Isolation、Identification And The Protein Synthesis Of Adult Primary Hepatocyte, And The Expression Of P450

Objective: To explore the hepatocyte isolation and the identification technology of adult benign lesions of liver resection operation of leaves, To study the changes of cell viability in the culture process of adult primary hepatocyte,and the Changes in protein synthesis and cytochrome P450 expressionMethod: Isolate hepatocytes from the livers of patients with benign lesion with surgical excision,using the trypan blue staining method to detect fresh liver cell survival rate;Periodic acid Schiff(PAS) attaining was performed to evaluate the status of hepatic glycogen storage,combining with the immunocytochemical staining targeting cytokeratin 18(CK-18) to identify the purity of hepatocytes;The hepatocyte activity in 7 days continuous cuture were detected by CCK-8; Fully automatic biochemical analyzer test albumin 、 urea synthesis and other related biochemical changes of adult primary hepatocytes in the process of cultivation. detection of adult primary liver cells in culture and biochemical change process; using Western blotting(Western Blot) to detect the hepatic cytochrome P450(biotransformation reaction first phase reaction) expression of the hepatocyte in vitro.Result:1. The quantify of fresh adult primary hepatocytes count up to more than109,viability detection of fresh adult primary hepatocytes with trypan blue staining was 90% or more.2. Glycogen preserved in hepatocytes was dyed purple using periodic-acid-Schiff staining.Through the PAS and anti-CK-18 immunocytochemical staining of hepatocytes,the purity of hepatocytes was more than 95%.3. The experimental results of CCK-8 showed that the cell vitality of hepatocyte were in the best state in the third days cultivation.4. In the 1 week process of cultivation of, liver cells have good synthesis function of glycogen, albumin and urea.5. in the process of cultivation,ALT, AST and LDH leakage rate rise after a downward trend, in 3 days,to reach the lowest, albumin secretion reached the highest in 3 days.6. In the 1-7 days cultivation,the hepatocyte were visible in the 80 KD espression of cytochrome P450,and it espressed the best in the third day.Conclusion:1. The improved collagenase perfusion chromatography for liver cells isolation is simple short, get large number of liver cells,and the cells have high purity and have perfect cell function.2. This research shows that in the process of adult primary liver cell cultivation, the cell vitality is the best in the third days,And the biochemical properties of liver cell is also the best at this time.3. In the process of 1 week in vitro cultivation, Adult primary hepatocytes have very good expression of cytochrome P450.