臺灣博碩士論文加值系統

神經醯胺是重要的脂質二次傳訊物(lipid secondary messenger). 媒介許多生長因子,
細胞激素, 或環境刺激(包括抗癌藥物等等) 的訊息傳遞. 有許多細胞分
子被發現和它有關,
例如: CAPK, Raf-1, ras, PKC等, 但神經醯胺引起細胞凋亡的詳細機制尚未被研究清楚.
在本實驗中, 我們使用人工合成的神經醯胺類似(C2-ceramide)及血癌細胞株嘗試了解, 它
引起細胞凋亡的機轉.
本實驗依(1)型態變化, (2)DNA階梯狀(DNA fragmentation), (3)細胞
週期中出現apoptotic
peak等現象判斷, 血癌細胞株 (HL60)的死亡是典型細胞凋亡(
apoptosis). 我們發現, 在細胞
凋亡的過程中伴隨poly-ADP ribosyl polymerase (PARP)的分解, 大
約2-4小時即可檢測到被
分解的片段(33kDa). 這個現象先於DNA階梯狀的發生, 所以我們判斷PARP
的分解和細胞凋亡
的發生可能相關. 而IAA(iodocaetamide)(nonspecific inhibitor of
cysteine protease)
和Ac-DEVD-CHO(specific inhibitor of CPP32/Yama/Apopain)的前處理
均可抑制神經醯胺引
起的細胞凋亡, Ac-DEVD-CHO亦明顯抑制PARP的分解. 這表示PARP的分解和細胞凋亡是由
CPP32/Yama/Apopain 的活化與否主導. 我們進一步發現細胞凋亡的過程
中伴隨CPP32/Yama/Apopain
的分解(活化), 大約2-4小時即可檢測到被分解(活化)的片段. 因此, 神
經醯胺引起細胞凋亡
的執行者乃是CPP32/Yama/Apopain, 至於PARP的分解是否為重要原因, 則
需要再進一步的實
驗證實.
我們嘗試了解細胞激素和生長因子, 對神經引起的細胞凋亡的調控. 在
IFN-gamma, GM-CSF,
TGF-beta 1, IL-6中, 只有TGF-beta1明顯抑制神經醯胺的效應.對於PARP的分解和
CPP32/Yama/Apopain的活化也有效地抑制. 同時也抑制神經醯胺引起的
BCL2減少. 雙重染色
的實驗中指出, 神經醯胺引起BCL2減少是全面的, 包括已走上凋亡和尚未走上凋亡的細胞.
而TGF-beta 1的抑制效果也是全面的, 雖然所有細胞的BCL2均未減少, 但
仍有部份走上凋亡.
而過量表現BCL2蛋白的血癌細胞也較耐受神經醯胺的毒性. 因此我們認為
TGF-beta1抑制神經
醯胺引起細胞凋亡的拮抗點, 應是BCL2蛋白.
我們也針對已報告的相關分子研究, 包括: c-jun mRNA, JNK, mapk,
發現神經醯胺引發的
c-jun mRNA 表現,JNK活性均被TGF-beta1抑制, 而神經醯胺引發mapk的活
性, TGF-beta1則不
影響. 因此, TGF-beta 1影響神經醯胺的效應是有選擇性的. 但c-jun
mRNA 和JNK 的表現和
活性的改變, 在TGF-beta1抑制神經醯胺引起細胞凋亡的相關性如何, 則
需要更進一步的實驗
證明.

Cdermide, a important lipid secondary messenger, was reported to be involved
in different signal transduction pathways in various cell lines. But the detail
mechanism of the apooptosis induced by ceramide was not well
understood. In this
report, we studied the mechanism by which artificial ceramide analogue, C2-
ceramide-induced apoptosis in human leukemic HL60 cells.
We demonstrated the apoptosis induced by ceramide by examination of (1)
morpholoical chnages, (2) DNA fragmentation, (3) apoptotic peak
viewed by FACScan
analysis. We found that the cleavage of PARP (poly-ADP ribosyl polymerase) was
coincident with the onset of apoptosis, i.e., the fragment of
PARP (33kDa) could
be detected with Western blot at the first 2 to 4 hours prior
to DNA fragmentation
Both IAA(iodoacetamide), a nonspecific inhibitor of cysteine
protease, and Ac-DEVD-
CHO, a specific inhibitor of CPP32/Yama/Apopain, prevent apoptosis induced by
ceramide in HL60 cells. Again, the activation of CPP32/Yama/
Apopain was correlated
to the cleavage of PARP and apoptosis. Accordingly, we
concluded that the excutor
of the apoptosis induced by ceramide was CPP32/Yama/Apopain,
and further experiments
should be performed to understand if the cleavage of PARP is a key step in this
process.
We woundered if there are some cytokines or growth factors can modulate the
apoptosis inudced by ceramide. To test this hypothesis, a wide
variety of growth
factors including IFN-gamma, GM-CSF, TGF-beta 1, IL-6 etc. were
used however, only
TGF-beta 1 significantly prevented the apoptosis induced by ceramide. Both the
cleavage of PARP and activation of CPP32/Yama/Apopain were
inhibited by TGF-beta 1
( 1ng/ml ).And the decrease of BCL2 by ceramide was also
inhibited by TGF-beta 1.
In the double-staining experiments analyzed by FACScan, ceramide downregulated
BCL2 was concomitantly observed in nonapoptotic cells and apoptotic cells. And
HL60/ws1 (bcl2 transfected) was more resistant to ceramide than
HL60. We proposed
that TGF-beta 1 inhibited the apoptosis induced by ceramide through preventing
the downregulation od BCL2.
We also studied other signaling pathways reported to be
associated with apoptosis,
including c-jun mRNA, JNK (c-jun N-terminal kinase), mapk
(mitogen-activated protein
kinase). Again, TGF-beta 1 atteuated ceramide-mediated JNK
activation as well as
c-jun mRNA increase, but it failed to affect mapk activity. But
the contribution
of c-jun mRNA expression and JNK activyti alteration were not
clear in TGF-beta 1
inhibiting ceramide-induced apoptosis, there should be more
experiments for uncovering
the detail mechansim.