The Effect Of Substrate Stiffness On Confluent Growth Behavior Of Hepatic And Hepatoma Carcinoma Cell

Objective To investigate the cause of differences on the confluent growth ofhepatic(L02) and hepatoma carcinoma(HCCLM3) by comparing the cell responses tothe substrates with different rigidity (0.5kPa、4kPa and glass) so as to understand thepopulation growth behavior of tumor cells.Methods The real-time photomicrography technique was used to observe themorphological characteristics, the Immunofluorescence microscopic technique wasadopted to detect the cytoskeleton conformation and the distribution of E-cadherin, theflow cytometry technique was employed to measure the expression of Integrinβ1, andthe expression levels of E-cadherin and phosphorylated Src kinase were tested byWestern Blotting.Results In different rigidity subsrates, the confluent growth behavior of L02andHCCLM3cells was different.(1) Confluent L02cells displayed a round or cubic shape,but HCCLM3cells showed a polygon shape. The morphology of HCCLM3cells weremore obvious spread and polarize than L02cells. With the increasing of substraterigidity, the changes of L02cells conform factor were smaller than HCCLM3cells withtime. After adding EDTA cells turned round with time, CF and spread area decreased.With the increasing of substrate rigidity, CF and spread area increased, and the changeof HCCLM3cells was more obviously than L02cells.(2) The cytoskeleton of confluentL02cells showed a ring-like conformation, and the E-cadherin protein co-located withcytoskeleton at the cell-cell contact sites. However, the E-cadherin of HCCLM3cellswas dispersed in cytoplasm, and HCCLM3cells could not assembled the cortical rings.The expression of paxillin was increased, and HCCLM3cells more than L02cells.Paxillin didn’t co-location with cytoskeleton.(3) The expression of E-cadherin wasdecreased with increasing substrate rigidity, while the level of p-Src and integrinβ1increased with the substrate rigidity, and the change of HCCLM3cells was moreobviously than L02cells. After adding EDTA, the expression of E-cadherin in L02cellswas decreased dramatically other than p-Src increased, but didn’t change significantlyin HCCLM3cells.Conclusions In two-dimensional environment (1) There are positive correlationbetween the distribution of E-cadherin and adjustment of cytoskeleton conformation.(2)The influence substrate rigidity on the balanced regulation of E-cadherin and Integrin adhesion systems of hepatocarcinoma cells shows more significant compared with thatof hepatic cells.