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研究生:蘇懿瑄
研究生(外文):I-Hsuan Su
論文名稱:藉由Metformin調節成骨細胞凋亡減緩發炎性骨吸收
論文名稱(外文):Metformin alleviates inflammatory bone resorption by modulating apoptosis in osteoblasts
指導教授:賴向華賴向華引用關係
口試日期:2017-03-29
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:臨床牙醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:48
中文關鍵詞:Metformin活性氧分子根尖病變成骨細胞細胞凋亡
外文關鍵詞:MetforminReactive Oxygen Species (ROS)apical lesionosteoblastapoptosis
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根尖病變(periapical lesion)所引起的發炎性骨吸收會形成一個缺氧的環境,此時粒線體內電子傳遞鍊部份會受阻而促進活性氧分子(Reactive Oxygen Species, ROS)的生成,此時過多的ROS不但會造成組織的傷害,也會引發成骨細胞的凋亡。Metformin是臨床上一廣為被使用作為降血糖的藥物,它同樣也具備抗發炎、減少ROS以及影響骨代謝的功能。在我們先前的研究已經證明,發炎反應時產生的活性氧和根尖的骨頭喪失有關,且牙根尖病變的範圍越大,成骨細胞的細胞凋亡數目越多。然而,其中詳細的機轉仍待闡明。在本次的研究中,首先我們觀察人類骨肉瘤細胞(human osteosarcoma cell)U2OS在缺氧刺激下,利用MitoSox試劑檢測細胞加入Metformin後是否影響ROS的濃度; 凋亡路徑的測定方面,我們利用西方點墨法去分析,前凋亡分子cytochrome C、凋亡蛋白酶caspase-9和“poly(ADP-ribose) polymerase 1” (PARP-1)片段的蛋白質表現,是否因Metformin的加入而改變; 另一方面也設計誘導根尖病變的大鼠模型,在經過標準的根管清創後給予Metformin治療,利用影像學分析以及組織免疫染色去探討對於根尖病灶的癒合狀況。
研究結果發現,缺氧刺激會提高ROS在粒線體中的濃度,讓粒線體釋放cytochrome C,且使caspase-9與PARP cleavage的表現增加,顯示細胞走向凋亡。但同樣的狀況下加入Metformin後,ROS在粒線體的濃度不但被降低,Cytochrome C的釋放和Caspase-9、cleaved-PARP的表現也同樣被抑制。動物實驗方面,從組織切片染色的結果,我們觀察到在根尖發炎病變的區域中,有明顯的細胞凋亡訊號。而影像學分析的結果顯示,Metformin以根管局部的投藥方式可以有效減小根尖病變的範圍。總結以上所述,在in vitro的研究中我們驗證”因缺氧所誘發的ROS上升使成骨細胞走向凋亡路徑”可被Metformin抑制; 而在in vivo的研究中,我們也發現局部投予Metformin能減緩大鼠根尖病變的發炎性骨吸收。
Inflammatory bone resorption in the periapical lesion causes a hypoxic environment, so the electron transport chain of the mitochondria is blocked and it promotes the production of reactive oxygen species (ROS). Accumulation of ROS not only results in tissue damage, but also leads to osteoblast apoptosis. Metformin is clinically widely used as a hypoglycemic drug, and it also attenuates inflammation, reduced ROS production, affects the function of bone metabolism. In our previous studies, it has been shown that the ROS generated during the inflammatory response is associated with bone loss in the apical region. The larger area of apical lesions, the greater number of apoptotic cells in osteoblasts. However, the detailed mechanism remains to be elucidated. In this study, the human osteosarcoma U2OS cells were incubated in hypoxic environment. MitoSox reagents was used to detect ROS concentration after adding Metformin. To determine whether Metformin affected the hypoxia- induced apoptotic pathway, we detected the protein expression of cytochrome C, apoprotein caspase-9 and poly (adenosine phosphate ribose) polymerase (PARP) fragments in the pathway by Western blott. Further more, the rat model with induced apical lesion were also designed. Metformin treatment was performed after standard root canal debridement, and the healing of the apical lesion was investigated by imaging analysis and tissue immunostaining.
