Studies On The Effect Of Neogenin/Netrin-1 On Biological Behavior Of Cytotrophoblasts Of Early Pregnancy And Placental Circulation Disorder

With blastocyst embedding into decidua, trophoblast fuse into trophoblast cell columns, then villous tree structure began to grow. With the development of pregnancy, some cells derived from the trophoblast cell columns, differentiating into extravillous trophoblast cells, invading dispersely nearly one-third of uterine decidua and myometrium, and remodelling the spiral arteries, then reducing the placental bed peripheral-vascular resistance. More blood then flows to the intervillous lacuna of placentavilli barrier-free space and ensure normal growth of the placenta and fetus. If the invasiveness of endovascular trophoblast cell decreased, "shallow implantation" in placenta will appear. The probability of spontaneous abortion in pregnancy increases, and the fetal growth restriction and pre-eclampsia increase in mid and third trimester of pregnancy. Then result in high maternal and perinatal morbidity and mortality during pregnancy. Recently, some studies have shown that angiogenesis-related factors being closely linked to invasion of extravillous trophoblast. Therefore, the study of possible mechanisms that angiogenesis-related factors play an importment role in "shallow implantation" is utmost importance.Netrins are laminin-related molecular family with highly conserved sequences. It has the dual role of attraction and repulsion during the migration of nerve cells. Recent studies have found that Netrin-1, as one member of the family, can have a conjunction with its specific ligand Neogenin to form Netrin-1/Neogenin axis. Netrin-1/Neogenin axis can mediate axon guidance growth.There are many morphological similarities between the nerve and vessel during their developmental process. Many scholars have found that Netrin-1 can mediate angiogenesis through inhibiting the apoptosis of endothelial cell, promoting proliferation of endothelial cell'and facilitating tube formation of endothelial cell. We also confirmed these through having tests in HUVECs. In addition, Neogenin also can promote the growth of rabbit corneal neovascularization as an angiogenic factor. As a newly discovered angiogenetic factor, it is unclear that Neogenin play physiological functions on trophoblast cells. In this study, we investigated the expression and distribution of Netrin-1 in maternal-fetal interface of normal pregnancy and in placenta of poor placetation.We study Neogenin how to affect the the biological behavior of human extravillous trophoblast cell line (TEV-1) in vitro. Then to explore the correlation between Netrin-1/Neogenin axis and dysvasiformation during pregnancy and elucidate the possible mechanism of the placentation. Even provide an experimental basis and theoretical basis for the occurrence of pregnancy and prevention of pathological pregnancy.Objective1.To observe the expression and distribution of VEGF, Netrin-1 in the human trophoblast cells (TEV-1)with serum abruption. And the proliferation, apoptosis, tube formation and the abilities of invasion in TEV-1 are monitored. Then explore the effect and potential mechanism of serum abruption on biological behavior of trophoblast cells.2 To observe the expression of VEGF, Netrin-1, RGMa, DAPK and Neogeninin the human trophoblast cells (TEV-1) with stimulations under different concentrations of Neogenin. And the apoptosis, invasion and tube formation in TEV-1 are monitored. Then explore the effect and potential mechanism of Neogenin on biological behavior of trophoblast cells.Methods:1 TEV-1 cell line was cultured and then subcultured in serum abruption medium or medium with 15% fetal bovine serum, Cell viability was measured by-MTT assay. TEV-1 cell apoptosis were assessed by flow cytometry (FCM) assay. In addition, we also observed the changes of tube formation.The expression of VEGF, Netrin-1 were monitored by Immunofluorescence staining and western blotting.2 TEV-1 cell line was cultured and then subcultured in serum abruption medium with different concentrations of neogenin (0,1,5,10,50 ng/ml). After 24h treatment, Cell viability was measured by MTT assay. TEV-1 cell apoptosis were assessed by flow cytometry (FCM) assay. AO/EB staining method of apoptosis were used to monitor the morphological changes in TEV-1 cell. In addition, we also observed the changes of tube formation, numbers through the Matrigel matrix membrane and the activities of Caspase-3 in TEV-1. The changes of RGMa, DAPK, Neogenin, Netrin-1, VEGF mRNA were detected by real-time PCR, and we also used western blotting to examine the protein changes in Netrin-1and VEGF.Results:1 Comparison of serum-containing medium, cell proliferation significantly decreased and total apoptosis of cells increased in serum-free medium. In addition, serum deprivation can significantly reduce Netrin-1mRNA in the TEV-1 cells (p<0.05), but there has no effect on the expression of VEGFmRNA (p> 0.05).2 After 24h treatment, Cell viability was measured by MTT assay. The OD values of all groups are 0.11±0.02,0.08.