Morioka Chikako, Komaki Motohiro, Taki Atsuko, Honda Izumi, Yokoyama Naoki, Iwasaki Kengo, Iseki Sachiko, Morio Tomohiro, Morita Ikuo
Department of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo, 113-8510 Japan.
Department of Cellular Physiological Chemistry, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo, 113-8510 Japan.
Inflamm Regen. 2017 Jan 16;37:1. doi: 10.1186/s41232-016-0032-3. eCollection 2017.
Periventricular leukomalacia (PVL) is a type of multifactorial brain injury that causes cerebral palsy in premature infants. To date, effective therapies for PVL have not been available. In this study, we examined whether mesenchymal stem cells (MSCs) possess neuroprotective property in a lipopolysaccharide (LPS)-induced neonatal rat PVL-like brain injury.
Human umbilical cord-derived MSCs (UCMSCs) were used in this study. Four-day-old rats were intraperitoneally injected with LPS (15 mg/kg) to cause the PVL-like brain injury and were treated immediately after the LPS-injection with UCMSCs, conditioned medium prepared from MSCs (UCMSC-CM) or interferon-gamma (IFN-γ)-pretreated MSC (IFN-γ-UCMSC-CM). To assess systemic reaction to LPS-infusion, IFN-γ in sera was measured by ELISA. The brain injury was evaluated by immunostaining of myelin basic protein (MBP) and caspase-3. RT-PCR was used to quantitate pro-inflammatory cytokine levels in the brain injury, and the expression of tumor necrosis factor-stimulated gene-6 (TSG-6) or indoleamine 2,3-dioxygenase (IDO) to evaluate anti-inflammatory or immunomodulatory molecules in UCMSCs, respectively. A cytokine and growth factor array was employed to investigate the cytokine secretion profiles of UCMSCs.
Elevated serum IFN-γ was observed in LPS-infused rats. The expression of IL-6, tumor necrosis factor-alpha (TNF-α), IL-1ß, and monocyte chemoattractant protein-1 (MCP-1) were increased in the brain by LPS-infusion in comparison to saline-infused control. LPS-infusion increased caspase-3-positive cells and decreased MBP-positive area in neonatal rat brains. A cytokine and growth factor array demonstrated that UCMSCs secreted various cytokines and growth factors. UCMSCs significantly suppressed IL-1ß expression in the brains and reversed LPS-caused decrease in MBP-positive area. UCMSC-CM did not reverse MBP-positive area in the injured brain, while IFN-γ-UCMSC-CM significantly increased MBP-positive area compared to control (no treatment). IFN-γ-pretreatment increased TSG-6 and IDO expression in UCMSCs.
We demonstrated that bolus intraperitoneal infusion of LPS caused PVL-like brain injury in neonatal rats and UCMSCs infusion ameliorated dysmyelination in LPS-induced neonatal rat brain injury. Conditioned medium prepared from IFN-γ-pretreated UCMSCs significantly reversed the brain damage in comparison with UCMSC-CM, suggesting that the preconditioning of UCMSCs would improve their neuroprotective effects. The mechanisms underline the therapeutic effects of MSCs on PVL need continued investigation to develop a more effective treatment.
脑室周围白质软化（PVL）是一种多因素导致的脑损伤，可致使早产儿发生脑瘫。迄今为止，尚无针对PVL的有效治疗方法。在本研究中，我们检测了间充质干细胞（MSCs）在脂多糖（LPS）诱导的新生大鼠PVL样脑损伤中是否具有神经保护特性。
本研究使用人脐带间充质干细胞（UCMSCs）。给4日龄大鼠腹腔注射LPS（15 mg/kg）以造成PVL样脑损伤，并在注射LPS后立即用UCMSCs、由MSCs制备的条件培养基（UCMSC-CM）或经干扰素-γ（IFN-γ）预处理的MSCs（IFN-γ-UCMSC-CM）进行治疗。为评估对LPS输注的全身反应，通过酶联免疫吸附测定法（ELISA）检测血清中的IFN-γ。通过髓鞘碱性蛋白（MBP）和半胱天冬酶-3的免疫染色评估脑损伤。逆转录聚合酶链反应（RT-PCR）用于定量脑损伤中促炎细胞因子水平，并分别检测肿瘤坏死因子刺激基因-6（TSG-6）或吲哚胺2,3-双加氧酶（IDO）的表达，以评估UCMSCs中的抗炎或免疫调节分子。采用细胞因子和生长因子阵列研究UCMSCs的细胞因子分泌谱。
在输注LPS的大鼠中观察到血清IFN-γ升高。与输注生理盐水的对照组相比，输注LPS使脑中白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和单核细胞趋化蛋白-1（MCP-1）的表达增加。输注LPS增加了新生大鼠脑中半胱天冬酶-3阳性细胞，并减少了MBP阳性区域。细胞因子和生长因子阵列显示，UCMSCs分泌多种细胞因子和生长因子。UCMSCs显著抑制脑中IL-1β的表达，并逆转LPS导致的MBP阳性区域减少。UCMSC-CM未能逆转损伤脑中的MBP阳性区域，而与未治疗的对照组相比，IFN-γ-UCMSC-CM显著增加了MBP阳性区域。IFN-γ预处理增加了UCMSCs中TSG-6和IDO的表达。
我们证明，腹腔大剂量注射LPS可导致新生大鼠发生PVL样脑损伤，输注UCMSCs可改善LPS诱导的新生大鼠脑损伤中的脱髓鞘。与UCMSC-CM相比，经IFN-γ预处理的UCMSCs制备的条件培养基显著逆转了脑损伤，这表明对UCMSCs进行预处理可提高其神经保护作用。MSCs对PVL治疗作用的机制仍需继续研究，以开发更有效的治疗方法。