The results showed that hypoxia stimulation increased the concentration of ROS in the mitochondria, the release of cytochrome C and increase the expression of caspase-9 and PARP cleavage, indicating hypoxia induced apoptosis in U2OS. However, adding Metformin under the same conditions not only reduce the concentration of ROS in the mitochondria, but also inhibited the release of cytochrome C and enhance the expression of caspase-9 and PARP cleavage. In the animal experiments, a number of cells apoptosis were observed in the apical inflammatory tissue. However, Metformin effectively reduced the area of apical lesion by local administration in the root canal. In conclusion, we found that osteoblast apoptosis induced by functionally-altered mitochondria in hypoxia can be suppressed by Metformin through inhibiting production of ROS. We also found that local administration of Metformin attenuated inflammatory bone resorption in rat apical lesions.
口試委員審定書-----------------------------------------------------------------------------------------i
誌謝-------------------------------------------------------------------------------------------------------- ii
中文摘要------------------------------------------------------------------------------------------------- iii
英文摘要------------------------------------------------------------------------------------------------- V
第一章 導論---------------------------------------------------------------------------------------------1
1.1 根尖病變之發炎反應及缺氧所引起的細胞凋亡------------------------------------1
1.1.1 發炎反應與缺氧----------------------------------------------------------------------1
1.1.2 缺氧與細胞凋亡----------------------------------------------------------------------1
1.1.3成骨細胞凋亡增加對根尖病變之發展的影響--------------------------------3
1.2 根尖病變的治療與根尖組織的修復----------------------------------------------------3
1.2.1控制發炎反應--------------------------------------------------------------------------3
1.2.2 促進組織修復------------------------------------------------------------------------ 4
1.3 Metformin與根尖病變-----------------------------------------------------------------------4
1.3.1 Metformin------------------------------------------------------------------------------ 4
1.3.2 Metformin抑制發炎與活性氧分子----------------------------------------------6
1.3.3 Metformin在牙科方面的應用-----------------------------------------------------7
第二章 實驗目的---------------------------------------------------------------------------------------9
第三章 實驗方法--------------------------------------------------------------------------------------10
3.1 缺氧誘發成骨細胞凋亡與Metformin對此路徑的影響--------------------------10
3.1.1 細胞培養------------------------------------------------------------------------------10
3.1.2 以MitoSox測定細胞內ROS的濃度-------------------------------------------- 10
3.1.3以西方點墨法測定粒線體/細胞質之Cytochrome C------------------------ 11
3.1.4以西方點墨法測定細胞質內凋亡marker: Caspase-9、cleaved- PARP---
----------------------------------------------------------------------------------------------13
3.2 誘導根尖病變動物模型--------------------------------------------------------------------15
3.3 免疫組織染色--------------------------------------------------------------------------------17
3.4 TUNEL檢測----------------------------------------------------------------------------------18
第四章 實驗結果-------------------------------------------------------------------------------------20
4.1 缺氧誘發成骨細胞凋亡------------------------------------------------------------------20
4.2 Metformin對缺氧誘發的細胞凋亡之影響---------------------------------------- 20
4.3 局部投予Metformin於根尖病變大鼠模型之療效------------------------------- 21
4.3.1 於2D以及3D影像分析之結果---------------------------------------------- 21
4.3.2 組織染色的結果------------------------------------------------------------------- 21
第五章 討論-------------------------------------------------------------------------------------------23
5.1缺氧誘發成骨細胞凋亡------------------------------------------------------------------23
5.2 Metformin可以抑制缺氧刺激所誘發的細胞凋亡--------------------------------24
5.3 Metformin於大鼠根尖病變的療效---------------------------------------------------25
第六章 結論 ---------------------------------------------------------------------------------------- 30
第七章 未來展望 ---------------------------------------------------------------------------------- 31
參考文獻 --------------------------------------------------------------------------------------------- 32
附件----------------------------------------------------------------------------------------------------- 37
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