臺灣大學: 臨床醫學研究所陳明豐Chen, Ying-HsienYing-HsienChen2013-03-272018-07-062013-03-272018-07-062010http://ntur.lib.ntu.edu.tw//handle/246246/253545研究背景 細胞之生死存亡的命運，可能與細胞所接受之外在環境刺激以及相鄰細胞所傳遞的訊號有關。而當中，細胞間隙接合可能是傳遞細胞間存活或死亡訊號的重要媒介。細胞自噬長久來被認為是一種細胞保護機制，透過細胞內部調控以達到清除壞損胞器或蛋白質的功能，進而促成延續細胞生存之目的。細胞自噬之研究者，莫不期望有朝一日能將調控細胞自噬化身為治療與預防疾病的利器。而目前有諸多研究顯示，細胞內部具同時調控細胞自噬與細胞凋亡的機制，細胞依據存在的環境狀況、所承受環境刺激之程度以及細胞壞損嚴重度，來決定細胞開啟何種生理機制；當中細胞內部的細胞自噬調控已被廣泛研究，但細胞間之調控研究卻付之闕如。反觀細胞凋亡之細胞間調控之研究，已證實細胞間隙接合可能是細胞間傳遞與溝通細胞凋亡訊號的重要窗口；為了讓細胞自噬之調控更為深入，進而運用這些調控方法於醫療使用，細胞間調控細胞自噬之研究的進行有其必要性。 研究目的 本實驗主要探討心肌細胞間隙接合對於心肌細胞於承受氧化壓力刺激引發細胞自噬中所扮演的角色。我們假設心肌細胞利用細胞間隙接合傳遞細胞之間的細胞自噬之訊號。 研究方法 我們使用H9c2作為實驗之模型，使用正庚醇(1-Heptanol)作為抑制細胞間隙接合之藥劑，並使用過氧化氫(Hydrogen peroxide, H2O2)作為氧化壓力源以誘發細胞自噬表現。細胞自噬偵測方式包括分析Light chain 3 (LC3)與Beclin-1之蛋白質與mRNA表現，同時利用流式細胞儀分析Acridine orange染色陽性比例。 本實驗進行，共分成三大部分: 實驗第一部分，計畫建立自身實驗室常模，探討H9c2細胞接受正庚醇以及過氧化氫之作用後之細胞反應，並尋求最適當的正庚醇與過氧化氫作用條件。 實驗第二部分，計畫合成pCMV-Lc3-Gluc質體，並將此質體以短暫轉染方式植入H9c2細胞內。同時檢驗經pCMV-Lc3-Gluc轉染之H9c2細胞能否作為偵測Light Chain 3 (LC3)表現之Luciferase Report System。 實驗第三部分，計畫在標準的細胞培養條件下我們將wild type H9c2細胞以及含Luciferase Report System之H9c2 (轉染pCMV-Lc3-Gluc之H9c2)各分成四大組包括:第一組(Control)、第二組(Heptanol)、第三組(H2O2)以及第四組(H2O2+Heptanol)。透過比較第三組(H2O2)與第四組(H2O2+Heptanol)，我們進而判斷使用細胞間隙接合抑制劑，對於細胞表現細胞自噬與細胞凋亡之影響。 實驗結果 第一部分實驗: 我們發現使用100至 400μM 過氧化氫處理超過六小時可誘發細胞自噬表現，而當過氧化氫作用濃度超過200 μM時，可能會同時誘發細胞凋亡，減少Connexin 43表現，以及降低細胞之存活率。使用正庚醇將不影響Connexin 43之表現，同時長時間使用正庚醇將不影響細胞之存活。 第二部分實驗，我們成功的合成pCMV-Lc3-Gluc質體，並將該質體成功轉染到H9c2細胞內以做為Gaussia Luciferase Report System，透過施予氧化壓力400μM 過氧化氫之刺激驗證發現，大約於作用8小時後，LC3表現量已明顯增加，作用至24小時後，LC3表現量增加38% (n=4, P<0.001)。 第三部分實驗，我們發現使用正庚醇抑制細胞間隙接合將使承受過氧化氫刺激之H9c2細胞減少24.2%之LC3 mRNA表現(P>0.05, n=3)；透過Gaussia Luciferase Report Assay，我們發現正庚醇於作用16小時後將使承受過氧化氫刺激之H9c2細胞減少20.6% LC3表現量 (P<0.001, n=4)。 結論與討論 我們研究發現抑制心肌細胞之細胞間隙接合，將使細胞自噬之表現減少。顯示細胞自噬，在可能需要透過細胞間隙接合達到傳遞、延續或增強細胞自噬表現。至於細胞間隙所傳遞的訊息為何? 細胞接受到細胞自噬訊號後究竟如何決定細胞之生或死的命運? 以及是否還有其他細胞間的調控機制，則需要進一步實驗來印證與回答。Study background: The gap junction intercellular communication may involve in signal exchange for both cell death and cell survival. The cell destiny depends on the cell status and environmental context of the cell that receives the signals. Autophagy was once considered as a protective mechanism in cells. Through removing the diseased organelles or protein, autophagy functioned in maintaining cellular homeostasis, nutrient or energy preservation and providing cell survival. Scientists who devoted in autophagy study are trying to transform autophagy concept into therapeutic tool enthusiastically. However, the best studied intracellular regulation of autophagy was known to be complex, and in the contrast the interaction with apoptosis was well-documented. In fact, the intercellular communication of autophagy signal between cells was never investigated and the role of gap junction in autophagy was unknown. Study purpose: We investigate the contribution of gap junction to cardiomyocyte autophagy which was produced by oxidative stress. We hypothesized that autophagy signal between individual cardiomyocyte was conducted through gap junction. Material and methods: We