颅内移植的人类骨髓间充质干细胞迁移至脾脏后对脑缺血后颅内外炎症反应的作用和机制研究

Objective: Inflammatory responses after ischemia stroke produce various cell death cascades that lead to further brain damage and poor prognosis. Stem cell therapy for neurological disorders is associated with modest survival of grafted cells within the local brain tissue. Interestingly, our recent study reported that human bone marrow mesenchymal stromal cells (hBMSC) transplanted directly into the rat stroke brain migrated peripherally to the spleen. However, the effects of intracerebrally transplanted hBMSC that migrated to the spleen on peripheral inflammatory responses remain unclear. The present study explored whether grafted hBMSCs abrogated peripheral inflammatory factors by increasing the proportion of regulatory T cells (Treg) in the spleen, thereby reducing both central and peripheral inflammatory responses. Additionally, we assessed whether hBMSCs transferred healthy mitochondria to damaged neurons in reducing inflammation-mediated cell death, altogether improving stroke prognosis..Methods: Adult Sprague-Dawley rats initially received sham or experimental stroke surgery via the one-hour middle cerebral artery occlusion (MCAo) model. At one hour after surgery, rats were intracerebrally transplanted with hBMSCs （3×105/9ul) or PBS treatment. Motor deficits were characterized before MCAO (baseline) and at days1, 3, and 7 after transplantation using the motor asymmetry elevated body swing test (EBST) and neurological tests. TNF-α, IL-1β, and IL-10 were measured in serial samples of peripheral blood serum collected at specified time points: day 1, 3, or 7 after transplantation. Serum samples and brain tissues were analyzed by enzyme-linked immunosorbent assay (ELISA) and RT-PCR. Isolated splenocytes from animals treated with hBMSCs or vehicle were analyzed by flow cytometry with Gallios Flow Cytometer. The splenocyte T cell populations were labeled using CD4 and CD8 antibodies, and Treg cells were labeled as CD4+/CD25+/FoxP3+. Mitochondrial changes in ischemic neurons were observed using immunofluorescence. In parallel, we performed mitochondrial transfer assays in co-cultured hBMSCs and damaged neurons. Cortical neurons were plated in 6-wells plates at 1x 106/well. The cells were then exposed to oxygen glucose deprivation (OGD) (in PBS, 5% CO2, 95% N2) for 90 minutes and control (in complete media, 5% CO2, 21% O2). Twenty-four hours after, the cells were stained using MAP2 and ATP5A antibodies. .Results: Our data revealed that surviving hBMSCs were detected in brain and spleen of transplanted stroke animals, but not in sham or PBS-infused stroke animals. We observed that hBMSC-transplanted stroke animals displayed an improved motor activity on EBST and better neurological performance on forelimb akinesia and paw grasp tests compared with PBS-infused stroke animals. TNF-α and IL-1β levels in blood and brain were significantly lower, while IL-10 levels were significantly higher in the hBMSC-transplanted stroke animals compared to PBS-infused stroke animals (p<0.05). In spleen and brain, the proportions of total CD4+ and CD8+ cells were significantly less, and the proportion of CD4+/CD25+/FoxP3+ Treg cells were significantly higher, among the isolated splenocytes derived from stroke animals treated with hBMSCs compared to those treated with PBS. Furthermore, in vitro data indicated that hBMSCs’ mitochondria could be detected in rat ischemic neurons coupled with better cell survival after OGD compared to non-hBMSC co-culture..Conclusion: This brain-to-spleen migration of hBMSCs likely contributed to dampened central and peripheral inflammation as evidenced by reduced T cells and increased proportion of Tregs in the spleen. Immunomodulation of the splenic response, as well as mitochondrial repair, via targeted migration of hBMSCs may aid in brain repair, providing a platform to develop novel therapies for stroke or other CNS injuries associated with aberrant inflammation.

