中文摘要
神經細胞在體內生長需靠neurotrophic factors及神經的傳入刺激才能良好生存，在體外培養的神經細胞則需使用含有血清的高鉀(25 mM K+)培養液，小腦顆粒神經細胞(cerebellar granule neurons, CGN)的培養模式，一直被用來研究神經細胞生長發育及凋亡的機轉，以往只知這和高鉀引起的胞內鈣離子濃度的持續升高有關，但對於維持生存的胞內鈣離子濃度的詳細變化及細胞內鈣貯池在維持神經細胞的發育上比較少被研究。本研究之目的在於使用Thapsigargin (TG)，一種內質網鈣離子幫浦拮抗劑，來探求此內鈣池與神經細胞內鈣離子濃度變化及相關之存活率。結果發現在 CGN培養初期有血清的存在下，不論有無高鉀的刺激，細胞內鈣離子濃度都會先持續地上升蓄積至一高峰值，到第3天 ( 780 nM) ，而第八天後卻降回（252.3nM）。之後漸下降而成一鐘形曲線。神經細胞培養於體外時，支持初期發育的胞內鈣離子濃度之蓄積，主要來自血清的刺激以及內質網鈣離子幫浦的運作，受胞外高濃度鉀離子的影響則較微小。因此發育初期培養於一般正常鉀濃度的未成熟期神經細胞可能因具有同樣的胞內鈣離子濃度變化而使其形態與生存率與高鉀培養液中的相近。這些培養初期胞內鈣離子濃度的持續蓄積無論高鉀的刺激存在與否皆可被TG幾乎完全阻止，劇烈地降低，且造成神經細胞大量凋亡。與一般預期將因造成胞內鈣離子濃度過度上升的結果相反。這說明了未成熟的(DIV=2)的神經細胞在失去高鉀狀態的環境能存活於是因為它可以維持細胞內的高鈣離子濃度，而這需要內質網貯存的鈣離子的參與。在細胞接近成熟時培養於一般正常鉀濃度 (5 mM K+) 的神經細胞內鈣離子濃度開始明顯低於培養於高鉀狀態下的，並伴隨著細胞的凋亡。當神經細胞在高鉀培養液中成熟後，TG對其胞內鈣離子濃度及生存率的毒性將大幅地降低，然而TG將內質網鈣離子的排空，卻可阻止成熟的神經細胞因失去高鉀及血清的刺激而導致的凋亡。 由本實驗的結果可推知，當神經細胞成熟後，維持存活的胞內鈣離子濃度，則主要來自胞外高鉀的刺激引起的外鈣內流。此時，經過充分發育成熟後的神經細胞，其內質網中的鈣離子對整體神經細胞內鈣離子濃度的作用角色也已大幅改變。
臨床上治療manic-depressive disorder常用的鋰鹽 (LiCl) 也和TG有著相同的作用，若鋰鹽LiCl存在於所換入的培養液時，則細胞內鈣離子濃度也會劇烈地降低，造成神經細胞大量死亡。當神經細胞成熟時，若是鋰鹽存在所換入的不含血清之正常鉀離子培養液培養液時，則神經細胞內鈣離子濃度亦會比不加鋰鹽的對照組更為降低，而生存率卻大幅地提升。由於鋰鹽已被報導會抑制磷酸肌存醇的訊息傳遞 (receptor-stimulated phosphatidylinositol (PI) second messengers) ，而內質網上又有著三磷酸肌醇 (IP3) 的受體可促使內質網貯存的鈣離子釋放到細胞質中。這更加證明內質網對形成此細胞內高鈣離子濃度的貢獻，以及對傳遞凋亡訊息的鈣離子的重要調控角色。
當小腦顆形神經細胞培養在 DMEM 時，TG 無法阻止失去胞外的高鉀濃度與血清引起的成熟神經細胞死亡。TG略為提升了因失去高鉀及血清而下降的神經細胞內鈣濃度 89.75 nM至 117.64 nM，但是卻會加重神經細胞的死亡，生存率由 494% 降至 282%。但若是有與神經膠細胞共同培養的神經細胞，此種因失去高鉀及血清刺激的神經細胞的死亡會加重，然而在此種神經膠細胞與神經細胞共同培養的狀態下，TG 對此種死亡的保護作用又會再現。處理TG又可以使神經細胞內鈣濃度更加排空降低而有效降低神經細胞的死亡。本實驗的結果顯示神經細胞周圍的神經膠細胞會顯著地影響神經細胞對外來刺激的反應，此種影響又可因神經細胞所受不同的刺激，將會有保護或加劇傷害神經細胞的相反作用。
在研究此 TG，鋰鹽及血清的劇烈作用的過程中，我們發現在原培養著神經細胞的培養液之中測細胞內鈣離子濃度時，可看見大不相同於一般使用緩衝溶液測量的結果，在緩衝溶液中的測量的結果無法可看上述的鐘形曲線，以及因此發育初期培養於一般正常鉀離子濃度的未成熟期神經細胞具有同樣於培養在高鉀培養液中的相近樣的胞內鈣離子濃度變化的現象，而來解釋使其與培養在高鉀培養液中的相近的形態與生存率。在緩衝溶液中的的測量的結果中TG對未成熟小腦顆粒形神經細胞內鈣離子濃度降低的幅度與其引起的毒性相比並不相稱，在原培養液中量測到TG對未成熟小腦顆粒形神經細胞內鈣離子濃度降低的幅度比在緩衝溶液人工腦脊液中所量到的大得多。如此我們可測得為何高鉀培養液可使神經細胞存活於體外的端倪及神經細胞成熟的過程中細胞內鈣離子濃度的真正變化。這可作為日後研究方法上的參考。
在本研究中，我們發現了內質網鈣貯池在調控神經細胞之存活的重要角色。其中在未成熟神經細胞中，支持其生存發育的胞內鈣離子濃度主要是內質網鈣離子幫浦的運作且主要受來自血清中的活性因子的刺激與調控。此論點不同於一般認為維持神經細胞存活的鈣離子濃度主要依賴細胞膜去極化引起的細胞外鈣內流之作用的觀念。
