PDGFR-β（+）早期周细胞移植促进脑缺血损伤修复的机制研究

PDGFR-β is highly expressed in early pericyte, which mediates pericyte proliferation, migration and inhabitation during embryo blood-brain barrier formation. After cerebral ischemia, the number of PDGFR-β(+) early pericyte increases not only in penumbra but also in peripheral circulation; however，the role and mechanism that PDGFR-β(+) early pericyte plays in cerebral ischemia injury recovery are still unclear. We have identified that injection of PDGFR-β(+) early pericyte into the ischemic basal ganglia of tMCAO nude mice could decrease infarction area and promote neurological function recovery (7 days).We also demonstrated that oxygen and glucose deprivation (OGD) promoted pericyte expressing and secreting microRNA-146a.Moreover, the conditioned medium (CM) of OGD pericyte could upregulate microRNA-146a in endothelial cell. On the basis of our previous results, the present study aimes to explore the following questions: ① Could PDGFR-β(+) early pericyte transplantation promote long-term neurological function recovery after cerebral ischemia (28 days)? ② Whether the therapeutic mechanism of PDGFR-β(+) early pericyte relates to its role on improving blood-brain barrier function, promoting neovascularization and promoting neurogenesis? ③ Whether microRNA-146a mediates the effect of PDGFR-β(+) pericyte in improving blood-brain barrier function after ischemia? Results of the present study might provide new theoretical basis for cell therapy of cerebral ischmia, as well as other cerebral injuries.

PDGFR-β高表达于早期周细胞，介导胚胎血脑屏障形成中周细胞的增殖、迁移和定居。脑梗塞后，半暗带及外周循环中PDGFR-β（+）早期周细胞增加，但其在脑缺血损伤中的作用还不清楚。我们研究发现，向tMCAO裸鼠缺血侧基底节注射PDGFR-β（+）早期周细胞能够减小梗死区域并促进神经功能恢复（7天）；糖氧剥夺（OGD）促进周细胞microRNA-146a表达和分泌增加，且其条件培养基（CM）能上调内皮细胞microRNA-146a表达。本课题拟在前期工作基础上探索：①PDGFR-β(+)早期周细胞移植能否促进脑缺血后较长期神经功能恢复（28天）？②PDGFR-β（+）早期周细胞的治疗机制是否与改善血脑屏障功能、促进血管新生和神经再生有关？③microRNA-146a是否介导PDGFR-β(+)早期周细胞改善缺血后血脑屏障功能的作用？本研究结果将为脑缺血及其他脑损伤的细胞治疗奠定新的理论基础。

缺血再灌注损伤导致血脑屏障结构和功能的破坏。改善血脑屏障功能，促进半暗带区新生血管形成，对于减轻缺血后神经损伤，挽救神经功能至关意义。周细胞是血脑屏障的重要组成部分，对于血脑屏障结构和功能的完整至关重要，PDGFR-β（+）早期周细胞增生和迁移能力较强。脑梗塞后，半暗带区血管内皮细胞大量释放PDGF-B，促进PDGFR-β（+）早期周细胞向病灶周围的迁移募集。我们的研究发现，PDGFR-β（+）早期周细胞移植组动物模型在不同时间点的改良NSS评分(14分制)、平衡木实验、Rotor-rod实验和转角实验均明显优于对照组，尤以28天时的行为学差异最为明显。与PBS组比较，移植组梗死范围明显缩小，缺血侧脑组织含水量明显降低，提示PDGFR-β(+)早期周细胞移植有利于改善缺血再灌注损伤后血脑屏障功能。对不同分子量示踪剂Evans Blue（961Da）、BSA-Alexa Fluor-555（66KD）和IgG-DyLightTM549（200KD）的研究进一步证实了移植组血脑屏障完整性优于对照组。进一步的机制研究发现，tMCAO后，PDGFR-β（+）周细胞移植组缺血半暗带区微血管周细胞密度和覆盖率高于PBS对照组。周细胞覆盖率的增加伴随微血管管径的改变及微血管密度的增加。在周细胞移植组，缺血半暗带区微血管密度高于tMCAO + PBS对照组。同时，周细胞覆盖率越高，微血管对示踪剂Evans Blue、BSA-Alexa Fluor-555及IgG- DyLightTM549的通透性越低。我们的研究还发现，PDGFR-β（+）早期周细胞移植促进内皮细胞紧密连接蛋白ZO-1、Occludin、Claudin-5的表达及血管基底膜蛋白Lama2、Laminin α4、Laminin α5的表达。于分子水平，我们证实PDGFR-β(+)早期周细胞移植促进tMCAO裸鼠缺血半暗带miRNA-146a水平增高, 并伴随其下游底物TRAK1、TRAF6的表达下调。提示PDGFR-β(+)早期周细胞移植可能通过miRNA-146a途径发挥其减轻脑缺血损伤的作用。我们的研究率先将周细胞移植引入缺血性卒中的治疗中，为减轻脑缺血再灌注损伤提供了可干预的重要靶点。