【作者】 段平；

【导师】 王一；

【作者基本信息】 第三军医大学 ， 眼科学， 2013， 博士

【摘要】 研究背景和目的：视网膜色素上皮（retinal pigment epithelial，RPE）细胞的变性、坏死和丢失可造成视网膜和脉络膜的损害，导致视网膜变性类疾病的发生和视力下降。目前尚无有效治疗RPE变性的方法。细胞移植是治疗RPE细胞变性的具有前景的方法，但是如何获得数量充足的、低免疫排斥和低成瘤风险的RPE细胞是目前细胞治疗RPE变性的所面临的主要问题。而在可用于视网膜变性疾病治疗的干细胞中，胚胎干细胞（embryonic stem cells，ESCs）因其全能分化特性（可分化为包括RPE在内的体内所有细胞类型）和无限增殖能力，被认为是可用于视网膜变性类疾病治疗的重要种子细胞。然而，ESCs来源的治疗细胞的免疫排斥和成瘤风险、以及不可控增殖、伦医学等问题限制了ESCs的临床转化。和ESCs相比，目前已在临床广泛应用的自体成体干细胞可避免免疫排斥和成瘤风险，且自体取材可规避伦理障碍。在本课题中，我们重点研究是否能将成体干细胞中的间充质干细胞（mesenchymal stem cells，MSCs）诱导分化为RPE样的细胞，并且具备天然RPE细胞的生理功能，并最终转化成为治疗视网膜变性类疾病的理想种子细胞。方法：1、hMSCs和猪RPE细胞的分离（1）原代分离和纯化获得hMSCs和猪RPE细胞，观察细胞形态；（2）hMSCs和猪RPE细胞标记检测、hMSCs成骨诱导分化钙结节茜素红染色、成软骨诱导分化甲苯胺蓝染色和成脂肪诱导分化油红-0染色鉴定hMSCs；免疫荧光鉴定猪RPE细胞。2、hMSCs和猪RPE的共培养及其向RPE细胞的分化（1）Transwell共培养法诱导hMSCs向RPE细胞分化；条件培养基法诱导hMSCs向RPE细胞分化；（2）hMSCs来源RPE细胞的鉴定细胞形态学观察、细胞内色素含量的定量分析、透射电镜检测、Real-time PCR、免疫荧光染色检测、吞噬实验和细胞断层扫描、Western Blot、验证MERTK蛋白在诱导细胞特异性吞噬功能中的作用和诱导细胞的神经营养因子分泌能力检测。3、hMSCs来源RPE细胞对急、慢性视网膜变性的修复作用（1）碘酸钠注射制备急性视网膜变性大鼠模型及鉴定：HE染色和F-ERG检测；（2）急性视网膜变性大鼠模型视网膜下腔移植分别于移植后4周、8周检测以下指标：细胞形态学观察、免疫荧光染色检测hESCs来源的RPE细胞、吞噬实验和细胞断层扫描、移植后免疫荧光染色检测、F-ERG和Western Blot。（3）选取21天龄的RCS白化大鼠进行视网膜下腔移植，分别于移植后4周、8周检测以下指标：外核层厚度的测量、移植后细胞的免疫荧光染色检测、F-ERG和Western Blot检测细胞在体的吞噬能力。结果：1、hMSCs的鉴定（1）hMSCs形态多为梭形成纤维细胞样外观，呈旋涡状排列；猪RPE细胞外形呈多边形，细胞内充满了棕黑色的色素颗粒；（2）hMSCs的表面标志CD73、CD90、CD105表达强阳性，而CD14、CD45、CD34阴性；茜素红染色后，细胞间出现致密的、圆形不透光团块状的红色结节；阿新蓝染色可见细胞外基质被染成蓝色，成条索状错综排列；油红-0染色细胞中含有被染成红色的脂肪颗粒；猪RPE细胞CRALBP、RPE65、ZO-1表达均为阳性。2、hMSCs向RPE细胞的分化（1）hMSCs共培养诱导后的RPE样细胞可观察到hMSCs中出现丰富的色素颗粒，含量接近于成体猪的RPE细胞，而采用条件培养基诱导法获得细胞色素含量较少；（2）共培养hMSCs-RPE细胞的色素含量与RPE细胞相比无统计学差异，条件培养基诱导hMSCs-RPE细胞内色素颗粒含量与共培养hMSCs-RPE细胞的色素含量相比具有统计学差异；（3）透射电镜观察见hMSCs-RPE细胞内可见成熟的色素颗粒，细胞顶端可见典型的微绒毛结构；（4）hMSCs-RPE细胞表达RPE细胞特异的基因MITF、OTX2、tyrosinase、RPE65、Bestrophine、PEDF、PMEL17、CRALBP、PAX6和诱导前相比都显著上调，且和原代分离的人RPE细胞的表达水平相类似；（5）hMSCs-RPE细胞可表达RPE细胞特异的蛋白CRALBP、RPE65和ZO-1；（6）FITC标记的POS和hMSCs-RPE细胞共孵育后，猪RPE细胞和诱导细胞内均可观察到绿色荧光，而未诱导的hMSCs内未见到绿色荧光；hMSCs-RPE细胞MERTK蛋白表达显著上调，αvβ5Integrin的表达也有所上调， MERTK抗体封闭掉该受体的功能后，吞噬POS的能力显著下降；（7）hMSCs-RPE细胞BDNF和GDNF的分泌量上调明显，但仍显著低于猪RPE细胞。3、hMSCs来源RPE细胞对急、慢性视网膜变性的修复作用（1）碘酸钠损伤模型的视网膜HE染色可见后极部RPE细胞几乎完全损毁，部分RPE细胞死亡后残留的色素团块残留于Bruch’s膜上，上方的感光细胞层呈波浪形；F-ERG显示A波和B波的幅值较正常大鼠均显著降低；(2）碘酸钠损伤的急性视网膜变性模型中，移植后4周、8周视网膜免疫荧光染色共聚焦扫描可见，hMSC来源的RPE细胞可表达mitochondrial和CRALBP；（3）碘酸钠损伤的急性视网膜变性模型中，移植后4周、8周F-ERG结果显示了hMSCs-RPE细胞移植后较伪手术组和移植未诱导分化的hMSC组，A波和B波的波幅明显升高，具有统计学意义；（4）碘酸钠损伤的急性视网膜变性模型中，移植后4周和8周视网膜总蛋白的Western Blot结果显示rhodopsin的表达量在移植hMSCs-RPE细胞组较伪手术组和移植hMSC组明显升高，具有统计学意义。（5）RCS慢性视网膜变性模型中，移植后4周和8周hMSCs-RPE细胞组较伪手术组和移植hMSC组大鼠视网膜外核层厚度明显增厚，具有统计学意义；（6）RCS慢性视网膜变性模型中，移植后4周和8周视网膜免疫荧光染色共聚焦扫描可见，hMSC来源的RPE细胞可表达mitochondrial和CRALBP，但在8周时间点移植细胞存活较4周明显减少；（7）RCS慢性视网膜变性模型中，移植后4周F-ERG结果显示hMSCs-RPE细胞组较伪手术组和移植hMSC组，A波和B波的波幅明显升高，，具有统计学意义；而在移植后8周，各组电生理均呈熄灭型，各组间幅值无统计学差异；（8）RCS慢性视网膜变性模型中，移植后4周、8周视网膜总蛋白的Western Blot结果显示，MERTK蛋白的表达在移植hMSC来源的RPE细胞组4周为阳性8周时呈阴性，伪手术4周、8周均为阴性；（9）RCS慢性视网膜变性模型中，移植后4周和8周视网膜免疫荧光染色共聚焦扫描可见，4周时RPE65阳性的移植细胞内存在rhodopsin阳性的感光细胞外节，而8周时未见RPE65阳性的移植细胞内存在rhodopsin阳性的感光细胞外节。结论：1、成功分离获得了hMSCs和猪RPE细胞，获得细胞的细胞形态和细胞表面标志符合hMSCs和猪RPE细胞的特性；2、通过Transwell构建hMSCs和RPE细胞的体外非接触共培养体系，诱导hMSCs向RPE细胞分化，诱导的RPE细胞的体外生物学特性和功能类似于原代分离的RPE细胞。3、碘酸钠尾静脉注射制造的急性视网膜变性大鼠模型，其视网膜组织和结构改变符合急性视网膜变性的改变；4、碘酸钠损伤的急性视网膜变性大鼠经移植hMSCs-RPE细胞后，对感光细胞具有更好的保护作用、对视功能有更好的维持作用；5、在RCS大鼠慢性视网膜变性模型中，移植hMSCs-RPE细胞后，具有吞噬感光细胞外节的能力；但在移植后8周存活细胞数量显著降低，少量细胞表达成熟RPE细胞的标记，未观察到移植细胞具有吞噬感光细胞外节的能力，对视功能的维持作用消失。更多还原