另外，神經細胞在高張溶液的刺激之下，縮小體積的同時亦會出現鈣離子濃度降低的現像，且甚至會蓋過細胞膜去極化引起的外鈣內流的效應。這性質的發現可使我們更了解鈣離子和神經細胞體積調節機制的關係。
本論文的結果所得的重要啟示是我們發現傳統在緩衝液測量細胞內鈣濃度，所得數據不但偏低，而且不能反應真正培養細胞所需血清的重要意義，而我們研究在原培養液測鈣所得之數值，除了數值較高之外，而且比較能正確反應血清所以能支持神經細胞正常生長及發育，是有賴於其能提高發育中的神經細胞的內鈣濃度日益升高的作用，除此之外，以此方法測量也可真正觀察到內質網鈣離子幫浦的運作對血清增加胞內鈣濃度的重大影響。在發現此內質網鈣蓄池對神經細胞生長的重要性之後，並且又發現臨床上常用，會干擾內質網鈣蓄池訊息傳遞物三磷酸肌醇 (IP3)代謝的鋰鹽其神經毒性是來自干擾神經內鈣濃度之調控，而神經細胞周圍的星狀膠細胞也對神經細胞內鈣濃度也有調節的作用，因此在面臨外界環境變化的壓力狀態(如成熟神經失去高鉀及血清之刺激)下，星狀膠細胞能協助TG 保護神經細胞，免因失去高鉀及血清而導致凋亡。總之，藉由我們改良測鈣的方法，使我們得以闡明血清之培養神經細胞不可或缺的機轉。也讓我們進一步瞭解內質網鈣儲池對神經細胞生長的調控的機制，進而瞭解臨床使用藥鋰鹽神經毒性與干擾細胞內鈣之關係。因此，由基礎對細胞內鈣濃度調節機制的研究，以至於對臨床藥物神經毒性的瞭解。這是本論文的重要成果，但引發許多引人入勝的問題，如內質網鈣儲池如何藉鈣傳達訊息至細胞核內，引發各種基因表現來影響細胞生長及發育的重要問題，有待今後繼續探求。

Abstract
The roles of intracellular calcium pool involved in regulating Ca2+ profile and neuronal survival rate during development were studied by using thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase in the cultured cerebellar granule neurons (CGN). Measuring the neuronal [Ca2+]i directly in the culture medium, we found a bell-shaped curve of [Ca2+]i versus cultured days in CGN maintained in medium containing serum and 25 mM K+. The progressive increase in [Ca2+]i of the immature granule neurons (1-4 DIV) was abolished by thapsigargin, which resulted in massive neuronal apoptosis. Lowering the [K+] from 25 to 5 mM, neither the progressive increasing [Ca2+]i nor the survival of immature granule neurons was significantly changed during 24h incubation. Similarly, thapsigargin exerted a dramatic decrease in [Ca2+]i and survival rate of these immature neurons switched to 5mM K+ medium. Following maturation, the granule neurons became less sensitive to thapsigargin both in [Ca2+]i and neuronal survival. However, thapsigargin can protect the mature granule neurons from the detrimental effect of switching to 5mM K+/serum free medium by depleting [Ca2+]i to an even lower level than that in respective thapsigargin free group. Based on these findings, we propose that during immature stage, thapsigargin-sensitive endoplasmic reticular Ca2+-ATPase play a pivotal role in progressive increase of [Ca2+]i which is essential for the growth and maturation of cultured granule neurons.
