評估間質幹細胞應用於治療母體發炎導致胎腦皮質發育異常之成效

Evaluation the potential therapeutic effect of mesenchymal stem cells on maternal inflammation induced developmental defects in fetal brain

吳鈞豪(Chun-Hao Wu)

指導教授 ： 曾嘉儀 ； 招名威

中原大學/工學院/生物醫學工程研究所/碩士(2020年)

https://doi.org/10.6840/cycu202000119

摘要

懷孕婦女於孕期受到細菌感染是造成早產及死胎的重要因素之一。流行病學研究表示，母體受感染在誘導神經發炎的反應中扮演著重要的角色，而這會導致胎兒大腦的神經病變和功能缺陷，包括學習及記憶方面。然而胎兒大腦發育異常的機制至今尚未明確。但研究指出神經發炎在病理學疾病及神經病學中有高度的關聯性，像精神分裂症、腦癱瘓、自閉症。間質幹細胞(Mesenchymal Stem Cell, MSC)為多功能幹細胞，可透過細胞分化與增生的功能修復組織，存在於多種組織中，包括骨髓、脂肪、皮膚、肌肉、血液、胎盤等。近幾年來的研究結果發現，間質幹細胞可以與免疫細胞作用，進而調節免疫系統。目前臨床上已成功使用間質幹細胞治療部分炎症，如成骨不全症、移植物抗宿主疾病(Graft versus-Host Disease, GvHD)、克隆氏症(Crohn’s disease)。因此，我們假設間質幹細胞可透過調節發炎反應或促使神經細胞新生來減緩母體發炎所引起的神經發育損傷。脂多醣(Lipopolysaccharide, LPS)為革蘭氏陰性菌外膜主要的成分，研究中常利用LPS注射至懷孕母鼠模擬臨床細菌感染導致的母體發炎。我們在母鼠懷孕第15天將MSC (1x105 cell/2µl)注射至胚胎後一小時腹腔注射LPS (25 µg/kg)，並於母鼠懷孕第19天犧牲後分析母體血漿及羊水中細胞因子表現量，並進行血液學及生理參數量測；蘇木精-伊紅染色觀察大腦皮質層厚度；最後利用免疫組織螢光染色檢測大腦皮質中神經細胞新生、分化及遷移狀況。實驗結果顯示，MSC不僅可降低LPS造成母體血漿中發炎相關細胞因子表達量還可提高胚胎存活率。MSC能預防LPS造成胎兒大腦皮質層分層混亂並增加胎兒大腦的神經新生。還發現MSC可抑制LPS造成的大腦皮質中神經膠細胞活化。總結，MSC可有效改善母體發炎反應並且降低胎兒腦中活化的神經膠細胞水平，並且可能透過神經新生的方式逆轉胎兒的腦部損傷。因此MSC可能在近期內成為對抗母體感染造成發炎且具潛力的臨床治療方法。

關鍵字

母體發炎 ； 脂多醣 ； 間質幹細胞

並列摘要

Maternal bacterial infections during pregnancy represent a significant risk factor. It might cause preterm labor as well as newborns with neuropsychiatric disorders and neurodevelopmental deficits, such as schizophrenia, cerebral palsy, and autism. Epidemiological studies suggested that infection caused downstream neuroinflammatory response would lead to neurodegeneration and functional deficits in fetal brain, which might affect the learning and memory capacity. However, the underlying mechanism remains largely unknown. Mesenchymal stem cells (MSCs) are pluripotent cells that are mainly responsible for cell differentiation and proliferation to tissue repair, they are present in multiple tissues, including bone marrow, adipose tissue, skin, muscle, blood, and placenta. It has been proved that MSCs can interact with cells of both the innate and adaptive immune systems. Thus it may act as a possible clinical therapy for immune-mediated diseases, such as osteogenesis imperfecta, severe Graft-versus-Host Disease (GvHD), and Crohn's disease. Through modulating the inflammatory response, we hypothesized that MSCs might attenuate neurodevelopmental defects caused by maternal infection. Lipopolysaccharide (LPS) is an endotoxin that can be found on the outer membrane of the Gram-negative bacteria. LPS administration is commonly used to model clinical bacterial infection in pregnant animals. Hence, MSCs (1x105 cell/ 2µl) were injected into the embryo of a pregnant rat on embryonic day 15 (E15), followed by an intraperitoneal injection of LPS (25 µg/ kg) to simulate intraperitoneal bacterial infection during pregnancy. Animals were sacrificed on E19. Maternal plasma and amniotic fluid were collected to analyze the level of cytokines and other physiological parameters. In order to observe the changes in the thickness of the cerebral cortex, Hematoxylin Eosin (H E) staining were used. The proliferation, differentiation, and migration of neurons in the cerebral cortex were examined via immunohistochemistry. Results showed that MSCs not only reduce LPS-induced increased inflammation-related cytokines level in maternal plasma, but also increases embryonic survival rate. MSC was able to prevent LPS-induced lamination failure of the fetal cerebral cortex and increased the neurogenesis of the fetal brain. MSC was also found to inhibit LPS-induced activation of glial cells in the cerebral cortex. In summary, MSC was able to effectively ameliorate maternal inflammation response and reduce activated glia cell level in fetal brain and might invert the fetal brain injury through the neurogenesis. Hence, MSC might be a potential clinical treatment against maternal infection shortly.

並列關鍵字

Maternal infection ； Lipopolysaccharide ； Mesenchymal stem cells

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