Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22634
標題: 建立綠螢光基因轉殖小鼠之羊水間葉幹細胞及其應用於高濃度氧肺損傷之修復作用
Establishment of Mouse Amniotic Fluid-derived Mesenchymal Stem Cells from eGFP Transgenic Mice and Its Application on the Repairs of Hyperoxia-induced Lung Injury
作者: 溫世濤
Wen, Shih-Tau
關鍵字: lung injury;肺損傷;hyperoxia;fibrosis;stem cell;高濃度氧氣;肺纖維化;幹細胞
出版社: 生命科學系所
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摘要: 
間葉幹細胞於現今的科學領域中被廣泛的研究,其因間葉幹細胞擁有分化為多種細胞的能力,在人類羊水中所分離出的間葉幹細胞,已經被證實在體內與體外都具有多能功能性分化能力。在目前的臨床醫學上,患者在肺部功能失調時,必須給予高濃度氧氣治療賴以維持生命,而常會伴隨著引發另外的肺損傷。我們藉由設計實驗以增加由於曝露於高濃度氧氣時所引發肺損傷的保護作用,去證明是否羊水幹細胞能夠改善經由高濃度氧氣曝露後受損的肺部。我們選擇於懷孕週期11至14天的綠螢光基因轉殖小鼠作為分離抽取羊水間葉幹細胞的目標,並且利用挑取外型類似纖維母細胞的克隆群落再加以持續培養,這些細胞會表現出高度的綠螢光以便其後追蹤,與藉由流式細胞儀檢測細胞表面抗原,其表現出CD44與CD105成陽性反應而CD117為陰性反應。為了證實羊水間葉幹細胞有分化的潛能,選擇了兩種體外分化的方式去鑒別之,其可以分化為脂肪細胞與硬骨細胞,並分別藉由Oil Red O與Alizarin Red染色去證實。我們也發現所篩選出的羊水間葉幹細胞,透過反轉錄聚合酶連鎖反應得知,其會表現出Oct-4與Nanog這兩種胚胎幹細胞專一表現之基因。在動物模式中,將小鼠持續曝露於高濃度氧氣,觀察得知其曝露於高濃度氧氣96至102小時時會達到致死率50%。當在給予小鼠於高濃度氧氣曝露24小時後,從尾靜脈注射5x105顆羊水間葉幹細胞回體內,可發現曝露在96至102小時的小鼠還高達9成的存活率,而在48小時後給予注射羊水間葉幹細胞,也還有7成5的存活率。爾後,選擇給予小鼠曝露於高濃度氧氣60小時,並在曝露高濃度氧氣後,將小鼠移放入正常的飼養環境給予其自行修復,並比較在曝露高濃度氧氣60小時之後,是否從尾靜脈注射給予羊水間葉幹細胞,對其肺臟的發炎環境與修復性作比較。在隨著曝露高濃度氧氣時間的增長,可以發現其肺部組織伴隨著肺水腫、肺血腫、發炎細胞浸潤於肺臟與少部分的細胞產生細胞凋亡的現象,當小鼠離開高濃度氧氣的環境後,其種種發炎現象會因為其自體產生修復作用而慢慢降低,而在修復的過程中,大部分的小鼠會在肺部產生纖維化的作用,並且隨著時間增加,其肺部纖維化的情形會日益嚴重,雖然產生肺部纖維化也是修復的一種方式,但是產生肺部纖維化會造成後續肺部的功能失調,影響其氣體交換作用的能力。若是在曝露高濃度氧氣60小時之後,給予羊水間葉幹細胞的注射回體內,可以發現發炎的環境會有顯著性的改善,像是肺水腫的情形會加速消失、浸潤於肺部的發炎細胞會相對消失減少許多,並且會促使肺部的組織結構快速的修復回正常。另外最重要的是,有給予羊水間葉幹細胞的小鼠離開高濃度氧氣的環境後,在自行修復的過程中,其肺纖維化的情形則改善降低很多,甚至幾乎找不到有肺纖維化的地方。另外,從將間葉幹細胞與經由高濃度氧氣導致肺損傷的肺臟組織液共同培養的實驗中,也觀察到羊水間葉幹細胞會被受損肺臟的組織液趨化之,這說明了可能受損的肺部會釋放出一些水溶性因子與細胞激素,去刺激間葉幹細胞的增生與遷移至該受損的區域中。因此我們認為藉由注射羊水間葉幹細胞回小鼠體內,可以有效的改善高濃度氧氣對於肺部所造成的損傷。而在後續的研究中,將持續的探討與追蹤這些羊水幹細胞在修復機制裡面所扮演的角色與提供的功能。

Mesenchymal stem cells were studied extensively because they have the capable to differentiate into multiple lineages. Human amniotic fluid derived stem cells (AFS) have been demonstrated that exhibiting broadly multipotential differentiation in vitro and in vivo in previous studies. In the present clinical, the patients were got lung injury due to oxygen therapy when it is necessary. We designed the experiment in order to improve the protection from hyperoxia demages. To determine whether amniotic fluid derived stem cells could ameliorate the lung injury induced via exposured in the 99% oxygen environment. Murine amniotic fluid-derived stem(mAFS) cells from Tg. eGFP mice in the 11~14 days of pregnancy were isolated. To characterize the mAFS cells, the selected clones showed a homogenous fibroblast-like morphology and all the cells express highly eGFP fluorescence. The mAFS cells were expressed positive for markers as CD44, CD105 and negative for CD117(c-Kit) by flow cytometry analysis. To