The Effect Of NGF On P75NTR Expression And Apoptosis Of HSC In Microenvironment Of Hepatocellular Carcinoma

ObjectiveThis study is designed to detect the expression level of p75NTR (low-affinity nerve growth factor receptor) and TrkA (tyrosine kinase A, high-affinity nerve growth factor receptor) in HSC under microenvironment of hepatocellular carcinoma, and to investigate the effect of NGF on the expression of these two receptors and on the cell survival, providing experimental data for deeper understanding of the interaction between hepatoma cells and interstitial cells.Methods:1. HepG2 culture medium of 12h, 24h, 48h, diluted to 1:8, 1:4, 1:2 respectively, as conditioned medium (CM) were added to HSC, and then MTT method was used to examine HSC proliferation under different conditions, CM with the strongest effect on cell multiplication was selected as CM for further experiment.2. Changes in cell cycle and apoptotic rate of normally cultured HSC, HSC treated with CM, HSC treated with CM in the presence of NGF at different concentration (1, 10, 100μg/L) were examined via FCM.3. IHC was applied to determine the expression level ofα-SMA, p75NTR, TrkA in HSC of three experimental groups as described above. Semiquantitative analysis was performed using a Computer Image Analysis System.4. Observe the ultrastructural changes of HSC of three experimental groups by TEM. Results:1. The result of MTT showed that the group treated with CM of 48h diluted to 1:2 was the most effective one in enhancing cell proliferation.2. The result of FCM assay showed that compared with the group of DMEM, a significant increase of proliferation index in the group of CM was observed, but the change of apoptosis rate was not remarkable. Apoptosis rate of HSC cultured in CM with NGF increased with increasing concentrations of NGF while the proliferation remained almost unaffected.3. The result of IHC showed that HSC of each group expressedα-SMA, P75NTR, TrkA. However the expression ofα-SMA, P75NTR and TrkA in HSC treated by CM of HepG2 increased at different levels when compared with the group of DMEM.. The expression levels ofα-SMA and P75NTR in HSC treated with CM of HepG2 and NGF had a tendency to increase along with increasing concentrations of NGF, however, the expression level of TrkA had a tendency to decrease when the concentration of NGF increased.4. The result of TEM showed that HSC cultured in DMEM remained intact and few apoptosis could be found. In CM group there were more active proliferation and mitoses was easily seen, no apoptosis was observed. But HSC at different stages of apoptosis could be observed in the group of CM with NGF, and the number of apoptosis increased with increasing concentrations of NGF.Conclusions:HSC expressed p75NTR and TrkA when cultured in DMEM in vitro. The expression levels of p75NTR and TrkA both increased in the CM group. The expression levels of p75NTR in HSC treated with CM of HepG2 and NGF increased in a NGF concentration-dependent manner. However, the expression level of TrkA decreased as the concentration of NGF increased, at the same time, apoptosis increased,.This results indicated that NGF could induce HSC apoptosis in microenvironment of hepatocellular carcinoma, which migth be due to increase the expression of p75NTR and inhibit the expression of TrkA by NGF. The results of this study provided us an experimental data for deeper understanding of the interaction between hepatoma cells and interstitial cells.