脑梗塞后的颅内外炎症反应引发各种级联损伤，进一步加重脑损伤，静脉注射干细胞能减轻脑梗塞后的外周炎症反应，改善预后。既往我们的研究显示颅内移植的人类骨髓间充质干细胞（hBMSC）可迁移至脾脏，但迁移至脾脏的hBMSC对外周炎性反应的作用及机制不清。本研究的目的是探讨迁移至卒中大鼠脾脏的hBMSC能否提高脾脏内调节性T细胞的比例，减少梗塞后颅内外的炎性因子，继而减轻炎症反应。此外，hBMSC能否将其线粒体转移给受损的神经元细胞，减少神经元的凋亡，进一步减轻颅内的炎症反应，从而改善预后。复制大鼠大脑中动脉栓塞模型，颅内注射hBMSC后，通过评估大鼠神经功能，RT-PCR及Elisa检测脑及血液IL-1β、IL-10、TNF-α，流式细胞仪对脾脏免疫细胞进行分析，观察神经元的凋亡情况，证实迁移至脾脏的hBMSC能通过调节免疫细胞，减轻颅内外炎症反应，本研究有望为缺血性卒中的防治提供潜在的治疗靶点。

缺血性脑血管病已成为全球死亡及致残的主要原因。缺血性脑卒中在我国每年有240万患者，110万患者死于脑梗塞，大约只有5％的患者能使用目前的治疗方法治疗。目前的治疗方法通常受限于梗塞治疗的时间窗及严重的出血并发症。缺血性脑卒中的特征性变化是脑动脉闭塞后导致神经元的急性丧失以及突触结构的破坏。由于神经细胞的更新能力有限且转换缓慢，内源性细胞替代不足以修复受损的神经元，因此干细胞疗法已成为治疗缺血性脑卒中最有前景的治疗措施。既往对干细胞经颅内移植治疗神经系统损伤性疾病时修复机制的研究焦点都集中在是移植的干细胞通过颅内的内源性修复机制来完成，研究发现：颅内移植的hBMSC优先迁移到脾脏，通过靶向作用于由脾脏产生的炎症反应，从而达到改善慢性脑梗塞后的神经功能。但其作用机制有待研究。.研究内容：1、构建缺血性卒中的细胞模型，观察OGD对神经元线粒体的损伤；2、观察hBMSC对受损神经元的作用及是否能将其线粒体转移给受损的神经元；3、hBMSC颅内移植后颅内的存活情况及迁移能力的评估；4、评估hBMSC移植后大鼠神经功能恢复情况；5、明确hBMSC移植后对颅内外炎性因子的作用；6、探讨hBMSC移植后对颅内外炎性因子影响的可能机制.重要结果：OGD能使神经元细胞中的线粒体受损，增加神经元细胞的死亡率。hBMSC能将其线粒体转移至受损的神经元内，降低神经元细胞的凋亡率。hBMSC使卒中大鼠TNF-α浓度明显降低。移植组中脾脏中Treg细胞明显升高。.关键数据及科学意义：hBMSC能将其线粒体转移至受损的神经元内，降低移植组神经元凋亡，OGD组活细胞数较正常组明显下降（P＜0.001），hBMSC+OGD组的活细胞数较OGD组明显提高（P＜0.001）。移植组中脾脏及脑组织内Treg细胞明显升高，IL-1β、TNF-α较对照组明显降低，有显著统计学意义。揭示了干细胞治疗缺血性卒中的新机制：通过转移健康的线粒体，提高神经元的存活率。转移至脾脏的干细胞能提高Treg细胞的数量，降低外周炎症反应，从而修复受损的神经功能。证实了从颅内移植的干细胞既能发挥颅内的修复作用，又能迁移至脾脏，阐明颅内移植的干细胞对缺血性卒中的治疗作用不仅局限于颅内，而且能迁移至外周的免疫器官发挥外周作用，为今后应用颅内移植干细胞治疗中枢神经系统损伤性疾病提供新的理论依据及防治靶点。