醫學與健康科學

Most neurological diseases are characterized by the presence of protein aggregates,\r suggesting that aberrations in protein homeostasis are associated with neuronal demise. In eukaryotic cells, protein homeostasis is maintained by the chaperone, ubiquitin proteasome (UPS) and autophagy systems. As age is a risk factor for several types of neurodegenerative diseases, the function of these various protein homeostatic systems could become compromised with age. To understand the events that occur during normal aging, we examined the expression of key markers associated with the aforementioned systems in mice aged 1, 3, and >18 months. We found that proteasome activity and the amount of proteasome-related structures remained unaffected with age. Interestingly though, an agerelated increase of a novel Hsp70 chaperone protein species (herein designated Hsp70*) was observed. The expression of Hsp70* is also increased markedly in cells treated with pharmacological agents that promote proteasome inhibition, suggesting a functional interaction between the chaperone system and the UPS. Taken together, our results suggest that there is some form of crosstalk between the chaperone system and the UPS involving the observed HSP70 species.

During the evolution of humankind, development of frontal cortex has played a critical role, where higher brain function like emotions, self-consciousness, decisions…etc, were all related to frontal cortex. On the other hand, thalamus is usually associated with relaying the sensory signals from peripheral receptors. In order to understand the functional role of frontal cortex, the signal processing mechanisms in the thalamo-frontal cortical pathway became an important research issue. The aim of this experiment was to find a method to dissect a brain slice that contains a connecting route in vitro between thalamus and frontal cortex with functional activity. Through nerve fibers tracking technique using fluorescent-dye (DiI), it was understood that the 3D-space connection between thalamus and frontal surface was an upward curve with a turn of about 110 degrees and bending inwards from the two sides. If a conventional horizontal section was performed, the route would be cut-off and its integrity lost regardless of the direction. To solve this problem, a novel section method was developed to retain the route. Based on the route direction shown by the fluorescent-dye, a piece of brain block was cut and flattened of about 110 degrees. Other sections were performed as control for studying the effectiveness of the sectioning on the plane of the route. Finally, electrophysiological methods were used to verify the connection route was complete and functional. Thalamus-evoked extracellular field potentials in the frontal cortex were observed by changing stimulation strength, adjusting slice temperature and prepared oxygen supply and administration of drugs like CNQX and picrotoxin in the 110 degree flattened slice but not the others. It was found that the reaction was essentially a neuronal response, indicating the pathway between thalamus and frontal surface was retained substantially. With this novel brain slice technique, we can assess the functional connection between thalamus and frontal cortex and investigate the cellular mechanisms of the signal processing in this connection pathway. It is anticipated that present technique provides an important step to further elucidate the functional role of the frontal cortex. 在人類的演化史上，前額葉的發展扮演了極為重要的角色，凡舉情緒、自我意識、決策等，皆與前額葉有關。而丘腦通常與視覺、聽覺及本體感受如痛覺、觸覺、溫度覺的訊息傳遞有關。要了解前額葉的功能，丘腦到前額葉的徑路及訊息處理機制，便成為一個很重要的研究課題。本實驗的目的是尋找一個方法能在離體的腦切片上維持具有丘腦到前額葉連結的徑路並且有功能的活性。經由螢光染料(DiI)神經束追蹤技術，了解從丘腦至前額葉路徑的三度空間連結為一先向下再向上約110度的角度轉折，並同時先由內向外再轉向內的曲線，若用一般水平之切片方法，無論何種方向，其路徑必定會被切斷，不能保持其完整性。為了解決此問題，發展出一種可以保存其路徑的腦切片方法。依照螢光染料所顯示出的的路徑走向，在腦塊的皮質上切一刻痕，將腦塊以110度的角度展平，使其路徑處在一個平面上再切片。最後利用電生理的方法來證明所切出的連結路徑是完整且具有活性。改變在丘腦的刺激強度、調整腦切片(腦脊髓液)溫度、氧氣的供給以及施予藥物CNQX、picrotoxin，觀察其前額葉之電位變化，發現其反應確實為神經反應，表示從丘腦至前額葉的路徑已在這種特殊的腦切片中被完整保存。藉由這個方法，將有助於研究丘腦至前額葉功能性連結，神經網路結構，及其訊息處理機制，並期待以這樣一個全新的方法將來有助於瞭解前額葉的功能。

