【作者】 郭淼；

【导师】 葛霁光；

【作者基本信息】 浙江大学 ， 生物物理， 2003， 硕士

【摘要】 生物信息动态学是二十一世纪生物学的主要研究阵地之一。目前国内外对离体器官的体外存活问题的研究主要集中在钙超载对离体器官缺血再灌注损伤的作用问题上，而对诱发离体器官早期损伤的主要因素细胞水肿没有足够重视。 本文承接本实验室一贯的研究思路和成果，采用自行研制的脉动式低温灌流系统对离体肾脏进行连续灌流，并运用电子显微镜、激光共聚焦显微镜等仪器结合显微摄影、酶活力测定、钙离子荧光染色等手段从宏观和微观两个方面对体外长期存活的肾脏的超微结构与功能，尤其是钙离子对缺血再灌注肾脏离体长期存活中的损伤作用开展了较为深入的研究。 本文研究发现，离体肾在人工生命维持系统中，经过120小时的连续灌流后，经ATPase活性测定，仍然保持良好活性，但已开始出现组织细胞损伤，经电镜反复观察损伤乃是主要来源于组织水肿的机械损伤所致，而钙超载引发的缺血再灌注损伤在该过程中并非主导因素。经过调整灌流液配方，可以延缓细胞损伤出现时间和缓解损伤严重程度。相对肾小球等其他肾组织而言，肾小管是肾脏中较易产生损伤的结构。低温连续灌流可以对离体肾脏组织的肾小管结构产生较好的保护作用，故可以认为对离体肾脏的长时间灌注和存活而言具有单纯的低温保藏不可比拟的优势。 同时，电镜研究结果显示钙超载在离体，肾脏的缺血再灌注损伤中的作用确实不可忽视。进一步采用激光共聚焦显微镜和X-射线微区分析的结果显示，低温适应过程中出现的钙离浓度升高主要来源于细胞内源性的钙库释放，而且这些钙库主要位于内质网等处。应用钙离子通道阻滞剂结合激光共聚焦显微镜共同研究的结果显示：内质网上可以被普鲁卡因选择性阻断的内质网膜Ryanodine受体系统所介导的钙离子释放可能为该过程中钙离子的主要来源。进一步的研究中本文发现，在低温适应10分钟至15分钟这一段时间内，钙离子释放的速度逐渐变缓。本文认为这一变化与钙离子动态分布的调控阻力增加，钙释放减弱以及荧光染色剂的淬灭效应等原因均有一定的关系。同时应该着重指出，但这时钙离子浓度及其微量、缓慢变化无法用激光共聚焦显微镜检测出来。这表明细胞损伤是多因素的，若控制钙离子超载，细胞损伤可以得到缓解和改善。 如何延长离体肾脏的低温存活时间和减少离体肾脏的缺血再灌注损伤是目前许多基础生物医学研究者关注的热点课题。本文的研究在这个方面取得了一些有益的进展，希望可以为今后的离体肾脏的调控机理研究提供了一些参考。更多还原

【Abstract】 Dynamic bioinformatics is the main field for the researchers major in Biology. Nowadays quite a many groups pay more attention to the injury caused by the calcium overload during the perfusion of the off-body organs, overlooking the tremendous effect Cell edema may have on them.Based on the theory and the results achieved by our group before, the experimenter perfused the off-body kidney with the pulse-modulated hypothermic machinary continuous perfusion developed by the group. Then the experimenter examined the ultrastructure & function of the off-body kidney, especially the injury induced by the calcium overload to the ischemic reperfused kidney, by electron microscope and Laser Scanning Confocal Microscope (LSCM) integrated with the testing methods like microphotographics, Enzyme activeties examination and calcium fluorescence techniques.The paper would like emphatically indicated that after off-body kidney existing in the artificial life maintain system by 120- hour continous perfusion, it still had good activities measured with ATP. But the cells of tissue had come to appear wounded as a result of electrion microscopic examination. That was cell-to-cell junction emerged injury changes caused by cell edema while calcium overload was not the dominant element. However, both cell-wounded time and degree could be delayed and mitigated by changing the perfusate prescription to protect. In contrary to the glomerular and other renal tissues, the proximal tubular are more easily injuried during the perfusion. The pulse modulated hypothermic machinary continuous perfusion could protect the proximal tubular more effectively than the simple cold prefusion. So the conclusion could be drew that the former has superiority in surviving of the off-body kidney.Photos taken by electron microscope displayed that calcium overload may have close relation with the ischemia-reperfusion injury. More results got through LSCM and X-ray Microanalysis of Microsections all showed that the rise of the calcium concentration during the cold adaption period came from calcium pools inside the cell mainly. These calcium pools mostly distributed in endoplasmic reticulum. Then the paper used calcium channel inhibitors and LSCM to study the calcium source. The following results displayed that the calcium pools in the endoplasmic reticulum induced by the Ryanodine receptor system may be the source. Fartherly, the calcium intensity showed a gradual fade out between the 10th and 15th minute during thesurviving. The factors such as the rise of the modulation resistance, the deduce of the calcium release and the fluorescence quenching all could owe to this result. Ca2+ concentration and its variation could not be detected only by LSCM at this period. This fact indicated us that cell wounded was caused by multiple factors and it could be mitigated if calcium overload was controlled.How to prolong the surviving time and its ischemia-reperfusion injury to the off-body kidney are the hotspot for many researchers studying biomedicine. This paper got some useful developments in this area, supporting a few referenced datas for the study of modulatory mechanisms of the off-body kidney.更多还原