摘要或動機

特定基因的表現與不同氧自由基的產生，已知會影響細胞的生長和死亡。我\r
個人有興趣利用間質幹細胞體外培養擴充，以為筋骨組織再生的可能應用。因此\r
利用轉植(transfect)突變ras 基因(Glu61Leu)進入骨髓間質細胞株(HS-5 cells)，來\r
控制ras 基因表現的高低，進而研究這些間質細胞隨著Ras 蛋白質表現的高低，\r
對氧自由基引導細胞生長與死亡之影響。結果發現ras 基因高度表現的間質細胞\r
生長減緩；相較於原生株平均減少62.4%。進一步研究其生長減少是否與細胞凋\r
亡有關，發現ras 基因高度表現的間質細胞凋亡確實比原生株高22.6%。探究其\r
凋亡原因，發現與caspase-3 有關但和粒腺體功能無關：因為caspase-3 有活化，\r
但是以粒腺體膜電位螢光追蹤劑JC-1 測得的膜電位卻沒有改變。追蹤ras 基因高\r
度表現的間質細胞其細胞內氧自由基的產量，發現Ras 高度表現株其細胞內氧自\r
由基明顯增加。當細胞外加入超氧根轉化酵素(SOD, 500 U/ml)去清除超氧根時，\r
對Ras 高度表現細胞的凋亡沒有影響；但是外加觸化酵素(catalase, 500u/ml)於培\r
養液中，卻可以抑制ras 基因高度表現的間質細胞內caspase-3 活化和細胞凋亡；\r
並且增加細胞生長循環促進分子Cyclin D1 的表現。從這些研究我們歸結出兩點\r
重要新發現: 1) ras 基因突變造成Ras 高度表現時，會促成細胞內特定氧自由基產\r
生，使得細胞生長減緩並進行細胞凋亡，只有特定抗氧化酵素(catalase)才能恢復\r
其異常； 2) Ras 蛋白高度表現而引導氧自由基產生的細胞凋亡與caspase-3 活化\r
有關，但與粒腺體功能無關。根據這些發現，未來我們或許可以朝調節特定氧化\r
還原反應或使用caspase-3 抑制劑去調控間質幹細胞的生長，以供筋骨組織再生\r
的應用。Certain gene’s expression as well as different species of oxygen radicals can\r
affect cell growth and apoptosis. We are interested in amplifying mesenchymal\r
progenitor cells for the application of musculoskeletal tissue regeneration. Thus, we\r
transfected a mutant ras gene (61Glu/Leu) to change Ras protein expression in the\r
mesenchymal progenitor cell line (HS-5 cells) and studied how ras expressing levels\r
influenced intracellular oxygen radicals, and its relationship to cell growth and\r
apoptosis. Results showed that Ras over-expressing HS-5 cells grew slower than those\r
with wild type ras HS-5 cells and revealed a higher apoptosis rate. The higher\r
apoptosis in Ras over-expressing cells was not related to mitochondrial dysfunction\r
since mitochondrial membrane potential was normal as determined by flow\r
cytometric analysis of JC-1 fluorescent staining assay. The higher apoptosis was\r
related to higher caspase-3 activation. Further studies showed that Ras\r
over-expressing HS-5 cells revealed a higher production of intracellular oxygen\r
radicals in comparison to those with wild type ras HS-5 cells. Addition of catalase\r
(500 u/ml) but not superoxide dismutase (SOD; 500 u/ml) specifically revived the cell\r
growth associated with increase of cyclin D1 expression, but decrease of apoptosis\r
associated with lower caspase-3 activation. Results from these studies demonstrated\r
two important findings: 1) the ras gene over-expressing in a ras-mutant HS-5 cell line\r
triggers a higher production of intracellular oxygen radicals resulting in higher cell\r
apoptosis; and 2) the higher oxygen radicals related cell apoptosis is mediated by\r
caspase-3 but not mitochondrial dysfunction. Based on these findings, we may\r
propose to regulate mesenchymal progenitor cell growth for musculoskeletal tissue\r
regeneration via modulation of redox reactions or caspase-3 inhibitors in the future.