【Abstract】 Background：The degeneration, necrosis and the final loss of RPE cells usually cause the damage ofphotoceptors and vision loss. Till now, there is no effective therapy to treat RPEdegeneration. The major issue in the cell therapy in the retina degeneration is how to getenough RPE cells because the cells are low immune rejection and tumorigenicity. Thedevelopment of stem cell technology provides a novel choice for the treatment of retinaldiseases. Especially, embryonic stem cells (ESCs) are considered to be important seed cellsfor the treatment of retinal degenerative diseases. However, ESCs usually have the risks ofimmune rejection and tumor formation. Compared with the clinical application of ESCs,the adult stem cells, autologous and non-ethical advantage, can avoid immune rejection andthe risk of tumor formation mesenchymal stem cells (MSCs) can be induced to differentiateinto RPE-like cells and may become the promising cells for the treatment of retinaldegenerative diseases. However, the biological characteristics and functions of the MSCsderived from RPE cells are still fully understood.Methods：1. Isolation and identification of hMSCs, porcine RPE cells1.1Isolation of hMSCs, porcine RPE cells1.2. FACS and identification of differention properties of hMSCs; Identification ofporcine RPE cells by immunofluorescence2.Inducing RPE differentiation of hMSCs2.1. co-culturing with RPE using transwell and RPE conditioned medium inducedhMSCs to differentiate to RPE like cells 2.2. Identification of co-culture induced hMSCs to differentiate to RPE like cells：cellmorphology, intracellular pigment content quantitative analysis, transmission electronmicroscopy detection, Real-time PCR, immunofluorescence staining, phagocytosis and celltomography experiments, Western Blot, verify MERTK protein in inducing cell-specificphagocytosis role and induce cell secretion of neurotrophic factors testing.3.Transplantation of hMSC-RPE in acute and chronic retinal degeneration model3.1. Building sodium iodide treated acute retinal degeneration rat model; identificationof the model: HE staining and F-ERG testing.3.2. Subretinal transplatation of hMSC and hMSC derived RPE cell in the acutesubretinal transplantation: cell morphology, immunofluorescence staining hMSCs derivedRPE cells, phagocytic assay and immunofluorescence staining, F-ERG and Western Blotafter transplantation.3.3. subretinal transplantation in RCS rats model: measuring the thickness of the outernuclear layer, immunofluorescence staining, F-ERG and phagocytosis assay aftertransplantation.Results：1. Isolation and identification of hMSCs, porcine RPE cells1.1. hMSCs morphology formed mostly spindle fibroblast-like appearance andvortex-like arrangement. Porcine RPE cells are polygonal in shape, and filled withbrown-black