The Research On The Role And The Mechanism Of ROCKⅡ In Invasiveness Of Human 1^st Trimester Cytotrophoblasts

IntroductionImplantation of the human concepts involves invasion of the uterine epithelium and the underlying stroma by extraembryonic trophoblast cells, which undergo a complex process of localization, adhesion, breakthrough and invasion. The cytotrophoblasts located at the tip of the villi attach hardly to the free surface of uterine epithelium, breakthrough the syntrophoblast, proliferate to form multilayered columns of cells which named extravillous cytotrophoblasts (EVCT),subsequently invade to uterine deciduas, uterine spiral arterioles, the upper third of the myometrium, and replace the endothelial lining and most of the musculoelastic tissue of the vessel wall. This arteriole remodeling leads to low resistance vessels that provide an adequate supply of maternal blood to the intervillous space necessary for fetal growth. If the invasion level of EVCT to uterine is excessively strong, it can cause placenta increta and trophoblast tumor. If the invasion level of EVCT to uterine is excessively weak, it can cause miscarriage, premature labor, preeclampsia and eclampsia, et al.The Rho GTPase, that is Ras homologus oncogenes, is one of the membranes of the small guanosine triphosphatase family. It works as a molecular switch in the intracellular pathway. Several putative target molecules of Rho have been isolated. Among these, a family of Rho-associated serine/threonine kinase isozymes, named Rho-associated coiled-coil forming protein kinase (ROCK), has been identified as the most important of Rho effectors. ERM is one of the downstream effectors of ROCK. They consists of ezrin,radixin and moesin, and interact with both cell membrane or cell membrane proteins and F-actin, resulting in the association of the actin cytoskeleton with the plasma membrane. The Rho/ROCK/ERM pathway have a vital function on the cell shape, movement, invasion, adhesion and polarity formation, so as to involve in many kinds of diseases such as malignant tumor, wound healing and so on. Human's placenta implantation is similar to the invasion process of the malignant tumor, so we suspect whether the Rho/ROCK/ERM pathway is involved in Human's placenta implantation. The suspect results in this study.ObjectiveThe objectives of this research are to determine: (1) Detect the location and expression of RhoA,ROCK II and ERM on human placenta of 1st trimester and serum-free cultured cytotrophoblasts;(2) Investigate the relationship of ROCK II and ERM in serum-free cultured cytotrophoblasts; (3) Explore the role of ROCK II in the growth, invasiveness and movement of serum-free cultured cytotrophoblasts. Thus we can know more about the etiology mechanism of placental disease caused by implantation abnormality of embryo, and supply the theory basis of clinical treatment.MethodsThis study determine the location and expression of RhoA,ROCK II,ERM on human placenta of 1st trimester by immunohistochemistry and Western blot technique, determine the location and expression of RhoA,ROCK II,ERM on cultured cytotrophoblasts by immunofluorescence cytochemistry, detect the change of growth, invasiveness and movement of serum-free cultured cytotrophoblasts under the stimulation of various density of ROCK inhibitor Y-27632 by MTT test, transwell test and motility test, investigate the alteration of ROCK II activity and phosphorylated ERM proteins expression in serum-free cultured cytotrophoblasts under the stimulation of various density of ROCK inhibitor Y-27632 by Rho kinase assay and western blot.Results1. In human placenta of 1st trimester, RhoA is located and expressed in plasma of cytotrophoblasts, ROCK II in plasma of both cytotrophoblasts and syntrophoblast, ERM in plasma of cytotrophoblasts and near to plasma membrane. In serum-free cultured cytotrophoblasts, RhoA,ROCK II and ERM are all located and expressed in cell plasma.2. In MTT test, after action of different concentration of Y-27632 in serum-free cultured cytotrophoblasts, A₄₉₂nm optical density of cells correspondingly decreased, that is that cell growth has been inhibited. The concentration was higher, the inhibition is stronger. When the Y-27632 concentration was more than 50μmol/L, the cell growth has been significantly inhibited, was considered statistically significant (P < 0.05). In transwell test, after 2 hours' action of 5,10å’Œ25μmol/L of Y-27632, the cell invasion index was relatively 0.87±0.08,0.75±0.09å’Œ0.58±0.03, showing concentration dependent relation. When the concentration was 10 and 25μmol/L, was considered statistically significant (P <0.05) . In cell motility test, the concentration of Y-27632 was higher, the motility index was lower. When the concentration was 5,10 and 25μmol/L, the motility index was relatively 0.83±0.07,0.68±0.03å’Œ0.54±0.06, was considered statistically significant (P<0.05) . 3. After action of different concentration of Y-27632 in serum-free cultured cytotrophoblasts, ROCK II activity of cells correspondingly decreased. The concentration was higher, the activity is lower, was considered statistically significant (P < 0.05). And under the action of different concentration of Y-27632, total ERM amount was no change, but phosphorylated ERM amount was decreasing, showing concentration dependent relation, considered statistically significant (P <0.05) .ConclusionsRhoA, ROCK II and ERM all express in human placenta of 1^st trimester and serum-free cultured cytotrophoblast. ROCK II can regulate cell growth, invasion and movement in serum-free cultured cytotrophoblasts probably through regulation of ERM, suggests that Rho/ROCK/ERM pathway maybe play a role in human placenta implantation.