BDNF基因转染脂肪间充质干细胞移植对视网膜色素变性的实验性干预及作用机制研究

No effective treatment is currently available for retinitis pigmentosa (RP), the leading cause of incurable blindness, due to its complicated heterogeneity. Brain-derived neurotrophic factor (BDNF) exerts its survival-promoting effects on photoreceptors and retinal ganglion cells, however, delivery systems with little-to-no side effect are needed to sustain its controlled release and long-term efficacy. Our previous studies demonstrated that adipose-derived mesenchymal stem cells (Ad-MSCs) are ideal delivery systems for gene therapy; moreover, Ad-MSCs present unique properties like migration to damaged tissue sites, immunomodulation and anti-inflammation. Herein, we propose to employ Ad-MSCs as the BDNF gene delivery vehicle, and the BDNF-transduced Ad-MSCs would be transplanted into the RP mouse model through intra-peritoneal injection for intervention and treatment. The hypothesis of this study is that Ad-MSCs not only effectively deliver the BDNF into the damaged retina, their anti-inflammation and multi-lineage differentiation capacities also synergize with BDNF treatment. Fluorescence microscopy will be used for the analysis of BDNF delivery efficiency; real-time PCR and western-blot for the expression of BDNF and its effect on the expressions of inflammation factors and other neurotrophic factors; Immunohistochemistry for the differentiation of Ad-MSC; HE and TUNEL staining for the measurement of the thickness of the retinal outer nuclear layer and the apoptosis rate of the photoreceptors respectively; and Cryo-TEM for the ultra-structure detection of photoreceptors. This proposal aims at exploring the treatment efficacy of BDNF-transduced Ad-MSC transplantation and clarifying the underlying mechanism.

复杂的遗传机制导致视网膜色素变性(RP)仍被WHO列为不可治盲症。脑源性神经营养因子(BDNF)对感光和视网膜神经节细胞具有保护和抗凋亡作用，但需要更安全有效、持续释放BDNF并可反复多次注射的给药系统。本项目应用易于外源基因转染的脂肪干细胞(Ad-MSCs)作为BDNF基因治疗的载体，通过腹腔注射将其移植于RP小鼠动物模型进行实验性干预，旨在利用Ad-MSCs向组织损伤部位定向迁移的生理功能，将BDNF有效地递送至病变视网膜发挥其神经细胞保护功能；同时也利用其抗炎特性和多向分化能力抑制RP的发展，对BDNF的治疗产生协同和叠加作用。本课题将通过对眼部组织BDNF、炎症和其他营养因子的表达，视网膜外核层厚度的变化，感光细胞凋亡阳性率及其超微结构的变化等的检测来研究Ad-MSCs的靶点递送效率、炎症抑制及细胞分化能力，阐释其作用机制。此给药系统前景广阔，可以广泛应用于其他神经系统疾病的治疗。

脑源性神经营养因子（BDNF）是一类具有神经调控能力的神经营养因子，对神经细胞存活及生长发育起促进作用；并且其人体内含量最高，受体分布最广，同时能促进其他神经营养因子的释放，增强神经细胞的生存能力。但是，目前应用BDNF治疗疾病存在限制：BDNF无法透过血脑或血眼屏障，因此全身给药后难以到达病变位置；而且其半衰期短，即使局部注射，也需反复注射。因此合适的传递载体是突破BDNF治疗限制的关键。脂肪间充质干细胞（ADSCs）属成体多能干细胞的一种，具有来源丰富、易于提取、无伦理道德问题及多向分化潜能等优点。研究表明ADSCs易于外源目的基因的转入，兼其向损伤部位定向迁移的特性，是.一种良好的传递载体。而且ADSCs的干细胞特性对疾病治疗起协同作用。因此本课题构建了持续表达外源BDNF基因的ADSCs细胞系，实现BDNF基因的持续表达，研究对疾病治疗的作用机制。.本课题采用胶原蛋白酶消化法，将获得的ADSCs分离纯化并进行其增殖、表面标志的鉴定及成脂成骨能力测试。ADSCs为长梭形细胞，贴壁旋涡式生长，具有多向分化潜能。表面标志鉴定结果表明其干细胞表面标志阳性表达，而阴性表达造血、吞噬和上皮表面标记物等，符合间充质干细胞鉴定标准。用携带增强绿色荧光蛋白（EGFP）基因和新霉素（Neo）基因为筛选因子的外源BDNF基因的重组慢病毒转染ADSCs,通过荧光筛选和G418药物筛选，得到稳定细胞株BDNF-ADSCs。流式测试、多向分化能力鉴定及菌落形成单位分析结果表明，BDNF转入未改变其干细胞特性及多向分化潜能。.通过ADSCs和BDNF-ADSCs生长曲线对比，转染细胞具有较长的细胞周期，分析主要因外源基因的表达增加了细胞增殖的负担。Real-time PCR测定结果显示BDNF mRNA 及相关基因mRNA表达均有不同程度提高，BDNF在体外细胞可以调控相关因子；Western-blot分析得NGF蛋白与BDNF蛋白表达水平均有提高，BDNF基因可以成功在体外ADSCs中表达并发挥促进其它神经营养因子分泌的作用。

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