微陣列基因分析法探討心肌細胞在機械性展延下的基因表現
高血壓所誘發的機械性展延是造成心肌肥大的基本因子，本實驗即藉由微陣列基因分析法同時大量的分析機械性展延所造成心肌細胞的基因表現。將新生鼠的心肌細胞施以 20﹪的機械性展延，抽取其 mRNA製作成 cDNA 探針與現成的 cDNA 晶片進行雜漬反應 （此晶片上包含了 480個如訊息傳遞、控制細胞生長週期、細胞骨架等的已知基因），在眾多有因為機械性展延而造成基因表現差異的基因中，我們選擇了 eNOS 基因（內皮細胞 NO合成?）進行西方墨點法及 NOS活性和 NO 產生量測定的實驗，進一步證實 eNOS 的基因表現量的確是增加的，此一結果與微陣列基因分析所得之結果不謀而合。 Mechanical stretch induced by high blood pressure is an initial factor laeding to cardiac\r hypertrophy. The use of cDNA microarrays has made it possible to simultaneously analyze\r stretch-induced gene expression in cardiomyocytes. Neonatal rat cardiomyocytes were cultured on\r malleable silicone dishes and were stretched by 20%. We compared the transcript profiles of\r cardiomyocytes under mechanical stretch for 60 minutes by hybridization of cell-derived cDNA to\r DNA probes immobilized on microarrays. The microarrays contained probes for 480 known genes\r including signal transduction, cell cycle regulators, cytoskeleton and cell motility, and so on. Eighteen\r genes were indentified that showed significantly differential expression in response to mechanical\r stretch in cardiomyocytes. Of the represented genes expressed, endothelial nitric oxide synthase (eNOS)\r genes was the most interesting one. Northern blot and western blot analysis further quantified the\r expression of eNOS gene. Mechanical stretch also increased constitutive NOS activity and NO\r production. Our results indicate that mechanical stretch induces eNOS gene expression thus increases\r constitutive NOS activity and NO production in cardiomyocytes.
The Neem Dream: "Nature vs. Malaria""
The purpose of investigation was to find and test an inexpensive and effective remedy against malaria. After a great deal of research, I learnt that an effective herbal remedy against malaria may be extracted from Neem (Azadirachta indica)leaves. This remedy is not very well known about all around the world, however, the Neem tree is found in many parts around the world and is therefore a very accessible and inexpensive remedy against malaria. The purpose of my project was also to inform people about the harmful effects of malaria on the human body, as well as to advise people on how to protect themselves.
Dioscorin 對塵?造成氣管上皮細胞傷害的保護性之研究
古籍上記載山藥益肺氣，養肺陰，且最近研究報告發現山藥含有珍貴的dioscorin，其為山藥貯存養分的重要蛋白質，有抑制胰蛋白水解?之活性。而塵?糞便、屍體中的消化蛋白?會破壞呼吸道上皮細胞緊密連接處引起過敏氣喘反應。我對此甚感興趣，進而利用山藥的萃取蛋白dioscorin、塵?粗萃取蛋白及呼吸道上皮癌細胞A549，藉細胞培養、免疫螢光染色、螢光顯微攝影來實驗山藥是否真能保護上皮細胞緊密連接處。再利用膠體電泳、西方墨點法中抗體的高專一性、二抗的高靈敏度來確知我之前的實驗。另外也用ELISA 來實驗dioscorin或塵?引起A549 發炎的情況是如何。由實驗得知，dioscorin 可抑制及預防塵?引起的過敏氣喘反應；而經由ELISA 實驗發現，dioscorin 對於塵?刺激A549分泌發炎物質Eotaxin 並沒有助長或是降低的效果。It was believed that Chinese yam have benefits in treating asthma. Recently, it was found that the valuable dioscorin can be isolated from Chinese yam. The major function of dioscorin is nutrition-storage, however, it also have activities in inhibiting trypsin-like protease. This inhibitory activities trigger my interests. Because the major allergens, mites, and their stool may destroy the tight junctions of airway epithelium cells through their trypsin-protease activities. I therefore carry out an in vitro study to identify whether dioscorin can be used in protecting epithelium from the attack of mites. My results showed that dioscorin can protect the tight junctions of A549 cells from the attack of mite crude extract protein. I believed that dioscorin can be a good candidate in pharmacology application. The genetic and proteomic information are my further focus. On the other hand, we also investigated the possible activity of dioscorin in inhibiting mites-induced inflammation through using A549 as a model and employing ELISA. We found dioscorin neither inhibited nor enhanced the eotaxin being secreted from A549 under the stimulation of mites.
