膽固醇是構成細胞膜的主要成分，亦是細胞維持功能正常不可或缺的物質。 目前有研究指出細胞內膽固醇含量異常與某些腦部疾病如：阿滋海默症及腦中風等腦部疾病之形成有關。

1. 膽固醇對腦細胞死亡之影響
Effects of Cholesterol on Cell Death in the Brain
林欣貝
Shin-Bey Lin
生理學研究所
關鍵詞:
膽固醇;
Keyword:
cholesterol;
摘要:
膽固醇是構成細胞膜的主要成分，亦是細胞維持功能正常不可或缺的物質。目前有研究指出
細胞內膽固醇含量異常與某些腦部疾病如：阿滋海默症及腦中風等腦部疾病之形成有關。本
論文實驗目的為利用海馬迴細胞培養的模式系統，來探究膽固醇對腦部細胞死亡時造成的影
響以及可能的調控機制。 現今已知神經細胞死亡是由於興奮性胺基酸 (如麩胺酸)，引發興
奮性毒性 (excitotoxicity) 作用所造成。首先我們利用海馬迴細胞培養與麩胺酸作用6小時或12小
時，再經8小時的復原期後，測量由細胞質中滲漏至細胞外的乳酸脫氫?含量，以作為細胞膜
破裂的指標。我們發現海馬迴細胞培養經過6小時麩胺酸 (2 mM) 處理後，會出現細胞膜受損情
形；而12小時的麩胺酸作用，不但會造成細胞膜的完整性受損，且細胞內的粒腺體亦會受到
傷害。以辨識神經細胞特有的 microtubule-associated protein 2 之抗體進行免疫細胞染色法，發現
經麩胺酸處理後的神經細胞存活數目降低，證實麩胺酸會引發興奮性毒殺作用。以星狀神經
膠細胞特有的 glial fibrillary acidic protein (GFAP) 抗體，進行免疫細胞染色的結果發現：經麩胺
酸處理的海馬迴細胞培養，其 GFAP 的表現會有增加的情形，此現象即為腦疾病所見神經膠樣
變性 (gliosis) 之典型特徵。 在 lovastatin 及 b-cyclodextrin 降低海馬迴細胞培養的膽固醇含量之
實驗結果，我們發現降低細胞膽固醇含量會加劇麩胺酸對粒腺體的傷害，並增加滲漏出細胞
外的乳酸脫氫?。上述這些因膽固醇含量降低所引起的變化，可被膽固醇的合成前驅物 mevalon
ate 所抵消。細胞內膽固醇含量的減少，會提高麩胺酸促使乳酸脫氫?釋放至細胞外的現象，
意味著膽固醇含量降低會使得細胞膜更加脆弱，因而導致細胞在遭受麩胺酸的毒殺作用時，
出現較為嚴重的死亡情形。除此之外，我們發現移除細胞的膽固醇後，便看不到麩胺酸所引
發的神經膠樣變性現象，由此推測膽固醇含量降低會透過阻礙神經膠細胞活化的機轉，加重
了麩胺酸毒殺細胞之作用。 當腦部受損後，腦細胞可利用膽固醇進行修補的工作。為驗證
膽固醇含量降低的細胞經麩胺酸毒殺作用後，是否仍具有修復之能力，我們將經過 lovastatin
及 b-cyclodextrin 前處理的海馬迴細胞培養，在麩胺酸處理時及8小時的復原期間均加入 lovastati
n 及 b-cyclodextrin ，發現其時所見之細胞死亡均較復原期間未以 lovastatin 及 b-cyclodextrin 處理
之細胞為高。由此可見，膽固醇降低的細胞在經麩胺酸作用後，仍可以利用膽固醇進行細胞
重塑。 現今已知阿滋海默症及腦中風等腦部疾病皆與細胞膽固醇代謝異常相關；本論文研
究利用海馬迴細胞培養的模式系統，證實降低膽固醇會加劇腦細胞的死亡，印證膽固醇在腦
部疾病的病理機制中扮演著很重要的角色。此模式系統也可應用於未來探討膽固醇對腦部疾
病影響詳細機轉的研究。
Abstract:
Cholesterol, the main component of cell membrane, is essential for normal cellular functions. Ample
evidence indicates that alterations in cellular cholesterol levels are associated with neurological disorders.
Using hippocampal culture as a model system, this study aims to investigate the mechanism by which
cholesterol modulates cell death in the brain. Given the excitotoxic mechanism of neuronal death,
hippocampal neuron/glia mixed cultures were treated with glutamate (Glu) for 6 or 12 hr followed by a
recovery period of 8 hr, in accordance with the delayed cell death occurred in brain diseases. Membrane
damage, as determined by the leakage of lactate dehydrogenase (LDH), was observed in cultures treated
with Glu (2 mM) for 6 hr. A 12 hr-incubation of Glu, nonetheless, resulted in membrane damage as well as
mitochondria injury. The Glu-induced neurotoxicity was verified by the reduced number of neuronal cells, as
determined by levels of the neuron-specific microtubule-associated protein 2. In contrast, the expression of
glial fibrillary acidic protein (GFAP), a specific marker of astrocytes, increased in cultures subjected to Glu
treatment, indicating the activation of gliosis as observed in injured brain. The impact of cholesterol on
Glu-induced cell death was studied in cultures subjected to cholesterol depletion by lovastatin and

2. b-cyclodextrin. Cholesterol depletion was found to exacerbate mitochondria injury and LDH leakage resulted
from Glu treatment for 6 and 12 hr. Removal of cellular cholesterol, nevertheless, reduced the expression of
GFAP, suggesting that cholesterol may take part in the activation of gliosis. The deleterious effect of
cholesterol depletion was reversed by mevalonate, a key intermediate of cholesterol synthesis. In view of the
elevated release of LDH, it likely that cholesterol depletion may impair the plasma membrane, thus
rendering cells more vulnerable to the excitotoxic insult. Much evidence suggests that cholesterol provides
substrate for neuronal repairing in injured brain. The issue that lovastatin and b-cyclodextrin affected the
repair of injured neurons after Glu treatment was also addressed. Cell viability was examined in cultures
subjected to cholesterol depletion followed by Glu treatment and an 8 hr-recovery period in the presence of
lovastatin and b-cyclodextrin. The leakage of LDH exceeded that released from cultures receiving no
lovastatin nor cyclodextrin during Glu incubation and the recovery period. It appears that the pretreatment
of lovastatin and b-cyclodextrin does not abolish the capability of injured cells to recover after Glu
incubation. However, the possibility that cholesterol deprivation aggravated delayed cell death cannot be
eliminated. As noted, alternated cholesterol metabolism has been linked to brain diseases such as cerebral
ischemia and Alzheimer's disease. Using hippocampal cultures subjected to cholesterol depletion as a model
system, results from this study further supports the notion that cholesterol plays an important role in the
pathogenesis of neurological disorders.