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Background and purpose: The placenta is an appendage of the fetus, it ranged between fetal and maternal metabolism, defense, endocrine and immune function, and therefore maintain fetal nutrition and development of vital organs. Placenta by the end of decidual lobulated chorionic and amniotic three-part structure. Late blastocyst implantation, trophoblast cells start to proliferate to form fluff. Fluff consists of two parts, the inner active cell mitosis trophoblastic trophoblastic form. Trophoblastic fit of the outer layer of executive function. New trophoblast cells can be fused into syncytiotrophoblast. The trophoblastic is the most important cells in the placental tissue. Placenta formation and development during trophoblast cell population first invasion of endometrial stromal contact with capillary invasion after entering the capillaries continue to migrate, and finally settled in the maternal spiral arteries and rebuild its structure to ensure fetal placental system adequate blood supply. Trophoblastic the biological behavior invasion, proliferation, differentiation and apoptosis, these acts are a variety of factors, growth factors, adhesion molecules, cytokines and hormones. A variety of extracellular stimuli through intracellular signaling to regulate the effect factor gene and protein expression, thereby regulating cellular activity. Subject to strict temporal and spatial regulation of the biological behavior of the trophoblast, once the regulatory mechanism of disorders, it may lead to miscarriage, preeclampsia, and fetal growth restriction, trophoblast cell tumors and other diseases occurred, and therefore the the gestational trophoblastic regulation link has become currently producing scientific research hotspot. With further research, trophoblastic development from pathological level to the level of molecular biology. Rho and its downstream signaling molecules ROCK (Rho associated colied coil forming protein) as a signaling molecule in many tissues and cells play a role, such as the role of RhoA in tissue such as colon cancer, breast cancer, gastric cancer, hepatocellular carcinoma of RhoC The increase in the expression level and activity level, and promote the proliferation of cancer cells, RhoB, having the role of anti-tumor activity. Back in 1992 it was found the ROCKII expressed in the placenta. In 2000, scholars have found that RhoA plays an important role in the development and differentiation of the embryo. Followed people began to pay attention to the role of Rho, ROCK trophoblastic. In subsequent studies found, expression of ROCKII in the placenta in preeclampsia compared with normal pregnancy increase in the course of the study of the past, we also found that the increase in RhoB expression levels in the placenta of preeclampsia compared with normal pregnancy . Visible Rho, ROCK expression levels during pregnancy is different, so we have the dynamic variation of animal experiments carried out research to find a theoretical basis to nourish the study of cell-associated diseases. Materials and methods: (1) the establishment of animal models and packet Wistar rats in the same cage at 18:00 pm by male and female 1:1, 8:00 observation cages tray next morning, found the vaginal plug conceive success (20 only), pregnancy and is set to 0. 20 successful pregnancy in female rats were randomly divided into five groups: group pregnant seven days, 10 days pregnant group, group 13 days pregnant, pregnant 15-day group and 18 days of gestation group. At room temperature 25 ° C, relative humidity of 40% -70% of the barrier system feeding, animal feed and water without restriction. Specimen collection seven days gestation group, 10 days pregnant group, 13 days pregnant group, pregnant 15-day group and 18 days pregnant rats respectively 7,10,13,15,18 days of pregnancy in Sumianxin new intramuscular anesthetic, by cesarean section. placenta, cold saline wash away the bloodstains, dry filter paper, balance weighing placental wet weight, and record the placenta the number and placental diameter size. The placenta in the sterile gauze try to suck the moisture, the portion of the placenta tissue placed in 4% paraformaldehyde fixed, all the rest of the placental tissue into the freezing tube and quickly put in liquid nitrogen filling save spare . Take complete specimens of the new dose of chase acceleration sleep after the rats were killed. Immunohistochemical detection of RhoA, B, C, and ROCK I, II expression placenta of pregnant rats weighing determination after a portion of the placenta placed in 4% paraformaldehyde fixed 24h; embedded in paraffin, sliced. RhoA, B, C, ROCKI, ROCKII expression using immunohistochemical ABC method for the determination of the placenta. The one anti: RhoA1: 1500, RhoB1: 1500, RhoC1: 1500, ROCKI 1:500, 1:1500 dilution ROCKII. Reference to immunohistochemistry reagents manual, in strict accordance with the steps. PBS instead of primary antibody as a negative control, DAB color hematoxylin light staining, mounted with neutral gum microscopy. Brown particles appeared to intracytoplasmic positive, Image-Pro Plus software analysis. 