| 摘要: | 腦神經細胞在出生之後即停止生長並分化為各種突觸細胞，最近的研
究顯示細胞的生長與細胞週期調控蛋白及細胞週期的進程有關，但腦部神
經分化的分子機轉大部分仍不清楚。在此實驗，我們使用兩種研究模式:
初代培養神經細胞(於胚胎期第18天解剖)，屬In vitro模式；出生前後不
同時期大腦皮質細胞取樣，屬於In vivo模式。在兩模式不同的時間點偵
測細胞週期調控蛋白與α-tubulin (microtubule的組成物)的表現，來了
解大白鼠出生後神經細胞生長的控制。除此以外，我們以一項新的技術-
差異顯示法，來顯示DIV 3 (培養皿培養3天) 與DIV 14兩者之間表現有差
異的基因。只於DIV 3而非DIV 14天表現的有8個基因片段，只於DIV 14而
非 DIV 3表現的基因則找到了7個。它們被選殖進入TA質體與pBS質體。以
PCR放大的基因片段做的引子，將用來驗證這些基因是否也會在In vivo模
式中不同的時間點表現。我們在In vitro與In vivo兩者偵測Rb、cyclin
D3、cdk4、p16與p21的表現，結果發現它們在這些時間點都沒有什麼顯著
變化。細胞週期抑制蛋白: p16、p21與p27在初代培養與出生前後的神經
細胞都只表現少量，可能是為了抑制神經細胞在胚胎期第18天之後的細胞
週期進行。剛出生幾天的老鼠，其大腦蛋白-α-tubulin在SDS-PAGE上可
看出表現為兩種形式。此較小分子量的形式在出生後開始出現到第16天消
失，而在出生前或出生16天之後，只有高分子一種型式存在。在初代培養
的細胞中，也只有高分子一種型式出現。由免疫沉澱法與Potato acid
phosphatase的處理，可知此蛋白之酪氨酸未被磷酸化。為了試圖了解如
何引發此現象的產生，我們將A23187、PMA、EGTA、KCl、NMDA、安非他命
、NGF與CRH加入初代培養5天之神經細胞，但沒有一種能改變α-tubulin
的表現形式。此項結果顯示α-tubulin在出生後二週會以低分子量的形式
存在，究竟此低分子量係去酪氨酸的α-tubulin或另一種變異型式，及引
發此型態出現的原因和影響，均有待進一步的研究證實。
Studies of Neuronal Cell Differentiation in Neonatal Rat Brain
Neuronal cells cease proliferating before postnatal stage. It is
knownthat cell cycle regulatory proteins control the progression
of cell cycle, but the molecular mechanisms involved in neuronal
differentiation in brain remain unclear. In the presence study,
we use primary cultured neuronal cells (in vitro model,
dissected on embryonic day 18) and pre- and postnatal rat brain
(in vivo model) to examine the expression of cell cycle
regulatory proteins and a-tubulin, the component of microtubule.
By the new technique " Differential Display ", we have
identified the genes that express differently between DIV 3
(days in vitro) and DIV 14. Eight gene fragments were found
expressing on DIV 3, but not DIV 14. Seven gene fragments were
found expressing on DIV 14, but not DIV 3. They were cloned into
TA vectors and pBS vectors. PCR amplified gene fragments will be
use as probes to verify the mRNA expression of the genes on
different days in prenatal and postnatal rat brain. We screened
the expression of Rb, cyclin D3, cdk4, p16 and p21 both in vitro
and in vivo. No significant expression of these proteins were
found during these time points. Cell cycle inhibitory proteins,
p16, p21 and p27 were found expressing at low level in both
primary cultured cells and pre- and postnatal neuronal cells,
presumably inhibiting the cell cycle progress of neuronsafter
embryonic day 18. a-Tubulin, migrated as a doublet in SDS-PAGE
at early postnatal rat brain. This low molecular weight form of
a-tubulin was neither expressed in the primary culture cell
system, nor were they expressed before birth or 16 days after
birth in vivo. This protein was not tyrosine-phosphorylated
demonstrated by immunoprecipitation and potato acid phosphatase
treatment. To investigate the cause of these phenomenom, A23187,
PMA, EGTA, KCl, NMDA, amphetamine, NGF and CRH were added to DIV
5 cultured neurons. None was found to change the expression form
of a-tubulin. Further characterization of the genes found in
differential display will help us understand the neuronal
differentiation in neonatal rat brain. |