pigment granules.1.2. Flow cytometry analysis revealed that hMSCs were strongly positive for CD73,CD90and CD105, but negative for CD14, CD45and CD34. After osteogenic induction,hMSCs stained by alizarin red exhibited matrix mineralization formation after21-days inculture. hMSCs were also able to differentiate towards an adipose phenotype after culturingfor21days with adipogenic medium. In addition, the cells could also be induced to be achondrogenic phenotype after four weeks in culture following the formation of ahigh-density pellet and a serum-free chondrogenic medium with TGF-β3, positive alcianblue staining. Porcine RPE cells were positive staining for CRALBP, RPE65, ZO-1.2. Inducing RPE differentiation of hMSCs2.1. hMSCs derived RPE-like cells can be observed some pigment granulesintracellular, the content of melanin pigment is closer to the adult porcine RPE cells, some of which origins from the pigment granules secreted into the extracellular RPE cells wereinduced phagocytosis into the cell.2.2. The pigment content is of no significant difference between hMSCs derivedRPE-like cells induced by co-culture and native RPE cells. The pigment content is ofsignificant difference between hMSCs derived RPE-like cells induced conditioned mediumand native RPE cells.2.3. Electron microscopy also revealed the presence of mature pigment granules andfurthermore, microvilli was clearly present in hMSC-derived RPE cells after14-dayco-culture2.4. Various RPE-specific markers in hMSC-derived RPE-like cells were determined.These genes were significantly up-regulated; MITF, OTX2, tyrosinase, bestrophin,PMEL17, RPE65, PEDF,PAX6and CRALBP after being co-cultured with adult pig RPEcells for14days compared with control hMSCs without co-culture induction. Furthermore,the expression levels of the above genes in hMSCs-derived RPE were comparable to thosein freshly isolated adult RPE.2.5. An immunofluorescence assay was used to assess the expression of typical RPEmarker proteins in order to confirm that the increased gene expression led to changes inprotein levels; these included CRALBP, RPE65and ZO-1.2.6. Green fluorescence was observed in both adult pig RPE cells and hMSCs-derivedRPE cells after24h in culture suggesting these cells had acquired the ability to phagocytoseextracellular elements of the POS. Green fluorescence was not observed in the non-inducedhMSCs. After14-day co-culture, the expression of MERTK in induced hMSCs wassignificantly enhanced as compared with hMSCs without co-culture induction. Meanwhile,the expression of αVβ5Integrin was also enhanced slightly. hMSCs-derived RPE cellsexposed to MERTK antibodies showed a significant reduction in the number of POSingested over a5h period compared to control cells.2.7. The results from the ELISA assay showed that there was significant enhancementof BDNF and GDNF in hMSC-derived RPE, relative to hMSCs without co-cultureinduction. However, these values were significantly less than the values seen for the porcineRPE samples. 