| During the development of human placenta, trophoblast cells (TCs) invasion of, and migration within the uterine vasculature is an essential process for the remodeling of the uterine vasculature, which is important in assuring an adequate blood supply to the developing fetal-placental unit. In vivo, the courses of cell migration are that TCs firstly invade into the capillary vessels, then migrate within uterine vasculature, and eventually reside within the spiral arteries. This indicates that the direction of TC migration within the uterine vasculature is against the blood stream. In order to understand the physiological roles of TCs in the pregnancy process and the mechanism of TC invasion into the uterine vasculature, a great amount of studies have been carried out. However, little has been known about the hemodynamic mechanism of TC migration against the blood stream. Apparently, local blood flow environment must be of paramount importance and involved in regulating biological behaviors and functions of TCs in the migratory process of TCs within the uterine vasculature. The effects of environmental factors on cell division/proliferation, differentiation, apoptosis and motility have become one of the hottest topics in cell biology and biomechanics. Using a 2-D flow chamber technique to mimic the local blood flow environment of the uterine vasculature, the present thesis studied the migratory behaviors of human TCs from the hemodynamic point of view and tried to understand what decides the direction of TC motility and what is the power or mode of TC motility against the blood stream. Meanwhile, the correlation between integrinβ1 and the migratory behaviors of TCs was analyzed on the adhesion molecule level.First, a simple and stable protocol for human trophoblast cell culture was established. The results revealed that the population of isolated human TCs were significantly augmented using the digesting method of trypsin and DNase at 37℃. The purification of TCs was carried out by serial progress, and cytokeratin positive of TCs was greater than 95%. Configuration of the TCs isolated and cultured were singularity and the growth was stable with stronger viability.Then, the effect of the flow-induced shear stress on the motility of TCs cultured on collagen I was studied. The results demonstrated that TCs were highly dynamic with continuous non-directional or dominant shifting with little net displacement in the direction of flow (x-coordinate) under the static conditions (0 dyn/cm2). Average cell migration velocity and net displacement of TCs in the direction of flow increased almost linearly with increasing shear stress. It was also found that the displacement of TCs in the perpendicular direction of flow is not zero. Remarkably, the velocity ratio, Vx/Vy was slowly decreased and the motility of TCs was stable with the cells'own initiative during the 6-12h period. Changes of cell membrane fluidity and interdiction of flow-sensitive ion channels regulated the migration of TCs under different shear stresses was studied. These results demonstrated that shear stress regulated the motility of TCs, however the migration direction of TCs was not against the flow direction but generally in the direction of flow.Then, the migration of TCs co-cultured with HUVECs under different shear stresses was studied. The results revealed that the average velocity and the absolute displacement of TCs in the direction of flow remained essentially unchanged at all levels of shear stress. Again, no apparent counter-flow migration of TCs was observed. These results suggested that the interaction between TCs and endothelial cells up-regulated the adhesion of TCs, hence enhanced their resistance against the displacement by flow. Meanwhile, under the shear stress of 15dyne/cm2, when monoclonal mouse anti-human integrinβ1 was used to block the action of integrinβ1, the average migration velocity of TCs was significantly reduced and their absolute displacement (x-coordinate) was increased when compared with the control group. However, there was no significant difference between the nonspecific antibody group and the control group. These results suggested that integrinβ1 played an important role in regulating the migration of TCs co-cultured with endothelial cells.Using a micropipette aspiration technique, the adhesive mechanical properties of TCs were measured. The results revealed that the adhesion forces of TCs to type I rat collagen increased with increasing concentration of collagen I. When contacted with HUVECs, the adhesion force of TCs was significantly enhanced. Moreover, from the point of view of adhesion molecules, the adhesive mechanical properties of TCs to collagen I was compared with those of TCs to HUVECs. The results suggested that trophoblast integrinβ1 played an important role in the adhesion of TCs.Finally, integrinβ1 expression of TCs on the tissue sections and the isolated TCs were analyzed. The results demonstrated that migratory TCs was accompanied by higher integrinβ1 expression when compared to the non-migratory TCs. Shear stress up-regulated the integrinβ1 expression of TCs, and expression of integrinβ1 of TCs significantly changed with time. TCs co-cultured with endothelial cells increased the expression of integrinβ1 when compared with the TCs cultured alone, suggesting again that trophoblast integrinβ1 played a role in the adhesion and migration of TCs.In conclusion, the present in vitro study demonstrated that by contact with endothelial cells, the adhesion force of human TCs might be regulated according to local flow environment to ensure a stable adhesion of TCs in uterus spiral arteries. So long as the adhesion force of the TCs can resist displacement by blood flow, there is a reason to believe that migratory trophoblasts may reach the spiral arteries not by the actual movement of the cells but by a mechanism of directional cell division/proliferation. |
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