The commonly used anti-manic depressive drug, LiCl has similar effects of TG on the immature neurons and the HK/serum deprivation induced death of mature neurons. This may be due to the effects of LiCl on the IP3 second messenger, which can trigger Ca2+ release from endoplasmic reticulum (ER). However, unlike TG, LiCl still exerts similar toxic effect on mature granule neurons.
In order to understand the differential roles of potassium and serum on this survival promoting intracellular Ca2+ level, [Ca2+]i, during the time course of development, we continuously monitored [Ca2+]i in the developing CGN under different culture conditions with various concentrations of serum and K+. We found a progressive increase of [Ca2+]i to a similar extent in the first 3 days with either HK or NK medium containing 10% serum. Lowering serum concentration to 2% and 0.5%, not only markedly reduced [Ca2+]i of the immature neurons in a concentration-dependent manner but also dramatically decreased the neuronal survival especially in the NK group. This effect of serum on [Ca2+]i level was further confirmed by acute application of serum and the serum deprivation. Following maturation, after 3-4 days cultured in 10% serum, the neuronal [Ca2+]i began to decline in both HK and NK groups. However, a faster decline rate accompanied with massive neuronal death was found in the NK group. Therefore, we concluded that the immature CGN survive by the trophic factors of serum mediated by elevating [Ca2+]i in the first 3 days. After 3-4 days cultured, the neurons differentiate to mainly depend on HK evoked sustained rise in [Ca2+]i.
Unlike that in BME medium, in the absence of co-cultured astrocyte, the protective effect of TG on the detrimental effect of HK/serum deprivation could not be observed in neurons cultured in DMEM. In astrocyte-poor cultures of mature CGN (10 DIV), the basal level of [Ca2+]i dramatically droped from 1845 to 89.75 nM 24 h after HK/serum deprivation. Although treatment with TG slightly increased the [Ca2+]i to 117.64 nM, the survival rate of the neurons was even worse, reduced from 494% to 282%. However, in the presence of co-cultured astrocyte, the protective effect of TG could be seen again. In the astrocyte-rich cultures, HK/serum deprivation 24 h still caused a profound reduction of neuronal [Ca2+]i from 1663 to 90.26 nM accompanied with an even more serious neuronal death. Contrarily, treatment with TG in astrocyte-rich cultures further lowered the [Ca2+]i to 652 nM but markedly improved the neuronal survival rate from 4.51% to 602% in a concentration-dependent manner.
Owing to the multiple regulatory mechanisms on [Ca2+]i levels, we try to examine the differences between [Ca2+]i measured directly in the culture medium and in the commonly used buffer solution. The results obtained clearly indicated that changes in the neuronal survival in HK or NK culture medium are well correlated with [Ca2+]i measured in the respective culture medium but not in the buffer solution. The [Ca2+]i measured in the culture medium is much higher [Ca2+]i than that in the buffer solution. The contribution of serum and medium in these differences are also demonstrated. Furthermore, the cytotoxicity of thapsigargin, a Ca2+-ATPase inhibitor of endoplasmic reticulum on immature granule neurons is also correlated well with its dramatic lowering [Ca2+]i measured in the treated medium. The implication of these findings is that the importance of Ca2+ in regulating cellular functions can be better assessed by monitoring [Ca2+]i changes right in culture medium rather than in the commonly used buffer solution. This study highlights the impact of the measuring medium on microfluorimetric Ca2+ measurement.
In addition to the agents applying to the neurons, the volume change induced osmolyte fluxes also plays an important role in many pathological situations. Many studies focused on the relation between Ca2+ flux and the regulatory volume decrease after hypotonic condition induced swelling. In contrast, we investigated the hypertonic condition induced change in [Ca2+]i. The results obtained indicated that no matter the following regulatory volume increase was present or not, the neuronal [Ca2+]i reduced after hypertonic condition induced cell shrinkage and this effect could even cover the depolarization evoked Ca2+ influx. This finding can provide information to clarify the role of Ca2+ in volume corrective signals.
The results obtained from this study highlight some new insights about the Ca2+ signals in manipulation neuronal development and survival.

Cover
目錄
Abbreviations used
中文摘要
英文摘要
Introduction
The Scheme of this study
Materials and Methods
Results
Part 1. Roles of thapsigargin-sensitive Ca2+-store in the survival of developing cultured neurons
Part 2. Altered intracellular calcium level in association with neuronal deathinduced by lithium chloride
Part 3. Differential roles of serum and high K+ in regulating [Ca2+]i and survival of developing neurons
Part 4. Modulation by astrocyte-coculture in altering the intracellular Ca2+concentration and neuronal survival induced 39 by thapsigargin
Part 5. Measuring[Ca2+]i in culture medium rather than the commonly used buffer
solution has a better correlation with the neuronal survival
Part 6. The Ca2+ flux during hypertonic stress in neurons and astrocytes
Discussion
Figures
References
Publications

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