猪的诱导性多潜能干细胞向视网膜感光细胞分化的体内及体外研究

发布时间：2018-07-09 09:24

本文选题：猪 + iPSC　；参考：《中南大学》2011年博士论文

【摘要】：目的：探讨猪的诱导性多潜能干细胞(piPSC)在体外分化成视网膜神经细胞的方法。研究piPSC移植治疗猪的视网膜损伤的效果。材料与方法：piPSC在放射处理后的SNL细胞上培养并传代。取第28代、第40代和第43代piPSC分别悬浮培养3天后形成胚胎体,再将胚胎体转至未稀释基质胶,1：10和1：20稀释的基质胶以及层粘连蛋白-纤维连接蛋白基质上粘附培养以使其分化。用免疫荧光染色和实时PCR技术检测piPSC以及分化细胞中干细胞标志物OCT4、ABCG2、Nanog、Sox2,神经细胞标志物TUBB3,感光细胞标志物RCVRN、NRL、RHO、IRBP、ROM1和ARR3以及视杆细胞型双极细胞标志物PRKCA的表达,计数表达上述标志物的细胞的比例以及在形态上具有外节结构细胞的比例。用带IRBP-GFP基因的慢病毒感染分化后的细胞,使视锥型和视杆型感光细胞表达绿色荧光蛋白(GFP)。碘乙酸(IAA)静脉注射建立猪视杆细胞损伤的动物模型,将感染GFP的piPSC分化细胞移植入的一侧病眼视网膜下,对侧病眼视网膜下注射DMEM/F12培养液作为对照。分别在用药前及细胞移植术后三周检测视网膜电生理反应(ERG)。术后3周摘取视网膜,切片,通过免疫荧光染色观察移植后的细胞在病变视网膜中的整合情况。结果：实时PCR和免疫荧光染色显示分化后的细胞失去干细胞特异性基因OCT4、Nanog和Sox2的表达,转而表达视网膜感光细胞基因RCVRN、NRL、RHO、IRBP、ROM1和cone-arrestin。其中有30.91±7.2%的分化细胞表达转录因子NRL,25.61±8.5%表达RCVRN,6.765±2.083%表达RHO,此外还有2.6±0.4%的细胞表达PRKCA。以层粘连蛋白—纤维连接蛋白为细胞外基质可以分化出更多的RHO阳性细胞,但其外形更类似普通神经细胞。以基质胶为细胞外基质可以分化出更多的具有视杆细胞形态的细胞,即有外节结构的细胞。用携带IRBP-GFP基因的病毒感染分化细胞后,约44%的细胞表达GFP。这些细胞已经定向分化成视网膜感光细胞前体细胞或成熟细胞。移植到视网膜下后可检测到RHO阳性的细胞在视网膜各层均有表达,其主要表达部位在视网膜外核层,即正常的感光细胞所在的位置。在这些整合入视网膜的移植细胞中,有一部分细胞具有外节结构。结论：piPSC能在体外高效地分化成视网膜神经细胞并且部分细胞具有与原代培养的视杆细胞类似的外节样结构。将这些分化的细胞移植入视杆细胞损伤的猪视网膜下方,移植细胞可以整合入受损视网膜。部分移植细胞在眼内分化出外节结构,这可能有助于视觉功能的恢复。
[Abstract]:Aim: to investigate the method of differentiation of porcine induced multipotential stem cells (piPSC) into retinal neurons in vitro. To study the effect of piPSC transplantation in the treatment of retinal injury in pigs. Materials and methods: PSC was cultured and subcultured on SNL cells after irradiation. In the 28th passage, the 40th generation and the 43rd generation of piPSC were cultured for 3 days, respectively, and the embryonic body was formed. The embryo body was then transferred to the undiluted matrix glue 1: 10 and 1:20 diluted matrix glue and laminin-fibronectin matrix adhesion culture to make it differentiate. Immunofluorescence staining and real-time PCR techniques were used to detect the expression of stem cell marker OCT4, ABCG2NogSox2, neuronal marker TUBB3, photoreceptor cell marker RCVRNNNRHORHON, IRBPROM1 and ARR3, and rod cell type bipolar cell marker PRKCA, respectively. The proportion of cells expressing these markers and the proportion of cells with outer ganglion structure were counted. The differentiated cells were infected with lentivirus with IRBP-GFP gene, and the green fluorescent protein (GFP) was expressed in the cone-type and rod type photoreceptor cells. PiPSC differentiated cells infected with GFP were transplanted into the subretinal of unilateral diseased eyes and DMEM- F12 culture medium was injected into the retina of the contralateral diseased eyes. Retinal electrophysiological response (ERG) was measured before medication and 3 weeks after cell transplantation. The retina was removed 3 weeks after operation and the integration of the transplanted cells in the diseased retina was observed by immunofluorescence staining. Results: Real-time PCR and immunofluorescence staining showed that the differentiated cells lost the expression of stem cell-specific genes OCT4Nanog and Sox2, and instead expressed the retinal photoreceptor cell genes RCVRN- RHOBPP-ROM1 and cone-arrestinin. Among them, 30.91 卤7.2% of the differentiated cells expressed the transcription factor NRLN 25.61 卤8.5%, and 6.765 卤2.083% of the differentiated cells expressed RHO.In addition, 2.6 卤0.4% of the cells expressed PRKCA. Using laminin-fibronectin as extracellular matrix, more Rho positive cells can be differentiated, but their appearance is more similar to normal nerve cells. Using matrix glue as extracellular matrix, more cells with the shape of rod cells, that is, cells with outer ganglia structure, can be differentiated. After infecting differentiated cells with IRBP-GFP, about 44% of the cells expressed GFP. These cells have been directed to differentiate into retinal photoreceptor cells, precursor cells or mature cells. The expression of RHO-positive cells was detected in all layers of the retina after transplantation to the retina, and the expression was mainly located in the outer nuclear layer of the retina, that is, the location of the normal photoreceptor cells. Some of the transplanted cells integrated into the retina have an outer ganglion structure. ConclusionTwo pipiPSC can effectively differentiate into retinal nerve cells in vitro, and some of the cells have an outer nodal structure similar to that of primary cultured rod cells. These differentiated cells are transplanted under the damaged porcine retina, which can be integrated into the damaged retina. Some of the transplanted cells differentiate into nodal structures in the eye, which may contribute to the restoration of visual function.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1

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