Remote Activated Chip-Based Drug Release System Using Nanoparticles as an Anti-Cancer Therapeutic
The pharmaceutical industry is constantly searching for methods that allow drugs to be delivered as a direct response to a specific stimulus, in which the locus of delivery is in the vicinity of required region. A unique thermo?reversible hydrogel,F127 modified with dimethacrylate (DMA), that can deliver drugs at physiological temperatures was synthesized. Nanoparticles which are specific for targeting human body cancer cell were absorbed by this hydrogel. The toxicity of nanoparticles with different diameters and coating was measured using the MTT assay. It was found that nanoparticles with smaller diameters and folate coating were most toxic to the cancer cells. The release rate of the nanoparticles from the hydrogel was measured as a function of temperature with the hydrogel releasing approximately 3 L nanoparticles per hour. Exposing this drug delivery system to cancer cells would effectively inhibit MCF7 cell proliferation. By grafting this nanoparticle?loaded hydrogel onto a thermoelectric module, the release of the drug would be controlled. Thus, a successful temperature sensitive hydrogel was synthesized that releases cancer?targeting nanoparticles which inhibit cancer cell proliferation, thereby engineering a controlled drug delivery system.
對抗無尺度流行病傳染之新方法
流行病的傳染過程如同一個無尺度網路，但較一般無尺度網路有著更多的變數而明顯差異，因此無法直接應用一般的無尺度網路模式來描述其傳染途徑。我建立一個新模式「無尺度流行病模式」，經由比較模擬結果與疾病管制局的數據，證實此「無尺度流行病模式」是正確與確切可用，且適用於短期暴發性傳染病與長期流行病。SARS案例研究結果，顯示影響SARS疾病傳染因子的大小是：ψ>m>γ。其中降低ψ值可使SARS確定病例至5月31日止降為143人(減少確定病例190人，相當於減少死亡21人)；僅提高防疫使5=γ，亦可使確定病例減至307人(減少確定病例26人，相當於減少死亡3人)。因此強化隔離措施以減少傳染天數最為重要，且可以有效控制每日SARS新增病例，避免發生高侵襲率的現象。HIV/AIDS案例研究結果，獲知採用ψ值來進行月份模擬，則至 2005年12月HIV(+)與AIDS分別為可減少2,715與285人。而進行年度模擬結果，則至 2014年底HIV(+)與AIDS分別為可減少41,936與5,328人。無尺度流行病模式可以協助所需警戒的程度與政策決定的計畫結果。因此無尺度流行病模式在幫助政府評估社會經濟成本與健康憂慮上的有用之工具。當面臨一個全然無知的新病毒的侵襲時，如何減少死亡與傷害人數？是本研究之最終目的。因此，本研究結合了流行病、無尺度網路與灰預測，建立面對病毒侵襲，一個確切可行的對抗無尺度流行病傳染新方法，並詳細說明運作流程。\r \r \r The course of epidemic infections resembles a scale-free network. However, they are different due to more variables in the epidemic infection. Therefore, the model of scale-free networks is not enough to satisfy the reality epidemic infections. In this study, I propose a new the Scale-Free Epidemic Model. Comparison of the simulation results with Taiwan CDC report data for SARS and HIV/AIDS cases show that the Scale-Free Epidemic Model is accurate and useful. This model can be used in the short-term outbreak of infectious diseases and for the longer-term epidemics. In the SARS case study, the results show that the sequence of effect of the epidemic factors was: ψ>m>γ. The SARS confirmed cases would decrease to 143 cases (reduced 190 confirmed cases or 3 death cases) calculated to May 31, 2003, if the average infection time was reduced to two days (an optimum value of ψ). Therefore, vigorous action in isolation quarantine and treatment for SARS cases is most effective policy; the number of new cases and the attack rate would also decrease. In the HIV/AIDS case study, the simulation results of the Scale-Free Model indicates that the reduced numbers of HIV(+) and AIDS in the monthly simulation calculated to December 2005 are 2,310 and 361 and the annual simulation by December 2014 are 27,161 and 3,710. The Scale-Free Epidemic model can help determine the level of caution needed and the projected results of policy decisions. Therefore it is a useful tool in assisting the government to balance socio-economic and health concerns. The fight against a new epidemic and how to reduce the number of deaths is the main purpose of this study. So, a new method to fight against epidemics is proposed. Detailed procedures of this method are explained.
