알츠하이머병 모델 동물에서 양막줄기세포 엑소좀풍부배양액의 인지기능 개선 및 뇌보호 효과

국문 초록 (Abstract)

알츠하이머 질환 (Alzheimer's disease, AD)의 병리학적 특징으로는 아 밀로이드 베타(Amyloid-beta, Aβ)의 침착, 콜린성 시스템을 손상시켜 아세틸콜린(acetylcholine, ACh)의 방출이 감소되어 결국에는 기억력을 감소시킨다. 현재 사용되고 있는 AD의 치료제는 항-Aβ 및 아세틸콜린 에스테라제(acetylcholinesterase, AChE)억제제를 사용하는 방법으로 알려져 있으나 질병의 진행을 늦추거나 역전시키지 않고, 기억력 결핍을 완화할 뿐이며, 부작용으로 인한 위험을 수반함으로 안전하고 효과적인 치료방법이 필요한 상황이다. 본 연구에서는 AD모델 마우스에서 양막줄 기세포(amniotic membrane stem cells, AMSCs)엑소좀풍부배양액 (exosome-rich conditioned medium, ERCM)의 인지기능장애 개선 효 과를 연구하였다. 기본 메커니즘을 밝히기 위하여 F3세포에서 Aβ제거 및 콜린아세틸트랜스퍼레이즈(choline acetyltransferase, ChAT) 발현 에 대하여 AMSCs-ERCM의 효과를 평가하였다. 콜린성 신경에 흡수되 어 독성을 나타내는 콜린유사체(Acetylcholine mustard aziridinium ion, AF64A)를 마우스의 뇌실내로 투여하고 ERCM을 정맥투여하였다. ERCM은 ChAT, ACh를 개선하였고, Aβ를 분해하는 효소인 네프릴라이 신(neprilysin, NEP)이 Aβ 를 감소시키고 풍부한 성장인자와 신경영양 인자로 인한 신경재생 및 보호를 통하여 AF64A 모델 마우스에서 인지 기능을 회복하고 신경보호를 활성화하였다.

다국어 초록 (Multilingual Abstract)

Accumulation of amyloid β (Aβ) peptides has been believed to be a main etiology of Alzheimer's disease (AD). Aβ-mediated cholinergic nerve degeneration causes depletion of acetylcholine (ACh), responsible for memory acquisition, leading to cognitive dysfunction. Since current AD treatments using acetylcholinesterase (AChE) inhibitors and Aβ antibodies do not reverse disease progression and cause adverse effects, more effective and safe treatments are required. An exosome-rich conditioned medium (ERCM) containing high concentrations of neprilysin (NEP) and growth factors (GFs) and neurotrophic factors (NFs) was collected via hypoxic culture (2% O2) of human amniotic membrane stem cells (AMSCs). CM-DiI- labeled exosomes readily penetrated normal and H2O2-damaged human neural stem cells (NSCs), and the ERCM not only showed NEP activity, but also protected against AF64A-induced NSC degeneration in vitro. The AD-therapeutic effects of ERCM were assessed in AF64A (a cholinotoxin)-induced memory deficit mice. Intracerebroventricular injection of AF64A (2 nM, 2 μL/mouse) induced severe cognitive dysfunction in passive avoidance and Morris water-maze performances in 3 weeks. However, intravenous injection of ERCM (1.2 x 109 - 1.2 x 1010 particles /100 μL/mouse) 4 times markedly recovered the learning and memory function. In the brain of mice treated with ERCM, hippocampal pyramidal cells were preserved, Aβ level was reduced, and ACh concentration was restored. The neuroprotective and anti-inflammatory effects of ERCM were also confirmed by the recovery of GFs/NFs and cholinergic nerve markers and the decrease in glial fibrillary acidic protein (GFAP)-expressing astrocytes, respectively. In addition, nestin-positive host NSCs and choline acetyltransferase (ChAT)- expressing cholinergic neurons increased following treatment with ERCM. These findings indicate that AMSCs-ERCM containing large amounts of functional molecules such as NEP and GFs/NFs can ameliorate cognitive deficit of AD animals via Aβ elimination as well as neuroprotection and neurodegeneration.

목차 (Table of Contents)

• Abstract · iii
• Abstract in Korean vi
• Ⅰ. INTRODUCTION · 1
• Ⅱ. MATERIALS AND METHODS · 6
• 1. Preparation of AMSCs · 6
• 1) Institutional Review Board (IRB) approval
• 2) Preparation of amniotic membrane stem cells
• 2. Preparation of ERCM and characterization 7
• 1) Collection of ERCM
• 2) Morphological analysis
• 3) Nanoparticle-tracking analysis (NTA)
• 4) Exosome marker analysis
• 5) Enzyme-linked immunosorbent assay (ELISA)
• 6) Assay of NEP activity
• 7) Assay of Aβ-degrading activity
• 8) Exosome-uptake assay
• 3. In vitro assay of neuroprotective activity 12
• 1) Cell culture
• 2) AF64A preparation
• 3) Cytoprotection assay
• 4. In vivo assessment of AD-therapeutic activity· 14
• 1) Establishment of AF64A-induced AD model
• 2) Experimental design
• 3) Assessment of cognitive function
• 4) Measurement of Aβ1-42 and acetylcholine in brain
• 5) Western blot analysis of GFs/NFs and cholinergic markers
• 6) Histopathology of the brain tissue
• 7) Immunohistochemistry in brain sections
• 8) Statistical analysis
• Ⅲ. RESULTS 21
• 1. Size distribution of exosomes 21
• 2. Exosome markers in NCM and ERCM 23
• 3. Concentration of GFs/NFs in CM and ERCM 25
• 4. Enzymatic NEP activity of ERCM 27
• 5. Aβ-degrading activity of ERCM 29
• 6. Exosome uptake in SH-SY5Y cells 31
• 7. In vitro neuroprotective activity 33
• 8. AF64A-induced AD model 35
• 9. Restoration of cognitive function by ERCM 37
• 10. In vivo neuroprotective effects 40
• 11. Recovery of brain Aβ and ACh levels 44
• 12. Restoration of neuroprotective and neuroregenerative factors
• 46
• 13. Anti-inflammation on astrogliosis 49
• 14. Restoration of host NSCs 52
• 15. Restoration of cholinergic neurons 55
• Ⅳ. DISCUSSION · 57
• Ⅴ. REFERENCES 62