Deficiencies in clearance of apoptotic cells predispose to the development of autoimmune disease. This is evident in mice lacking the receptor tyrosine kinases Tyro3, Axl, and Mer that mediate uptake of apoptotic cells. Deficient mice exhibit an increased abundance of apoptotic cells in tissues and manifest diverse autoimmune conditions. To test these mice for the presence of autoantibodies to apoptotic cells, we generated spontaneous splenic B cell hybridomas and used microscopy to screen for clones reactive with apoptotic Jurkat cells. From hybridomas secreting IgG antibodies reactive with apoptotic cells, we selected one that recreated the major serum specificity for apoptotic cells. The antibody, LHC7.15, bound to an antigen that is differentially distributed between the nucleus and the cytoplasm in live and apoptotic cells. In late apoptotic cells, the antigen coalesces into aggregates that form blebs at the cell surface. Immunopurification of the antigen, followed by mass spectrometry, identifed a protein of 69kD whose partial sequence matched hnRNP P2. This multi-functional protein binds DNA, RNA, and several known RNP autoantigens. Our observations suggest that an RNP complex, formed and translocated to the cell surface in apoptosis, participates in the induction of linked sets of anti-RNP autoantibodies. Our results also implicate hnRNP P2 as a potential novel antigen involved in initiating and sustaining systemic autoimmune diseases.

本研究首度發現人類肺癌A549細胞會促進其所浸潤的樹突細胞分泌Resistin，而更深入地探究獲悉Resistin會透過活化WHSC1/Twist途徑促進肺癌A549細胞惡化，此惡化過程包括誘導癌細胞上皮間質轉化(epithelial-to-mesenchymal transition; EMT)及提升癌細胞的移行(migration)和入侵(invasion)能力。為確認Resistin在臨床的重要性，透過肺癌病患檢體分析發現，相較於健康捐贈者，肺癌病人的血清可測的較高濃度的Resistin；更甚之，比較非腫瘤組織部位之CD11c+樹突細胞，浸潤於腫瘤組織部位之CD11c+樹突細胞會呈現高量的Resistin。接續探討Resistin對肺癌細胞的影響機制，實驗結果發現Resistin會增加A549細胞表現histone methyltransferase WHSC1的表現，而WHSC1在Twist啟動子的H3組蛋白lysine 36位置進行dimethylation修飾，並降低H3組蛋白lysine 27位置的trimethylation進而促進Twist的表現，促使A549細胞進行EMT和增加癌細胞移行和入侵。因此，Resistin可作為肺癌診斷分子及藥物發展的重要標靶。

我們的研究工作有兩項重要成果：首先，根據物理學的彈性體振動模型發現：主動脈硬化的定量分析訊息可由測量主動脈相對於心臟運動的延遲時間明確得知，而且可用目前臨床使用中的心臟超音波儀器直接進行此一測量。在對23 個樣品、每個樣品分析大約50至100 次心跳的初步研究中發現，此一延遲時間是確實存在的，而且延遲時間超過大約0.17秒時，樣品就可能具有動脈硬化的現象。其次，在比照過10 組樣品的數據後發現，上述之延遲時間確定可由體外的胸前超音波掃描（TTE）準確測得，而不需要採用侵入式的經食道超音波掃描（TEE），以免除受測者的不適，及避免副作用的風險，使得此一新方法更具有方便、普遍的優點。而且此一方法也可能由數據的分佈發覺心血管之其他病徵。未來仍需應用此一方法對較多樣品進行研究以進一步確定此診斷方法之可靠性。 Two important results were achieved in this research. Firstly, according to the physical model of elastic oscillation, we found that the qualitative analysis of aorta stiffness could be obtained by a measurement of the delay time of the aorta motion relative to the cardiac motion. This measurement could be carried out with the conventional echocardiography. A preliminary analysis based on 23 samples, with 50 to 100 heartbeats per sample, confirmed the existence of this delay time. Also, a delay time of greater than 0.17 sec might imply the symptom of aorta stiffness. Secondary, after a comparison of 10 samples, the aforementioned delay time could be measured with TaransThoracic Echo (TTE) instead of TransEsophadeal Echo (TEE), in which the later is invasive while the former is not. Therefore, the discomfort of the patient and the risk of invasive operation can be avoided, making this new method more convenient and more common to accomplish. This method could also clearly discern some abnormal cardiac performance. A large-scale study with this method should be conducted in the future.