Mechanism of the subcellular localization of the actin binding protein adducin
Adducin蛋白在細胞骨架的調節上扮演著重要的角色。然而，近來有許多研究指出，骨架蛋白也會出現在細胞核並參與轉錄調控，因此本研究的目的即在探討adducin蛋白是否會進入細胞核中，並參與轉錄調控或具有其他功能。在本研究中，我們將綠色螢光蛋白(GFP)標示的adducin質體DNA，利用轉染技術送入老鼠纖維母細胞株NIH3T3中表現。NIH3T3細胞原本並無adducin蛋白的表現，在共軛焦顯微鏡下觀察，野生型的GFP-adducin蛋白會表現於細胞核與細胞質中。由於adducin蛋白尾端序列攜有可能往核內運輸的訊號，於是將位在此一訊號中的離胺酸718及離胺酸719進行突變，結果發現此一突變株只能在細胞質中表現。此外，蛋白磷酸脢C(protein kinase C)已知能磷酸化adducin蛋白在絲胺酸716及絲胺酸726的位置，於是假設其磷酸化是否與其在細胞內的分布有關。將adducin的絲胺酸726置換成丙胺酸，並不影響其在細胞內的分布。然而將絲胺酸716置換成丙胺酸後，則完全只在細胞核中表現。由於adducin可分布於細胞核，因此我們懷疑adducin蛋白可能與細胞分裂有關，於是本研究利用流式細胞儀分析adducin轉染後NIH3T3細胞的細胞週期。流式細胞儀的分析結果顯示，攜有GFP-adducin或其突變株的細胞與未經轉染的NIH3T3細胞的細胞週期並沒有顯著差異。其次，為了避免因轉染的效率不高而造成統計上的誤差，我們利用顯微鏡追蹤技術觀察攜有GFP-adducin的細胞株，結果顯示攜有adducin突變株的NIH3T3細胞株仍能正常分裂。再者，因為adducin能與細胞骨架中的肌動蛋白結合，所以adducin不同的分布位置可能影響細胞附著與細胞展延的效率。細胞展延試驗的結果顯示，adducin及其突變株對細胞附著與細胞展延的效率並無明顯的影響。本研究的結果證明，adducin的確帶有往核內運輸的訊號，其在細胞質中的分布可能也同時受到絲胺酸716磷酸化的影響。然而adducin的功用似乎與纖維母細胞的分裂與展延無明顯的關聯性。Adducin, an actin binding protein, is known to play an important role in the regulation of the membrane cortical cytoskeleton. More and more evidence indicates that proteins involved in the cytoskeletal regulation could also reside in the nucleus and participate in gene regulation. Thus, the goal of this study is to examine whether adducin is expressed in the nucleus and involved in certain nuclear events. In this study, adducin and its various mutants were fused with green fluorescent protein (GFP) and transfected into mouse NIH3T3 fibroblasts which do not have endogenous adducin for monitoring their subcellular distribution under a laser scanning confocal microscope. The wild-type GFP-adducin was found to be present both in the nucleus and in the cytoplasm. The COOH-tail of adducin contains a motif analogous to the nuclear localization signal (NLS). Mutation of two lysine residues (lysine 718 and lysine 719) located within this motif abolished the nuclear localization of adducin. Moreover, adducin is known to be phosphorylated by protein kinase C at serine 716 and 726. Substitution of adducin serine 726 with alanine had no effect on its subcellular localization. In contrast, substitution of adducin serine 716 with alanine led to only nuclear expression. Nuclear localization of adducin renders it possible that adducin may be involved in the regulation of cell division cycle. For cell cycle analysis, flow cytometry was applied. The results of flow cytometry indicated that expression of adducin and its mutants in NIH3T3 fibroblasts did not affect their cell cycle progression. To further examine the effect of adducin on cell division, NIH3T3 cells transiently transfected by adducin were