调控局部补体激活对人类干细胞源性视网膜色素上皮移植治疗AMD的影响

Human embryonic stem cell derived retinal pigment epithelial (hESC-RPE) cell has created a potentially unlimited source for replacing dead or dying RPE for age-related macular degeneration(AMD). Transplantation of such cells into the eye has been shown to rescue visual function and thus initiated many clinical trials ongoing. Nevertheless, many problems has yet to be overcome before ESC-based therapies can be used in standard procedures, among which the impact of the pathological change in local environment of transplantation and the immunogenicity of ESCs remains completely unknown. RPE, as an important local regulatory cells to immune reaction including complement activation, its injury may lead to decreased expression of complement factor H, an inhibitor in alternative pathway in complement activation. As s result, abnormal complement activation may contribute to the pathogenesis of AMD, as well as damage of immune privilege of subretinal space. In this study, we aim to investigate the effect of local activated complement on the survival and function of a hESC-RPE cell line(MA09-hRPE). We will take the advantage of an in vitro co-culture system to evaluate the cytotoxicity of activated complement on MA09-hRPE. In addition, aged CFH+/- mice fed with high fat diet will serve as an in vivo AMD model to further evaluate the role of activated complement at the site of transplantation, with/without inhibiting complement activation. These studies will help clarify the mechanisms by which administered ESC-RPE are lost and not functioning well in the recipient, provide rationales for translating these simple strategies into the clinic in the near future as new approaches for improving outcome of ESC-RPE transplantation for treating AMD.

人类干细胞源性视网膜色素上皮(hSC-RPE)移植对于治疗年龄相关性黄斑变性(AMD)尤其是晚期RPE地图样萎缩显示出巨大的潜力，并由此带动很多临床试验的启动。然而许多问题有待深入探索解决，如宿主微环境的病理状态和免疫特性改变对移植细胞的存活及功能影响目前尚未清楚。RPE作为局部免疫和补体调控的重要细胞, 其损伤可引起补体因子H(CFH)表达下降导致补体异常激活，视网膜下腔的免疫赦免特性改变。本项目旨在探索AMD局部补体异常激活的微环境对干细胞源性RPE移植的影响，以及如何改善提高移植的效力。我们将采用体内外模型,摸拟补体异常激活, 研究补体激活的微环境对人类胚胎干细胞源性RPE的存活和功能影响。 明确局部补体异常激活的微环境对移植细胞存在的危害性, 并希望通过调控局部补体激活, 改善和提高干细胞移植治疗的有效性和持续性。研究结果对进一步的临床试验和转化医学有着非常积极重要的作用和影响。

人类干细胞源性视网膜色素上皮(hESC-RPE)移植治疗RPE退行性疾病的临床试验，已在全世界逐渐开展起来，给年龄相关性黄斑变性(AMD)特别是伴有地图状萎缩的患者带来了希望。然而由于补体基因尤其是补体H因子(CFH)基因突变、视网膜局部补体异常激活、RPE损伤萎缩是AMD发病机制中的重要组成部分，移植的hESC-RPE在此病理状态的视网膜下是否能够抵御补体攻击，并且正常存活而发挥功能，目前尚不清楚。本研究从人胚胎干细胞分化并鉴定了hESC-RPE，检测了补体调节蛋白的表达情况，发现正常人血清中的补体系统对hESC-RPE无明显杀伤作用，但在体外模拟AMD补体异常激活的模型中，hESC-RPE则受到补体替代通路的攻击。在加入已进入临床试验中的补体抑制剂药物后，补体对hESC-RPE的异常激活明显抑制，提示hESC-RPE虽然具有一定的补体调控能力，但可能仍难以耐受补体失调的微环境，而调控补体的异常激活，则可能有助于维持hESC-RPE的存活。此外，本研究通过CRISPR/Cas9构建了Cfh基因敲除小鼠，并通过高脂饮食和吸烟处理，建立了具有AMD样病理改变的小鼠模型。该小鼠模型视网膜下补体异常激活，补体激活产物沉积，小胶质细胞激活迁移，视网膜电图显示视功能损伤，视网膜转录组分析提示与炎症通路相关基因表达上调。在进一步给与补体抑制剂治疗后，模型小鼠视网膜下补体激活产物减少，这与目前补体抑制剂临床试验中的结果相一致。本研究结果提示，AMD局部补体异常激活的微环境对移植的hESC-RPE细胞存在潜在的风险，而抑制补体激活，可能改善移植细胞的生存，同时也能延缓AMD发病进展。因此，我们开创性的提出，补体抑制剂联合hESC-RPE移植治疗，可能有着巨大的临床应用前景，该研究结果有望指导进一步的转化医学和临床试验。

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