Molecular Mechanisms Study Of β-NGF And Its Receptors On The Effects Of Perineural Invasion Of Pancreatic Adenocarcinoma

Objective:To study the beta-nerve growth factor and its receptor's effects on the mechanism of pancreatic carcinoma perineural invasion from operation tissue specimens,cell culture ex vivo and animal experiment in in vivo levels,especially elucidate the pancreatic carcinoma MIA Paca-2 cell's biological characteristics of migration,invasion,PNI and mutual relationship ofβ-NGF-NGFR biological axis in the tumor tissue and nerve fibre,enrich the basic theory of perineural invasion.Methods:Expression and distribution ofβ-NGF,TrKA,P75^NGFR,E-cadherin and MMP-2 were detected in operation tissue specimens of pancreatic carcinoma with immunohistochemistry.The effect of cell membrane surface and cytoplasm ultrastructure of MIA Paca-2 cell cultured ex vivo were observed byβ-NGF with SEM and TEM.Expressions ofβ-NGF,TrKA,P75^NGFR,MMP-2 and E-cadherin were detected in MIA Paca-2 cell ex vivo with immunohistochemistry.Effect ofβ-NGF on MIA Paca-2 cell cultured proliferation was evaluated by MTT and Flow Cytometry.Adhesion assay of MIA Paca-2 cells was studied by microplate reader.Migration,invasion assay and chemotaxis assay were studied by imposing exgenousβ-NGF and TrKA antagonist K252a on MIA Paca-2 cell with Transwell chamber methods in order to observe the motility and invasion.MIA Paca-2 cells were subcutaneously injected and orthotopically transplantated into nude mice to establish neural invasion animals models and neural invasions were observed in nude mice by 4wks,6wks and 8 wks using HE and silver nitrate staining.Tumor cells and nerve fibre's ultrastructure were observed by TEM.Expression ofβ-NGF,TrkA and p75 was detected in MIA Paca-2 cells in vivo by immunohistochemistry,RT-PCR and Western Blotting.The relations ofβ-NGF with MMP-2,MMP-9 and E-cad were analyzed.Results:Expression ofβ-NGF,TrKA,P75^NGFRand MMP-2 in the well-,medium- and poor-differentiated PAC groups lie in the cytoplasm of tumor cell,higher than normal group(P<0.05).However,E-cadherin expression are lower than normal group(P<0.05).The ratio of PNI found was 56.25%(45/80).β-NGF,TrKA and MMP-2 were correlated with perineural invasiveness.MIA Paca-2 cell presents epithelial form and have rich pink cytoplasm,prone to adherance growth.After the passage,adherence, growth and splitting could be found by light microscope,and the circle or ellipse nest-like structures were formed.Following,the detatched cells in the culture medium gradually increased.Ultrastructural observation:With the SEM,the villus were found decreased and became thick on the adjacent sides after the cells contacted,accompany with the decreasing contact area with other adjacent cells and the cultivate surface,Middle cells in the nest-like structures became round gradually and became detached cells at last.The apoptosis body was rare.In theβ-NGF group,the culture cell density and the round cells with intensive stain were more than the control group.With the SEM,most of the cell colony were irregularity and the cell surface villus and microvillus were more and thicker between the cells inβ-NGF group than those in control group.With the TEM,canaliculus become more,longer and thicker inβ-NGF group than those cells in control.MIA Paca-2 expressedβ-NGF,TrKA and P75^NGFR,especially TrKA,also expressed E-cadherin and MMP-2 with climb pieces.β-NGF may promote MIA Paca-2 proliferation and growth which were markedly inhibited by K252a(P<0.05)using MTT and FCM.Transwell chamber results showed that MIA Paca-2 cell's chemotaxis and invasion were remarkably enhanced with the concentration ascending of exogenousβ-NGF by concentration dependently,when we administer 100ng/ml K252a to MIA Paca-2 cells ex vivo,its chemotaxis and invasion were markedly inhibited.The attachment ability of MIA Paca-2 cells could be degraded byβ-NGF.Masson staining also show that there was an evident matrix degradation circle around tumor cells.The orthotopic animal model of pancreatic carcinoma were established.Perineural invasion rate of the orthotopic animal model of pancreatic carcinoma is 50%,80%and 60%at 4 wks,6wks and 8wks respectively in nude mice.By immunohistochemistry,RT-PCR,real-time PCR and Western Blot,we found thatβ-NGF,TrKA and P75^NGFRexpression of in PAC samples of 6wks are higher than the others(P<0.05).β-NGF and TrKA expression in PNI groups are higher than those non-PNI groups(P<0.05).β-NGF,TrKA's gene sequence were measured.Conclusions:MIA Paca-2 cell can expressβ-NGF and TrKA,mainly TrKA,meaning have same biological Characteristics between in vitro and ex vivo.Nerve cell can also expressβ-NGF and TrKA,but mainlyβ-NGF.β-NGF and TrKA may have mutual effect in signal transduction leading to MIA Paca-2 cell' proliferation and migration.Moreover,Multiple process and phase synergistic effects may result in perineural invasion,like accelerating MMP-2/9,reducing E-cadherin,affecting ultrastructure and proliferation.Neurotrophin mimicry(NM)theory may play important role in perineural invasion of pancreatic carcinoma.