HA/CD44信号通路在视网膜前体细胞移植后体内功能性整合的作用及机制研究

Stem cell based therapy can be a new treatment for the age-related macular degeneration, which drew a lot attention of the researchers. However low efficiency of the integration refrains its translation into the clinic, which is due to their disability to migrate to the disired location and make contact with host. Our results showed that chondroitinase ABC could enhance the migration and survival rate of transplanted retinal progenitor cells into the subretinal space. We also found CD44 was elevated in the recipients. Therefore we speculated that HA/CD44 played a vital role in the chondroitinase-promoted migration of the RPCs. This project is about to investigate the effect of HA/CD44 on the proliferation, differentiation and migration of RPCs and to elucidate the mechnism of HA/CD44 on the migration via Nanog and miR-21 pathway and HA/CD44 on the proliferation and differentiation via ROK and PI3K pathaway by transwell assay, shRNA transfection, RNase protection assay, and subretinal transplantation and etc. In conclusion, we provides a new perspective about the role and mechnism of HA/CD44 on the chondroitinase-enhanced migration and differentiation of the engrafted RPCs, and tends to come up with a new way and an ideal seed cell for the cell-based therapy.

干细胞移植治疗老年性黄斑变性成为眼科领域最新的研究热点之一，然而目前制约其向临床转化的主要因素是干细胞移植的整合率不高，主要是细胞未能迁移到理想位置与机体整合。我们的研究结果显示软骨素酶能够显著提高视网膜前体细胞（RPCs）在视网膜内的迁移率和存活率，且CD44在软骨素酶联合RPCs移植中表达增高，因此我们推测HA/CD44参与了软骨素酶促进RPCs定向迁移的过程。本项目采用transwell实验、shRNA技术、RNA酶保护试验以及视网膜下腔移植等方法深入研究HA/CD44在体外和体内对RPCs增殖、分化和迁移的作用，阐明HA/CD44通路是否通过Nanog信号诱导miR-21的生成来促进RPCs的迁移，以及通过ROK和PI3K信号通路来调控RPCs的增殖和分化，从新的角度揭示HA/CD44参与软骨素酶对RPCs迁移和分化的调节机制，为提高移植疗效提供新的方法和更理想的细胞。

老年性黄斑变性是最常见的神经致盲性眼病，干细胞替代治疗成为近年来研究的热点。目前的主要障碍就是移植细胞的整合率较低，细胞未能迁移到理想的位置，分化为靶细胞。因而，细胞的定向迁移和分化成为治疗的关键所在。项目通过体外实验检测HA/CD44信号通路在视网膜前体细胞（RPCs）迁移和分化能力的作用，明确HA/CD44调控RPCs迁移和分化的作用和机制。同时联合移植软骨素酶和RPCs移植至 C57BL/6J (B6)野生型小鼠视网膜下腔，研究视网膜微环境的影响以及HA/CD44信号通路的改变，以此阐明HA/CD44信号通路在软骨素酶促进RPCs在小鼠眼内定向迁移和分化的机制。. 实验结果显示HA处理可以增强RPCs细胞的粘附和侵袭能力，CD44、PKC和Nanog的反义寡核苷酸以及miR-21 antagomir处理后细胞粘附和侵袭能力明显减弱。HA可以明显上调miR-21的表达，CD44抗体、PKC和Nanog的反义寡核苷酸、miR-21 antagomir处理可明显下调miR-21的表达。采用反义寡核苷酸下调下调ROK的水平，细胞内p-Gab-1水平以及Akt信号活性显著下降，靶基因CyclinD1水平也明显下降，表明HA促进增殖是通过CD44/ROK Gab-1/Akt介导的。 HA处理的条件下，ROK/Gab-1/Akt信号增强，notch下游信号减弱，p-Gab-1、p-Akt水平增加，Hes1水平下降，细胞神经元方向分化能力增强，神经胶质方向分化能力减弱，表明CD44/ROK/Gab-1/Akt信号在HA促进的神经元分化过程中发挥了促进作用。体内软骨素酶通过降解胶质屏障GFAP、Vimentin、CSPG和CD44促进RPCs的迁移和分化，同时激活HA/CD44途径来促进细胞迁移和整合。. 综上所诉，HA/CD44通过PKC/Nanog/miR-21途径调控RPCs定向迁移， HA/CD44通过CD44/ROK/Gab-1/Akt途径调控RPCs的增殖和分化，体内软骨素酶通过降解胶质屏障GFAP、Vimentin、CSPG和CD44促进RPCs的迁移和分化，同时激活HA/CD44途径来促进细胞迁移和整合。此研究结果有利于提高干细胞移植体内整合的效率，加快干细胞替代治疗进入临床运用的速度。