本研究探討斑馬魚胚胎發育的不同時期，妊娠中藥對胚胎及仔魚的影響。在受精後第0小時（初期）、12小時（中期）或24小時（晚期）加入不高於人類用量的保產無憂方、當歸芍藥散與芎歸膠艾湯。每12小時觀察胚胎發育的情形；待仔魚孵化，比較其差異。結果顯示：(1)初期給予當歸芍藥散、芎歸膠艾湯不會造成胚胎死亡，但保產無憂方隨濃度增加而死亡率增加。(2)晚期給予保產無憂方，胚胎發育略有延遲，但全數存活，且劑量越高效果與控制組越相近。(3)三味藥均使發育延遲，尤以初期加入高劑量最為明顯。(4)中期或晚期給予安胎方皆造成仔魚體型縮短。(5)低劑量當歸芍藥散和芎歸膠艾湯使孵化後第四天仔魚運動總距離增加。我們建議，當歸芍藥散和芎歸膠艾湯在初期可用，但保產無憂方為胚胎發育後期之安胎順產藥劑，不適於早期之使用。

桂枝?樟科植物肉桂之嫩枝，依古籍記載具解熱、鎮痙、抗菌及對消化液分泌有抗拮作用，藥理作用廣泛。近年來，國內外學者對其進行了大量的研究，且達到了較高的水平，查閱國內大量文獻，發現研究者對桂枝的成分、藥理等方面進行了深入的研究，其中較大比例的研究是對桂枝中有效成分桂皮醛(Cinnamic aldehyde)的討論，而我們希望能用新技術、新方法探討其藥理作用機制。

The apricot kernel is believed to have a great medicinal value in many cultures. However, literature and research indicates that this belief still remains extremely controversial and conclusions regarding the medicinal value are ambiguous due to the presence of cyanide in the kernel. The focus of this research was to evaluate two objectives through the use of several integrated technologies and modified methods: (a) To successfully remove the cyanide from the apricot kernel using an adapted method; (b) To determine the effects of the cyanide free apricot kernel extracts on Helicobacter pylori and Streptococcus pyogenes bacteria. Procedures The apricot kernels were removed from the pits and then ground using a food processor. The kernels were then tested for cyanide using a cyanide test kit and Cyantesmo test tape. These tests indicated that cyanide was present. A novel approach was devised to remove the cyanide and when retested, the kernels tested negative for cyanide. This result was confirmed with Infrared Spectroscopy. The cyanide free kernels were then extracted using a Soxhlet Extractor with methanol for 24 hours. In addition to the methanol extraction, three other techniques were used to obtain kernel extracts: (a) Celite filtration, (b) Infusion Method A, (c) Infusion Method B. The Kirby Bauer method was modified for the microbiology aspect of this project. The Helicobacter pylori and Streptococcus pyogenes bacteria were plated using a 0.5 McFarland Standard. Paper filter discs containing 20µL of each extract were placed onto the inoculated plates in replicates of nine. After 48 hours of incubation, the zones of inhibition were read for each plate. Data The results were extremely encouraging and therefore to ensure the accuracy and preciseness of the data collected, four statistical analyses were completed. These include Confidence Intervals (CI), Standard Deviation (STDEV), T-Tests, and Chi Tests. The methanol extract was significantly different from the control in all trials. The Chi test also yielded a Chi Square value of 223, which was significantly greater than the critical value of 15.507, indicating that the results observed were not due to coincidence. Conclusion Literature evidence has indicated that the apricot kernel has been and is still used for medicinal purposes. Studies have shown that the presence of cyanide and the risks associated with this compound outweigh any benefits gained from the kernel. It has also been suggested that previous bacterial testing resulting in positive inhibition may have been due to the presence of cyanide. However, in this study, I was able to remove the cyanide through an innovative method to prove that the biological activity observed was highly unlikely to be due to cyanide. This indicates that there are other compound(s) in the apricot kernel that have specific antibacterial properties. The potential to improve the quality of life through the application of the apricot kernel appears to be supported, and further studies justified at this time.

