2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658820摘要：目的此三年計晝的主要目的是希望能夠發展出另一種的腦保存液，如同心肌保存液來改善 低溫血循停止時之腦傷害，更進一步延長手術時間。現況HTK保存液已經大規模使 用於器官保存，因此也許有可能使用在腦保護。此計晝便是設計三方面的實驗未來驗 證：一為細胞，二為小動物-老鼠，三為大動物-豬。第一年細胞首先來評估HTK及EPO對細胞缺氧或再灌流傷害之耐受度與保護能力。使用的細胞是老 鼠之大腦細胞及NG108-15細胞。以UW、HTK、及HTK + EPO以％,沁，1/8之比 例取代原有之培養液(21%氧氣含量）。缺氧則是以2%之含氧量之環境。細胞培養從0, 1, 2, 4, 8, 24, 48, 72小時來觀察其中的變化。以TUNEL、LDH及各種immunoblots研究來 看各種溶液(UW, HTK, HTK+EPO)及不同比例下之表現是否有所不同。同時，同一測試 標準也在不同溫度下測試(4, 25, 37 C)，來了解溫度之角色。第二年老鼠模式缺血這是以大鼠中腦動脈阻塞模式來測試HTK與HK+EPO對腦栓塞之保護效果。在左總頸動脈做10分鐘之血流阻斷，而後以不同保存溶液加以灌流（saline, HTK, HTK+EPO)，再經過90分之缺血後各組的變化及差別。在第一，三、七天之時間點來做 分析。（每一組用10隻）分析重點在(1)栓塞面積大小，以TTC stain (2)EEG記錄（3)neurological score (4)組 織學切片分析（5) RT-PCR for quantification of TRPV or EPOR mRNA expressions (6)lmmunostaining for detection of protein expression.第三年豬模式低溫血循停止用5-10公斤的新生豬行deep hypothermic circulatory arrest實驗。豬隻以心肺機降溫到20 °C，進行90分鐘之血流停止。其中分成三組：（1)控制組（2)HTK:以HTK在血流停止前 行腦部灌流（3) HTK + EP0:以HTK + EP0在血流停止前行腦部灌流。觀察時 間點為6小時、48小時及7天(n= 5 for each group, total 15 )。其檢測之重點為(1)栓塞面 積大小，以TTC stain (2)EEG記錄（3)neurological score (4)組織學切片分析（5) RT-PCR for quantification of TRPV or EPOR mRNA expressions (6)Immunostaining for detection of protein expression.<br> Abstract: Adjuvant Brain Protection Solution - “Solutions” as an adjuvant in deep hypothermic circulatory arrest effectPurposeThe main purpose of the 3-yea program is designed to develop the possibility of adjuvant brain protection solution in deep hypothermic circulatory arrest. We tried to identify the feasibility by applying HTK and EPO for the neuron protection. Three parts of study are designed. One is for cell level, on eis for rat model, and the final is for the pig model.In the FIRST year:The purpose of first-year study is designed for the evaluation of the effect of the HTK and the combined effect of HTK and EPO to the cultured neurons in response to severe hypoxia or 丨R.Primary culture of rat cortical neuronsCulture of NG108-15 cellsDirect effect of HTK or HTK plus EPO on cell viability will be accessed by replacement of culture medium with UW, HTK, or HTK+EPO at the portion of 1/2, 1/4, or 1/8 as showed in Figure 1. The cultured neurons will be divided into different groups. For the control, the culture will be placed in a 21% O2 and 5% CO2 environment in an incubator. For SH treatment, the cultures will be exposed to 2% oxygen for 0, 1, 2, 4, 8, 24, 48, and 72 h, unless stated otherwise. Five |aM of EPO will be added in culture medium to test its effect as an adjuvant based on our previous observation. Ischemia-reperfusion protocol was designed and several different temperature setting were planned (4, 25 and 37 C).The apoptosis parameters (TUNEL) were evaluated. Multiple immnoblots wee detected as well to evaluate the effect of HTK, EPO and HTK and EPO on the neuron cell.HTK保存液器官保存液神經細胞NG108-15細胞Histidine-tryptophan-ketoglutarate solutionOrgan preservation solutionNeuronNG108-15 cells