| The pseudopod is the primary locomotory organelle of free-living amoeboid cells as well as in a variety of mammalian cells.Various evidence supports the notion that pseudopod formation is governed by cytoskeletal reorganization,especially that of actin filaments.The pseudopod appears in the light microscope as a vesicle-free zone of protrusion,sometimes highly flattened along the substratum(as in lamellipodia)and sometimes more rounded or off the surface(as in lobopodia).Both fluorescence microscopy and electron microscopy demonstrate that this region invariably contains a dense meshwork of F-actin filaments and few if any microtubules.In living and permeabilized cells,actin polymerization has been shown to occur predominantly near the extending edge of the cell,by the addition of new actin monomers between the filament end and the membrane.Inhibition of actin polymerization by cytochalasins blocks cell motility and pseudopod extension,providing direct evidence for the role of actin filaments in movement.Although the involvement of actin filaments in pseudopod formation is more or less accepted,there are still conflicting ideas about the mechanism by which force is generated.Models invoking forces derived from the gelation of F-actin networks have been proposed to account for the outward expansion of the pseudopod. The materie is hepatocellular carcinoma cell in this text,and it is treated with chemoattractants targeting different receptors,and the study adopted many methods,such as micropipette technique,immunocytochemistry and Boyden chamber to study the relation of pseudopod protrusion of HCC cells to both the functional status of integrin receptors and cytoskeletal structures.Studying the chemotactic behavior is important for preventing and curing the invasion and metastasis of malignancies nowadays. The following are the main contents and results of the present study: Firstly, The study adopted single micropipette technique to research the chemotactic pseudopod protrusion of HCC cells in response to different chemoattractants(type IV collagen,Laminin,Histamine) targeting different receptors. We observed that the length of chemotactic pseudopod protrusion of a HCC cell will increased with increasing concentrations of chemoattractants until 400 μg/ml for type IV collagen (Col IV) or 200 μg/ml for Laminin (LN). The length of chemotactic pseudopod protrusion of a HCC cell is 6.35 ±1μm and 7.3±1.08μm respectively for Col IV of 400 μg/ml & LN of 200 μg/ml. Beyond this concentration, the chemotactic pseudopod protrusion saturated until up to 400 μg/ml for Col IV & 200 μg/ml for LN. Histamine(Ht) did not induce the chemotaxis of HCC within the concentration ranges of 0.05 ng/ml~500 ng/ml;The adoption of novel concentration-asymmetric as well as different chemoattractants filling both of pipettes. in which two pipettes filled with same concentration of ColIV respectively but Ht solution is filled with micropipette just in single side. While the concentration of Ht in one side was increaseding, the length of pseudopod protrusion was declining accordingly, and it has a saturation state for Ht of 5 ng/ml. In addition,it cannot weake the length of pseudopod in another side. Secondly, in order to discuss the influence of cytoskeleton on the formation of pseudopods of HCC cells, we have put the cytochalsinD into a single-pipette and observed the growth of pseudopod dynamically. We observed that the length of chemotactic pseudopod protrusion of a HCC cell will be decreased with increasing concentrations of interferential substance until 20μg/ml for CD or 3.0 μmol/l for Ca2+. In addition,we adopod the immunocytochemistry and electromicroscope technique. With increasing concentrations of CD until 20 μg/ml or Ca2+ until 3.0 μmol/l, the most parts of cytoskeleton were inordinate,and the fraction was dissolved and disappear. The results showed: cytochalsinD destroyed the microfilament,and then blocked the form of pseudopod.Calcium influx and efflux can induce the deploymerization of filamentous actin,and blocked the form of pseudopod of HCC accordingly. Thirdly, 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 observed the growth of pseudopods dynamically. We observed that the length of pseudopod protrusion cell was almost indeclinablede with increasing concentrations of chemoattractants until 20 μg/ml for Anti-CD49a,then added Anti-CD49d moloclonal antibodies to the micropipette, the same as the length for Col IV in single micropipette.But the length of pseudopod protrusion a HCC cell will decrease with increasing concentrations of Anti-CD49b moloclonal antibodies until 20 μg/ml, then 5~20 μg/ml Anti-CD49c moloclonal antibodies was filled into the same pipette sequentially, the length of pseudopod protrusion will decrease evidently. The results showed: integrinsa2 & a3 are main receptors mediating HCC cells chemotactic pseudopod protrusion to Col IV in vitro.Finally, We have combined Boyden charmber with the results of micropipette experiments of formation of chemotactic pseudopod protrusion. In the experiments, different concentrations of Col IV can induce the movement of HCC. The numbers of HCC cell movement is 541.06 ±40,. 720.36 ±60,924.21 ±71 when the concentration of the Col IV is 200 μg/ml,400 μg/ml,600 μg/ml. Beyond this concentration, the chemotactic pseudopod protrusion saturated until up to 600 μg/ml for Col IV. Histamine did not induce the chemotaxis movement of HCC within the concentration ranges of 0.05 ng/ml~500 ng/ml; The effects of blockade of cytochalsinD and Histamine on chemotactic migratition of cells population were evaluated with Boyden chambers. We show that CD can inhibit the form of pseudopod protrusion with the range concentration of 0.5 μg/ml~2.0 μg/ml,but the inhibition is the most remarkablely with HCC for CD of 2.0 μg/ml. Histamine can inhibit the movement of HCC cells with the concentration ranges of 10-10,10-9 ,10-8,10-7 mol/L. The cell numbers of movement of HCC cell is minimal when the concentration of histamine is 10-8 mol/L.( p<0.01 )。CD inhibited the movement of HCC cells by destroyed the cytoskeleton;Histamine may inhibit HCC chemotaxis by means of altering receptor activation,calcium flux,or antin polymerization. The results confirmed the above results within the second chapter and the third chapter.
|
PDF Full Text Request