許多難纏的疾病，如神經退化性疾病與糖尿病，在1998年建立人類的胚胎幹細胞後，開始露出一線曙光。但就算科學家已經從胚胎幹細胞，成功引導出特殊種類與功能的細胞，預備做細胞治療時，需透過細胞核移植技術，使排斥的情形大大減少。所以當今年3月，韓國透過細胞核移植技術成功作出人類的胚胎幹細胞株時，轟動全世界。但是，他們從242顆卵，只做出一個胚胎幹細胞株，浪費這麼多卵子就牽涉到醫療倫理的層面了。既然，每一個人都要訂製自己的幹細胞株，那麼卵子的來源就很重要了。理論上，胚胎幹細胞可以分化成所有細胞與組織，當然也應該含生殖細胞在內。去年5月底，賓州大學就利用小鼠胚胎幹細胞，體外培養出型態上非常像卵子的細胞。所以，今年暑假利用兩個月時間，有個機會參與了台大醫學院婦產科的胚胎幹細胞研究團隊，稍稍瞭解了胚胎幹細胞製造成生殖細胞的過程。我們採取一種方法，先從胚胎幹細胞株，用類胚胎體(EB)的立體環境，加上 SSEA-1的篩選，與維他命A的刺激，引導至初始生殖細胞，第二步利用細胞重組的方法，利用小鼠胚胎的性腺組織，來支持這些選擇過的細胞，希望可以培養出小鼠的卵子或精子。我們從EB得到SSEA-1細胞約為全體細胞的10%，然後經過維他命A的刺激，得到SSEA-1細胞約為全體細胞的5%，這時的細胞理論上是初始生殖細胞 (PGC)，但是之後的體外單獨培養或與胚胎性腺重組培養，發現成果並不是很理想。到最後，也許受限於時間還有能力，沒有得到明確的小鼠生殖細胞，但是過程中，我學習到生殖細胞的生理，胚胎幹細胞的特性，基本實驗室的細胞培養與組織染色，這些都令我印象深刻。我們認為，如果有一個適當的基因轉植的胚胎幹細胞株，例如GFP放進生殖細胞相關的基因裡頭，讓達到終點，或邁向目標的細胞群，可以自我顯現，實驗會更容易達成目標。Scientists discovered ways to obtain or derive stem cells from early mouse embryos more than 20 years ago. Many years of detailed study of the biology of mouse stem cells led to the discovery of human embryonic stem cells in 1998. Through years of experimentation scientists have established some basic protocols or "recipes" for the directed differentiation of embryonic stem cells into some specific cell types. One major problem that must be solved before human stem cell therapy becomes a reality is the threat of rejection of the transplanted cells by the host's immune system. One way to avoid the problem of rejection is to use stem cells that are genetically identical to the host. This could be achieved by the same techniques of somatic cell nuclear transplantation (SCNT) that produced Dolly. Low efficiency less than 0.5% (1/242) in Korea this March brought some controversies in ethical issues. Where and how to procure the large amount of oocytes that will be required? One source of eggs could come from the generation of oocytes from embryonic stem cells (ESCs), as described by Hubner et al. (2003) in the mouse. By the selection of primordial germ cells (PGCs) and re-aggregation with their stromal cells, we hope to derive mature germ cells such as oocytes and sperm from ESCs in this study. In this study, we have demonstrated some PGCs after sequential selection by SSEA-1 antigen and optimal culture conditions. We also proved some FE-J1 positive cells but no further fertilization was obtained after all. Further investigation about this mechanism may be needed to derive these germ cells from ESCs.