use H9c2 cell line as study model, 1-Heptanol as gap junction uncoupler, and hydrogen peroxide as oxidative stresser for autophagy induction. Autophagy activity was investigated by reverse-transcription polymerase chain reaction (RT-PCR) and Western blot for light chain 3 and Beclin-1 detection. Quantity analysis of autophagy was determined by densitometry by Alpha Innotech and Gaussia luciferase report assay after transfecting pCMV-Lc3-Gluc to H9c2 cell. Our study composed of 3 main parts. The first part of the study was designed to establish the laboratory model. We test the effects of 1-Heptanol and hydrogen peroxide on H9c2 and measure the change of autophagy expression, connexin 43 expressions and survival. The second part of the study included constructing pCMV-Lc3-Gluc plasmid and transfecting the plasmid into H9c2. The whole system was taken as Gaussia luciferase report system. We determined the effect of transfection on autophagy and testify the feasibility of Gaussia luciferase report system for the role of reporting LC3. During the third part of study, we divided wild type H9c2 and Gaussia luciferase report system transfected H9c2 into 4 groups, including the Control group, the Heptanol group, the H2O2 group and the H2O2+Heptanol group. In the H2O2+Heptanol group, 1-Heptanol was administered for gap junction blockade along with hydrogen peroxide for generating oxidative stress-induced autophagy. In the Heptanol group, we use 1-Heptanol only. We also setup negative control (the Control group) using DMEM culture medium and positive control with hydrogen peroxide only (the H2O2 group). Results: We found that 100 to 400μM hydrogen peroxide could induce autophagy in H9c2 when treatment duration is longer than 6 hours. However, higher concentration of hydrogen peroxide (>200μM) might decrease H9c2 survival measured by MTT assay. When hydrogen peroxide treatment was longer than 14 hours, H9c2 Cx43 expression might reduce. Heptanol when used at 0.5mM will not decrease Cx43 expression. Blocking gap junction by 1-heptanol (H2O2+Heptanol group), we found there is about 24.2% reduction of LC3 mRNA expression when compare to H2O2 group which use hydrogen peroxide (P>0.05, n=3). Using Gaussia luciferase report assay, the LC3 expression was attenuated by 20.6 % when compare to H9c2 cell that receive 1-Heptanol pre-treatment before hydrogen peroxide stressing (P<0.001, n=4). Conclusion: Blocking the gap junction inter-cellular communication showed trends in decreasing oxidative stress induced autophagy activity. It’s possible that autophagy regulation required complex machinery which involved autocrine, paracrine and probable gap junction intercellular communication. Further studies are required to figure out the exact signal molecules, the true effect of autophagy flux and the existence other intercellular regulation.3025646 bytesapplication/pdfen-US細胞自噬氧化壓力間隙接合過氧化氫正庚醇autophagyoxidative stressH9c2gap junctionHeptanolhydrogen peroxide connexinhttp://ntur.lib.ntu.edu.tw/bitstream/246246/253545/1/ntu-99-P97421017-1.pdf