Study on neovascularization and establishment of microcirculation for the function of hepatic stem cells transplanted to the liver

1)台盼蓝拒染法检测肝上皮细胞存活率，发现肝上皮细胞在缺氧18小时内仍能保持95%以上的存活率，而成熟肝细胞的存活率随缺氧时间的延长而降低。也就是说，在整个观察时间内，肝上皮细胞在低氧条件下能够保持或提高其活性，而成熟肝细胞的MTT值随着低氧时间的延长而降低。此外，肝上皮细胞的数量在低氧培养9h后才发生变化，之后数量有所增加。3)检测低氧培养过程中肝上皮细胞HSP72和HO-1的表达，并与正常肝实质细胞进行比较。缺氧6h后，LEC对HSP72和HO-1的表达均增加。正常肝细胞在低氧应激条件下不表达HSP72。缺氧条件下存活的正常肝细胞仍表达HO-1。另一方面，死亡细胞可以表达较少的HO-1。因此，应激蛋白的快速产生可能对LEC在低氧条件下的良好存活起到一定的作用。结论肝上皮细胞对低氧具有耐受性，在低氧条件下，肝上皮细胞甚至增殖。HO-1和HSP72等应激蛋白可能是这种耐受的机制之一。

1)Cell viability test by trypan blue exclusion ability showed that liver epithelial cells could keep their viability over 95% up to 18 hours in hypoxia, whereas the viability of mature hepatocyte decreased its viability as the time elapsed under hypoxia.2)Viability test using MTT assay corroborated the result of trypan blue exclusion test. That is, liver epithelial cells could keep or rather elevate their viability in hypoxia throughout the observation time, whereas the MTT values of mature hepatocytes decreased with hypoxic time. In addition, the number of liver epithelial cells did not change until 9 hours under hypoxia, but the number increased thereafter.3)Expressions of HSP72 and HO-1 of liver epithelial cells during hypoxic culture were tested in comparison with normal hepatic parenchymal cells. After 6 hours, LEC increased the amount of both HSP72 and HO-1 in response to hypoxic stress. Normal hepatocytes did not express HSP72 under hypoxic stress. Survived normal hepatocytes in hypoxic condition expressed HO-1 expression as ever. On the other hand, dead cells could express less HO-1. Therefore, rapid production of stress proteins may play some role for the good survival of LEC under hypoxia. On the other hand, HIF-α expression was not increased during hypoxic culture.Conclusively, liver epithelial cell is tolerant against hypoxia and even proliferates under hypoxia. Stress proteins like HO-1 and HSP 72 might paly some role as one of the mechanisms in this tolerance.