monitored by time lapse video microscopy. The video clearly showed that the cells with GFP-adducin underwent cell division to generate two daughter cells. Since adducin is well known to bind to actin and thereby regulate microfilaments, we wondered that expression of adducin in NIH3T3 cells might affect their adhesion and spreading onto extracellular matrix proteins. The results of cell spreading assays showed that adducin appeared not to affect cell spreading. In conclusion, our results demonstrate that the subcellular distribution of adducin is likely regulated by two signals, one is the nuclear localization signal and the other is the phosphorylation status of the serine 716. However, enforced expression of exogenous adducin in fibroblasts such as NIH3T3 cells does not alter their cell cycle or cell spreading on fibronectin.
探討茶液成分受光及貯存時間之影響
Tea is the most widely accepted and consumed beverage worldwide due to its characteristic aroma and taste. Recent studies have provided the strong scientific basis for understanding the health promoting effects and cancer preventive actions of tea. The components of tea especial the catechins are varied with the conditions of making tea. To understand and determine the chemical composition of tea is very important. Some investigations of the parameters on the storage and making of tea were carried on in this study. The kinds of tea studied were including black tea, oolong tea, green tea, and instant tea bags. A high performance liquid chromatograph combined with UV detector and mass spectrometer used to analyze the components of tea. The results showed that the composition of tea solution is dependant of the exposure of light. One of the components of tea, (-)-epicatechin methylgallate (ECMG) was oxidized to quino form. The concentrations of (-)-catechin gallate (ECG) decreased and one of new compound (M.W. 442) produced with increasing the storage time. From the results show tea made with cold water is better than that made with hot water. The ingredients in green tea were changed faster than those in fermentative black tea and oolong tea. The components of tea can be kept unchanged for a long time at low temperature. In refrigerator, the time can be extended to overnight. 茶由於其具有特殊的芳香氣味及口味，廣泛地被世人用為飲料，近年來的研究證據顯示茶具有促進身體健康和防癌功效，但茶中成分之變化，尤其兒茶素隨茶的種類、茶沖泡保存方式及置放時間而有所不同，因此對於茶中成分及沖泡方式的認知是一門重要的課題。 本實驗主要是探討茶沖泡時間及貯存條件，對於茶液所含兒茶素變化的影響，探討的茶包含紅茶、烏龍茶、綠茶及其茶包。並使用質譜儀及高效能液相層析質譜儀配備紫外光偵測器分析茶液中之成分，實驗結果顯示成分變化速率以未發酵的綠茶較發酵的紅茶和烏龍茶快；低溫儲存時，亦可延緩茶液成分的變化，例如加蓋並存放於冰箱，則茶置放至隔夜其成分均未改變；同時結果顯示成分產生變化者，主要為兒茶素氧化，如將多酚類氫氧基經氧化變成?（quino） ，其次為斷裂再產生聚合其中綠茶以不照光變化較大。本研究並經實驗發現如果以冷開水沖泡綠茶20~30分鐘，咖啡因的溶出量雖略多於沖泡2分鐘的熱茶，但各種兒茶素的溶出量卻遠高之，尤其置放至隔夜茶液成分仍無氧化現象。