Studies On Dynamical Behaviors Of Hepatoma Cell Pseudopod Protrusion In Response To Extracellular Components

The present research has been carried by selecting hepatocellular carcinoma (HCC) cells as the object of study through biomechanical, cell biological as well as immunofluorescence methods & technology, etc. With a micropipette technique, we study on dynamical properties of HCC cells chemotaxis to ECM, which was based on pioneering works on hepato-baliary rheology in our institute and Dr. Dong et al. in Pennsylvania State University, USA. This method is subtle in that it enables direct visual observation on single-cell level and thus higher degree of quantification. And a more strict comparison between pseudopod protrusion on a single cell can be achived through monitoring the pseudopods on both sides of the cell. Meanwhile, the method set-up can provide a new experimental means to study metastasis of tumor & anti-chemotactic effects of anti-metastatic agents.The following are the main contents and results of the present study:(1) Chemotactic pseudopod protrusion of a HCC cell was evaluated using a single-pipette set-up, in which a pipette filled with different concentration of type IV collagen (Col IV) solution & laminin (LN) solution respectively was positioned in close contact with the cell and pseudopod protrusion into each pipette was viewed dynamically. Along the increment of concentration within Col IV of 400μg/ml & LN of 200μg/ml, the length of chemotactic pseudopod protrusion of a HCC cell will increase. But it attained saturation length up to Col IV of 400μg/ml & LN of 200μg/ml.(2) The adoption of novel concentration-symmetric and concentration-asymmetric as well as different chemoattractants filling both of pipettes dual pipette methods revealed the interactions & intensity of pseudopod protrusion. Chemotactic pseudopod protrusion of a HCC cell was evaluated using a dual-pipette set-up, in which two pipettes filled with different concentration of Col IV solution & LN solution respectively were positioned in close contact with the same cell and pseudopod protrusion into each pipette was viewed dynamically and recorded with a tape recorder. The result showed the length of pseudopod in higher side of chemoattractant concentration is longer than that of lower chemoattractant. While the concentration of chemoattractant in a side was declining, the length of pseudopod was been weakening in the side with the more evident pseudopods protrusion in another side. Meanwhile, the maximum length & growth velocity of pseudopod filling LN of 200μg/ml is longer than thatof Col IV of 200μg/ml.(3) The integrin subunits on the surfaces of HCC cells were analyzed by flow cytometry system (FACS). Expression of integrinα₁,α₃ & β1 was up to 95.07%, 95.55% & 95.78% for the cell line (SMMC-7721) chosen in the experiment. And expression of integrinα₅ was 34%[58]. Expression of integrinα₂,α₄ &α₆ was only 23.17%, 2.47% &1 4.29%, respectively.(4) In order to discuss the influence of integrins on the formation of pseudopods of HCC cells, we have put the moloclonal antibodies into a single-pipette and observe the growth of pseudopods dynamically. And the effects of integrin blockade on pseudopod protrusion was also investigated by using a symmetric methods to investigate the interaction of pseudopods on both sides of the a same cell, in which both pipettes were filled with same concentration of chemotactant. The results showed: â‘  Upon addition of Anti-α₂, Anti-α₃, Anti-β1 to one of the pipettes in Col IV of 600μg/ml, pseudopod protrusion was blocked evidently while protrusion into the opposite pipette became more evidently with larger maximum length. And it indicated integrinsα₂β1 & α₃β1 are main receptors mediating HCC cells chemotactic pseudopod protrusion to Col IV in vitro.â‘¡ Upon addition of Anti-α₃, Anti-α₆, Anti-β1 to one the pipettes in LN of 200μg/ml, pseudopod protrusion was blocked completely while protrusion into the opposite pipette became more evidently, with larger maximum length. And it indicated integrinsα₃β1 & α₆β1 are main receptors mediating HCC...