validate the mAFS cells differentiation potential, two in vitro differentiation tests have been that done, they have capability to differentiation into adipocytes that were assayed by Oil Red O staining at intracellular lipid droplets and osteogenesis was assessed by the mineralization of calcium accumulation by Alizarin Red. We also found that mAFS cells expressed Oct-4 and Nanog by RT-PCR. In animal model, mice were placed in a sealed plexiglas chamber and exposed to 99% oxygen for 24h, 48h, 60h and 72h. The LD50 was observed after the mice exposure in high oxygen for 96~102h. A number of 5x105 mAFS cells were injected into hyperoxia induced lung injury mice through tail intravenous infusion in 24h and 48h after hyperoxia. The mice group which injected mAFS cells after 24h hyperoxia showed high survival rate then the group without injected mAFS cells at 96~102h after hyperoxia. We chose a high concentration of oxygen exposured 60h time point to observe whether injected mAFS would affect the lung repair after 60h hyperoxia in 7 days chased. Lung tissues were harvested at 1, 3, 7 days after hyperoxia treatment. With the time increased of exposure hyperoxia, we found that the lung tissues accompany edema, hemorrhage, cellularity, inflammatory cells infiltration and a few cells arise apoptosis after hyperoxia. With the departure of a high concentration in oxygen exposured environment to give the mice repair themselves. The mice lung tissues will be accompanied by the case of pulmonary fibrosis. Although the occurrence of pulmonary fibrosis is an act of repair condition, it will lead to pulmonary dysfunction. If injected the mAFS cells after hyperoxia, we found that the inflammatory environment will be a significant improvement. The effects of the pulmonary edema situation quickly dissipated, infiltration of inflammatory cells rapid decrease in the lung and accelerate the repair of lung structure. In addition the most important thing is, injected mAFS cells would reduce or ameliorate pulmonary fibrosis after the mice leave the high concentration in oxygen exposured environment. We also observed that mAFS cells would be attracted by the hyperoxia induced injured lung tissue fluid by co-culture experiment. It indicated that injured lung tissue might production of mAFS cells chemoattractants like cytokines that stimulate mAFS cells migration and proliferation to injured place. The improvement of lung injury against hyperoxia damages has been observed via infusion the mAFS cells. We are now trying to figure out the role and function in the part of these mAFS cells.
URI: http://hdl.handle.net/11455/22634
其他識別: U0005-0901200912341000
Appears in Collections:生命科學系所

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