Study On The Action And Blot Effect Of GDNF On Neural Invasion Of Pancreatic Carcinoma

Objective:(1) Build a co-culture model of human pancreatic cancer cell line MIA Paca-2and rat DRGs. provide a new quantifiable in-vitro model for the study of pancreatic cancer neural invasion.(2) Observe the action and blot effect of Glial cell derived neurotrophic factor (GDNF) on neural invasion of Pancreatic Carcinoma. explore the possible mechanism of induced nerve invasion.Methods:(1) Culture the human pancreatic cancer cell line MIA Paca-2and detect the expression of the RET gene by RT-PCR, get the rat dorsal root ganglia (DRGs) and detect the expression of the GDNF gene by RT-PCR. MIA Paca-2cells and DRGs were cultured in the same culture dish, in this way we build the MIA Paca-2/DRGs co-culture model. Observe the growth and interaction of the pancreatic cancer cells and DRGs neurite, observe the dynamic process of neural invasion of pancreatic cancer cells.(2) MTT assay were used to detect the effect of PD98059-the block of MAPK path and LY294002-the block of PI3K path on the survival rate of pancreatic cancer cell MIA Paca-2under different drug dosage and different reaction time. Different cytokines (GDNF、anti-GDNF antibody、PD98059、LY294002) were added in the lower chamber of the boyden chamber. Count the number of trans-membrane cells of each group and analyzed statistically. And then detect the chemotaxis of GDNF on pancreatic cancer cell and the blocking effect of PD98059and LY294002by Trans-well device.Results:(1) RET and GDNF genes are expressed in the MIA Paca-2and DRGs respectively. In the MIA Paca-2/DRGs co-culture model. Pancreatic cancer cells form more colonies than cultured alone. And more neurite grows around the ganglion. The chemotaxis growth is appeared between pancreatic cancer cells with neurites. Pancreatic cancer cells migrate to the direction of the ganglion; and then grow along around the ganglion eventually enveloping the ganglion.(2) In the MTT assay, pancreatic cancer cell morphology did not change significantly treated with different concentrations of PD98059and LY294002in different reaction time. No significant difference in the survival rate of each group. The overall level is mostly maintained between90%to100%; and there was no significant change regularly with different action time or drug concentration. In the Trans-well assay, Treated with GDNF, the number of trans-membrane cell in boyden chamber increases while reduces with PD98059. The difference is significant (P=0.014). LY29400also reduce the number, the difference is no-significant (P=0.221). The difference is the most obvious with two blockers(P<0.001).Conclusion:(1) Construct the co-culture model of human pancreatic cancer cell line MIA Paca-2/DRGs successfully. Pancreatic cancer neural invasion is the result of the interaction of nerve cells and pancreatic cancer cells. Provide a new quantifiable in-vitro model for pancreatic cancer neural invasion research.(2) GDNF can enhance the ability of invasion and metastasis of MIA Paca-2. The mechanism of the function may relate to MAPK path and PI3K path.