
Adenosine triphosphate (ATP) is the most abundant and primary carrier of the required energy for various functions in cells. Prolonged ischemia, reperfusion, anaerobic metabolism and lactate accumulation can lead to a dramatic decrease of ATP, cell swelling, cell rupture, and finally cell death by necrotic, necroptotic, apoptotic, and autophagic mechanisms. Due to drastic hydrolysis of ATP in vivo by ectoenzymes and poor cellular penetration, the direct delivery of ATP to the ischemic tissues is difficult.
To increase delivery of ATP to the tissues and protect from enzymatic degradation, encapsulation in liposomes has been proposed and demonstrated in various models of ischemia [1,2]. Studies on myocardial [1,3,4], liver [5-8], retina [9] and wound healing [10-12] ischemia have shown the ability of liposomal encapsulated ATP to prevent cell death and tissue dysfunction following ischemic events.
The encapsulation of ATP in liposomes markedly promotes its effectiveness by preventing the hydrolysis by extracellular enzymes, increasing ATP circulation time and enhancing its intracellular penetration. Depending on the type of the cell line and the target organ various types of liposomes with different surface charges such as anionic, cationic and neutral has been studied by various groups. Moreover, ATP encapsulated liposomes has been demonstrated to improve energy state and function of the cold-stored liver [6,7,13].
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请具体点,把联系方式和价位都说一下好吗?
Clontech的非常好, 感觉. 效果不好可能是RNA问题, 或RTase的问题. 因为目前clontech的RTase是Takara的产品,而非原来clontech的产品,可单购。
步骤:1,RNA抽提(同RT)
2,逆转录,使用合成的oligo
3,p内参
4,使用根据已知序列设计的上游引物和提供下游引物做PCR,注意设计上游引物是退火温度与下游引物相差不大。
5,产物切胶回收,TA克隆测序
这个oligo和下游引物是我根据Takara和BD公司的manual自己设计的,经过我的使用效果很好。具体操作参照takara的3-racemanual。你先做3-race,了解熟悉下,成功后再做5-race。5-race要买试剂盒做,较为复杂!