博士
國立臺灣大學
解剖學研究所
95
Successful implantation of embryo relies on the delicate control of invasion for extravillous trophoblasts. However, current studies on the phenotypic differentiations of invasion ...trophoblast and their relevant motile behaviors are still lacking. In addition, the precise role of invasive trophoblast in decidual vascular remodeling is not well-understood, either. In the first part of this dissertation, we prospectively collected the decidual sampling of the first and third trimester pregnancy, and processed with cryostat sectioning and immunohistochemistry. The aim of observation is to determine the phenotypic differentiations of invasive trophoblasts at different times and location, and postulate the inference for the relevant behavior of their specialized transformation.
After extensively inspection of decidual specimens containing invasive trophoblasts and vessels, we found the unipolar, spindle-shape trophoblasts extensively infiltrated in maternal decidual stroma. The density of distribution, however, presented an inverse proportion with the depth of invasive pathway. Interestingly, those trophoblasts invading into myometrium at the third trimester transformed into stellate architecture. Meanwhile, stellate trophoblasts also existed in the surroundings of decidual spiral arteries, or even penetrated and harbored inside the tunica media of the vessel. Besides, the existence of stellate trophoblasts also perturbed the arrangement of actin fibers, or resulted in clear lacuna inside the tunica media. From these observed data, we suggested that the stellate transformation might explain the controlled invasion of invasive trophoblasts during pregnancy, and probably play an upstream role in the initiating the decidual vascular remodeling.
My second experiment is to establish the in vitro model of living trophoblast observation by time-lapse video microscopy. First we used the cell line from SV40-transformed trophoblasts from first-trimester pregnancy for the transwell invasion assay. We seeded the cells to the upper chamber of transwell, and observed the cells staying in the upper or invading into lower chambers at 6, 24, 48, 72 hours. We found that cell invasion into lower chamber reached a plateau at 24 hour, and there was a trend of type I receptor of TGF beta (TbRI) downregulation for cells in upper chamber chronologically. Most importantly, TbRI expression in trophoblasts at lower chamber (representing invasive cells) remained low throughout the studied period. The data indicated that lower TbRI expression was associated with higher invasion capacity of trophoblast. To prove this notion, we further added selective TbRI inhibitor (SB431542) or TGF-beta3 in the trophoblast culture at upper chamber. The results yielded clear evidence that inhibited TbRI expression did increase the invasion ability of trophoblasts, while TGF-beta 3 supplement had an inhibitory effect of invasion, on the contrary. Besides, both the TbRI inhibitor and TGF-beta3 demonstrated a dose-related effect on invasion.
The trophoblast culture was further evaluated with cine images recording by time-lapse video microscopy. We conducted 3 groups of experiment – supplemented with TbRI inhibitor or TGF-beta3 as experiment groups, and normal culture condition served as control group. At the end of 24-hour culture, we snapshotted the cell image and analyzed by routine of integrated morphometric analysis provided by Metamorph software. The result showed treatment with TbRI inhibitor significantly decreased the cellular area and dragged the cell into spindle shape, whereas the effects of TGF-beta3 were completely opposite. In addition, motion analysis also demonstrated that treatment with TbRI inhibitor significantly increased the angular vector of trophoblast motion but decreased migration velocity. Tracing the migration path of those with high angular vector, a persistent directionality of motion can be consistently found. We postulated it could promote the ability of trophoblast to penetrate membranous pore of transwell or the cell-cell or cell-matrix along the invasive pathway in vivo. We also investigated the in vivo specimen. Those trophoblasts in cell column demonstrated TbRI up-regulation, while trophoblasts in the interstitial area downregulated their TbRI expression. We concluded that, downregulation of TbRI expression is probably an important factor that induces the trophoblast detached cell column and start to invade maternal decidual stroma.