4. Western-blot detection RhoA, B, C and ROCK I, II protein extract protein BCA Protein Assay line protein electrophoresis, electro transferred onto PVDF membrane, cut the corresponding protein electrophoretic bands, 3% BSA at room temperature closed 2h, add the appropriate primary antibody (RhoA1: 300, RhoB1: 300, RhoC1: 300 ROCKI 1:1000, ROCKII 1:1000) overnight at 4 ° C TBS buffer, the membrane was washed three times, adding HRP conjugated secondary antibody , 37 ℃ incubated 1h, the membranes were washed 3 times; ECL (electrochemiluminescence law) developing and using the Quantity-one4.62 software (Bio-Rad Laboratories) electrophoresis analysis of gray value, with reference to the target band within Article The ratio of the belt represent the expression level of the target protein. ELISA kit for detection of Rho / ROCK activity in strict accordance with the kit instructions, measured Rho / ROCK activity. Results: 1. General in the whole pregnancy process, each group of rats spirit state is better, no death, abortion. With increasing gestational age, the rat placental weight and diameter growth in line with previous studies. Immunohistochemical results, RhoA, RhoB, of RhoC, ROCKI ROCKII in pregnant of 7,10,13,15,18 day group placental tissue expression, and is mainly expressed in the cytoplasm of trophoblastic. 3.RhoA, B, C, ROCKI II expression in normal pregnancy rat placenta tissue RhoA expression levels in pregnant 10,13,15,18 days group was significantly higher than pregnant 7 days (P <0.05), and pregnant The expression levels of 13, 15-day group compared with 7,10-day group there are very significant differences (P <0.01). With the increase of gestational age, the upward trend in the level of expression of RhoA. RhoB was no significant difference (P> 0.05) in the expression levels of the pregnancy period. RhoC expression levels in pregnant 10,13,15,18 day group was significantly higher than the pregnant 7 days (P <0.05), and pregnant expression levels of 13, 15 days than 7,10 days group there was a significant difference (P <0.01). With the growth of the gestational age, the level of expression of RhoC upward trend. The normal pregnancy rat placental tissue ROCKI, ROCKII in pregnant 18 days group was significantly higher than the progesterone the 7,10,13,15-day group (P <0.01). With the increase of gestational age, ROCKI, ROCKII of expression showed an upward trend. RhoA, B, C, ROCKI II activity levels of normal pregnant rat placenta tissue RhoA activity levels in pregnant 10,13,15,18 days group was significantly higher than 7 days pregnant group (P <0.05), but 10,13,15 days pregnant group activity level is no significant difference (P> 0.05). With the increase of gestational age, the upward trend in the level of RhoA activity. The RhoB activity levels in pregnant 7,10,13,15,18 days between the groups was no significant difference (P> 0.05). RhoB activity did not change significantly with increasing gestational age. RhoC activity levels in pregnant 15 and 18 days group was significantly higher than the pregnant 7,10,13 day group (P <0.05), but no significant differences in activity levels among pregnant the 7,10,13-day group (P> 0.05) . With the growth of the gestational age, the level of activity of RhoC upward trend. ROCKI activity levels in pregnancy 13, 15 days group was significantly higher than 7,10 days pregnant group (P <0.01), and 10 days of gestation group than seven days pregnant group (P <0.05). With the growth of gestational age, the the Rocki level of activity in increasing. ROCKII activity levels in pregnant 18 days group significantly higher than the progestin of 7,10,13,15-day group (P <0.01), 13 and 15 days of gestation group activity levels higher than 7,10 days pregnant group (P lt; 0.05) , 7 and 10 days of gestation group activity level was no significant difference (P gt; 0.05). With the growth of the gestational age, the activity level of ROCKII increase during late pregnancy increased the most obvious. Conclusion: RhoA, RhoB, of RhoC, ROCKI ROCKII in the rat placenta tissue expression, and expression mainly in the trophoblast cell cytoplasm; 2. RhoA, RhoC expression in rat placental tissue and activity levels were upward trend, while RhoB did not change significantly. First trimester the role of RhoA of RhoC involved in trophoblast invasion during late pregnancy may be involved in to nourish cell proliferation and apoptosis, the role of which RhoC trophoblast apoptosis behavior than RhoA is more obvious. RhoB during normal pregnancy may not be involved in trophoblast invasion, proliferation, differentiation and apoptosis; with increasing pregnancy ROCKI expression level and activity level of ROCKII is increased, and the increase during late pregnancy The most obvious, especially ROCKII most significant. In the early pregnancy stage ROCKI, ROCKII expression, likely along with RhoA, RhoC regulation of trophoblast invasion. In late pregnancy during collaborative regulating trophoblast proliferation and apoptosis, promoting the maturation of the placenta, so that pregnancy can be carried out smoothly. The role which ROCKII most obvious.
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