3. Transplantation of hMSC-RPE in acute and chronic retinal degeneration model3.1. Hematoxylin and eosin staining of retinal section from NaIO3injected rat showsthat compared to the confluent RPE monolayer in the uninjured eyes, the NaIO3-treatedeyes demonstrate a paucity of RPE cells with only very occasional rounded RPE cellsremained on the underlying Bruch’ s membrane. Compared with normal rats the sodiumiodate rats display significantly lower F-ERG in A wave and B wave amplitude.3.2. Confocal images showed that hMSC-RPE cells could express mitochondrial andCRALBP after transplantation tested by immunofluorescence staining.3.3. F-ERG results show that there was signifincantly increased in A wave and B-waveamplitude after transplantation of hMSC-RPE cells compared with hMSC transplantationand sham operation.3.4. After transplantation in the model of acute retinal degeneration, Western Blotshowed that rhodopsin expression was higher in hMSC-RPE cell transplanted rats thanhMSC cell transplanted rats.3.5. RCS rat retinal outer nuclear layer thickness was significantly thicker after hMSCs-RPE cells transplantation.3.6. Confocal images showed that hMSCs-RPE cells could express mitochondrial andCRALBP after transplantation tested by immunofluorescence staining.3.7. F-ERG results show that there was signifincantly increased in A wave and B-waveamplitude4weeks after transplantation hMSCs-RPE cells in RCS rats model.3.8. Western Blot results show MERTK expression in the retina after transplantaionwere positive in4weeks after transplantaion hMSC-RPE cells and negative in4weeks aftertransplantaion hMSC-RPE cells and both sham operation.3.9. Confocal images showed that rhodopsin positive POS could be found in PRE65positive transplanted cells4weeks after transplantation, but could not be found8weeksafter transplantationConclusion：1. We successfully obtained hMSCs and pig RPE cells. The morphology andphenotype of these two cells are consistent with hMSCs and porcine RPE cellscharacteristic. 2. We used transwell system to establish hMSCs and RPE cells non-contact co-culturesystem in vitro to induce differentiation of hMSCs to RPE cells. The induced cells showedbiological properties and functions similar to native RPE cells.3. In the intravenous injection of sodium iodate rat model of acute retinal degeneration,the changes of organization and structure are consistent with acute retinal degenerationchanges.4. The rats with acute retinal degeneration induced by sodium iodate injury weretransplanted by RPE cells derived from hMSCs had potent protective effects on visualfunction and maintenance.5. In the RCS rat model of chronic retinal degeneration, followed by transplantation ofRPE cells derived from hMSCs, the rats had phagocytic ability of photoreceptor outersegment. However, after8weeks of transplantation, the number of viable cells wassignificantly decreased, and only a small amount of cells expressed markers of RPE cellsmature. In addition, we did not observe the transplanted cells with phagocytic ability ofphotoreceptor outer segments as well as the maintenance of visual function.更多还原