修复细胞主要为神经胶质细胞。人ADSCs玻璃体腔内裂孔部位移植,促进了受体自身细胞增殖,并促使受体眼修复细胞向感光细胞或双极细胞分化。在兔眼内视网膜损伤的环境中ADSCs亦可向感光细胞或神经胶质细胞或双极细胞分化。
Purpose:To investigate the morphological and differential changes of retinal hole repair tissue after human adipose-derived stem cells(ADSCs) transplantation in rabbit retinal hole models.And to investigate the differentiation of ADSCs in retinal hole eyes.
Methods:20 New Zealand white rabbit were used.Pars plana vitrectomy was performed in the left eye.And a 1.5mm-diameter retinal hole was made with an extrusion needle.In one group of rabbits We injected 20μl/10μl human ADSCs suspension solution into the eyes at the surface of retinal holes. In another group phosphate buffered saline(PBS) was injected as control. To assess the changes of retinal holes,at 2、4、8、12、20、32 days postoperatively eyes were examined with a 3D-OCT.2D-OCT,3D-OCT and fundus images were obtained.At 12 and 32 days after surgery,eyes were enucleated and prepared in paraffin sections for observation by light microscopy and immunofluorescence detect with antibodies to human nuclei,glial fibrillary acidic protein(GFAP),Opsin and Protein kinase C(PKC)
Results:3D-OCT showed that repairs occurred at the 4~(th) day after operation in both group.In the control group repair tissue proliferated as time passed by,and the maximum was reached at 20 days postoperatively.During the observation period,repair tissue was still thinner than the normal retina.And immunofluorescences showed that the area of retinal defect was covered with cells which were positive for GFAP only.In the ADSCs transplantation group the repair tissue grew more rapidly and generously, making a small bulge at the retina defect area.A proliferation membrane was formed In a 20μl transplantation group eye.Immuno-fluorescences showed that the area of retina hole was covered with cells which were positive for GFAP or Opsin or PKC.A few cells were positive for anti-human nucleus antibody(HuNu) in the recipient eyes,and also positive for GFAP or Opsin or PKC.
Conclusions:These findings suggest that in this model retina holes repair and close spontaneously.Glial cell is the main cell type in the repair process.Retina hole repair process is promoted by Human ADSCs transplantation. The human ADSCs also stimulate differentiation of repair cells, which express markers of sensory retina cell or bipolar cell.And human ADSCs in the micro-environment of retinal injury obtain the ability of differentiation to retina cell types.
方法:新西兰白兔20只,取左眼,行玻璃体切除,制作直径约1.5mm的视网膜裂孔模型。一组动物于玻璃体腔内裂孔表面移植ADSCs 20μl或10μl;另一组注入等量生理盐水作为对照。分别于术后第2、4、8、12、20、32日行三维OCT检查,观察裂孔修复情况。术后第12日、32日取出兔眼,行组织学及人细胞核、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、视紫红质N端残基表位Opsin、蛋白激酶C(Protein kinase C,PKC)抗原的免疫荧光检测。
结果:三维OCT及眼底检查示两组视网膜裂孔在术后第4日有修复组织出现,对照组修复组织随时间延长逐渐增多,至20日后不再变化,裂孔中心组织最终厚度薄于正常视网膜。免疫荧光染色显示裂孔处细胞主要为GFAP阳性细胞,Opsin,PKC抗原阴性。ADSCs移植组裂孔修复组织生长迅速,修复组织量大,至术后12日不再变化,20μl ADSCs移植组裂孔组织明显隆起,远高出正常视网膜,其中一眼有增殖膜生成。10μl ADSCs移植组,修复组织稍隆起,无增殖膜出现。免疫荧光染色显示裂孔处修复细胞主要为GFAP抗原阳性细胞,尚有少量细胞Opsin或PKC抗原阳性。受体眼内有抗人细胞核单克隆抗体(anti-human nuclei monoclonalantibody,HuNu)染色阳性细胞存在,并可表达GFAP或Opsin或PKC抗原。
结论:玻璃体切除兔眼视网膜裂孔模型,术后短期裂孔能够自我修复,修复细胞主要为神经胶质细胞。人ADSCs玻璃体腔内裂孔部位移植,促进了受体自身细胞增殖,并促使受体眼修复细胞向感光细胞或双极细胞分化。在兔眼内视网膜损伤的环境中ADSCs亦可向感光细胞或神经胶质细胞或双极细胞分化。
Purpose:To investigate the morphological and differential changes of retinal hole repair tissue after human adipose-derived stem cells(ADSCs) transplantation in rabbit retinal hole models.And to investigate the differentiation of ADSCs in retinal hole eyes.
Methods:20 New Zealand white rabbit were used.Pars plana vitrectomy was performed in the left eye.And a 1.5mm-diameter retinal hole was made with an extrusion needle.In one group of rabbits We injected 20μl/10μl human ADSCs suspension solution into the eyes at the surface of retinal holes. In another group phosphate buffered saline(PBS) was injected as control. To assess the changes of retinal holes,at 2、4、8、12、20、32 days postoperatively eyes were examined with a 3D-OCT.2D-OCT,3D-OCT and fundus images were obtained.At 12 and 32 days after surgery,eyes were enucleated and prepared in paraffin sections for observation by light microscopy and immunofluorescence detect with antibodies to human nuclei,glial fibrillary acidic protein(GFAP),Opsin and Protein kinase C(PKC)
Results:3D-OCT showed that repairs occurred at the 4~(th) day after operation in both group.In the control group repair tissue proliferated as time passed by,and the maximum was reached at 20 days postoperatively.During the observation period,repair tissue was still thinner than the normal retina.And immunofluorescences showed that the area of retinal defect was covered with cells which were positive for GFAP only.In the ADSCs transplantation group the repair tissue grew more rapidly and generously, making a small bulge at the retina defect area.A proliferation membrane was formed In a 20μl transplantation group eye.Immuno-fluorescences showed that the area of retina hole was covered with cells which were positive for GFAP or Opsin or PKC.A few cells were positive for anti-human nucleus antibody(HuNu) in the recipient eyes,and also positive for GFAP or Opsin or PKC.
Conclusions:These findings suggest that in this model retina holes repair and close spontaneously.Glial cell is the main cell type in the repair process.Retina hole repair process is promoted by Human ADSCs transplantation. The human ADSCs also stimulate differentiation of repair cells, which express markers of sensory retina cell or bipolar cell.And human ADSCs in the micro-environment of retinal injury obtain the ability of differentiation to retina cell types.
引文
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