Study On The Correlation Between Akt2 And HBV DNA Levels And Its Mechanism

Objective:The main purpose of this study was to explore the relationship between Akt2 gene and HBV load in patients with chronic hepatitis B;.To explore the effect of HBV DNA on the expression of Akt2; To construct the sh RNA interference lentivirus vector for Akt2 and evaluate its effects in humen Hep G2.2.15 cell mediated by lentivirus.Methods: Serum samples of 240 patients with CHB were collected and the HBV DNA loads were tested by fluorescence quantitative PCR. According to the serum HBV DNA loads, patients were divided into three groups: A high-load group with DNA loads higher than 107copies/ml, a medium-load group with DNA loads between 105copies/ml and107copies/ml, and a low-load group with DNA loads lower than 105copies/ml. The serum levels of Akt2 in four groups were quantified by ELISA. The expression differences of Akt2 in the four groups were compared. Hep G2 and Hep G2.2.15 were well cultured,when the two hepatocytes grew to 90%,the RNA of the two hepatocytes were extracted and then were reversely transcribed to c DNA. The expression levels of Akt2 m RNA in two kinds of cells were detected by real-time fluorescence quantitative PCR; RNAi sequences for Akt2 were designed using bioinformatics methods. The construction of Akt2 sh RNA vectors were using lentiviral vector, which were expressed in E.coli and packaged by 293 T cells. Comparsion of target sequences interfere effects were measured using real-time fluorescence quantitative method. Finally, the expression of Akt2 in Akt2-RNAi and control group were detected by ELISA. Results :The level of serum Akt2 was 292 + 15ng/mll in the 80 healthy control, The level of serum Akt2 was 378 +21ng/ml in the 80 low-load group; The level of serum Akt2 was 537±18ng/ml in the medium-load group, and the high-load group was 622±12ng/ml. The levels of serum Akt2 showed statistically significant differences among the groups with different viral loads and healthy control,(P1,2,3<0.001).The expression of Akt2 m RNA in Hep G2.2.15 cells were high than that in Hep G2 cells and the difference was statistically significant(P<0.05); the levels of Akt2 in Hep G2.2.15 cell culture medium were significantly higher than the Hep G2 cells. Three Akt2 targeting sequences were constructed and their corresponding sh RNA lentiviral vectors were screened for efficiency. One Akt2 sh RNA lentiviral vector was screened by transient transfection(interference efficiency reached85%) as the best efficiency and its working condition was established as well.In the supernatant of cell culture, the DNA HBV viral load of Hep G2.2.15 cells in the experimental group was 3.63 * 106 copy /ml.While in the control group, the supernatant DNA HBV viral load was 5.17*106 copy /ml. The difference between the two groups was statistically significant, P<0.05. Conclusions: The levels of serum Akt2 in patients with chronic hepatitis B were closely related with HBV DNA, and HBV was able to increase the expression of Akt2 in hepatocellular carcinoma cells in m RNA and protein levels;Low Akt2 expressioncan can decreased the secretion of HBV DNA in the cells. In clinical level and cell level, Akt2 is closely related to the replication of hepatitis B virus.