±0.01,0.12±0.02,0.14±0.07 and 0.12±0.09. The results of MTT indicated that neogenin has no effect on the proliferation of TEV-1 cell (P >0.05)3 The results of FCM indicated neogenin can induce the apoptosis of TEV-1 cell in a dose-dependent way. The total apoptosis of TEV-1 cell under 50 ng/ml Neogenin was up to 29.6%, which increased significantly than 6.8% in control group (P<0.01). The relative activity of Caspase 3 kept a level the accrescence of Neogenin, and the level were 1.021±0.00,1.06±0.00,0.93±0.02,1.04±0.01 times than control groups (P>0.05)4 After 24 hour's treatment, the expression amount of RGMa mRNA varied to 0.62±0.04,0.97±0.04,0.90±0.03 and 0.75±0.03 times than control group; the expression amount of DAPK mRNA varied to 0.68±0.02,0.74±0.01,0.51±0.01and 0.33±0.01 times than control group; the expression amount of Neogenin mRNA varied to 1.45±0.03, 1.22±0.08,1.47±0.00, and 1.14±0.00 times than control group.5 Immunoreactivity for netrin-1 and VEGF was observed in cytoplasm of the trophoblast. After 24 hour's treatment, the expression amount of VEGFmRNA,Netrin-1mRNA increased in all groups. When the concentration of neogenin was up to 50ng/ml, the expression level of netrin-1 mRNA was 44.84 times than control group(P<0.01). But the similar trend could not be seen in netrin-1 protein.6 After 24 hour's treatment, tube formation can be seen in trophoblast, and serum deprivation and Neogenin does not affect the tube formation of trophoblast.7 Neogenin can inhibit the invision of TEV-1 cell.Conclusion:1 Serum deprivation can inhibit the proliferation and induce the apoptosis of TEV-1 cell, and the expression level of Netrin-1mRNA decreased significantly. Serum deprivation can regulate the proliferation and apoptosis through the effect of the expression of netrin-1 in trophoblast. Serum deprivation does not affect the tube formation of trophoblast.2 Neogenin does not affect the proliferation of trophoblast. Neogenin can induce the apoptosis of TEV-1 cell in a concentration dependent manner. Neogenin does not affect the tube formation of trophoblast but inhibit the invision of TEV-1 cell.3 With the concentration of Neogenin increasing, the mRNA expression of DAPK is down but Neogenin is up, and the mRNA expression of RGMa is almost invariant. Neogenin possibly control the apoptosis of TEV-1 cell through DAPK pathway.4 Free Neogenin can decrease the concentration of Netrin-1 in medium, then enhance the expression and secretion of netrin-1 through the Netrin-1/Neogenin signal transduction pathway.Objective:1 To detect microvessel density (MVD) in villus and decidua during the first trimester, term pregnancies and in pregnancies complicated by preeclampsia. Then to confirm that angiogenesis is participative to pregnancies.2 To determine the concentration of VEGF in corresponding groups, and inspect the expression of VEGF and Netrin-1 in the villus (placenta) and decidua of pregnancies in corresponding groups. Then explore the effect of Netrin-1, VEGF on angiogenesis and the correlation between Netrin-1, VEGF and preeclampsia.3 To detect VEGF and Netrin-1 in placentas of normal term pregnancy and placental circulation disorder, then explore the possible mechanism during pathologic pregnancy.Methods:1 Immunohistochemistry was used to detect the MVD in villi and deciduas from 16 cases of normal early pregnancy. The same methods were used to monitor the MVD in feto-maternal interface in 72 cases (27 term pregnancies,21 cases of mild and 24 cases of severe preeclampsia)2 The concentrations of VEGF in corresponding serum were detected by ELISA. Immunohistochemistry and optical density analysis were used to examine the expression of VEGF and Netrin-1 protein in corresponding pregnancies.3 Western blot was used to detect the expression level of VEGF and Netrin-1 in 24 cases (6 cases of term pregnancies,5 cases of mild,4 cases of severe preeclampsia,3 cases of fetal growth restriction,3 cases of fetal growth restriction with mild preeclampsia and 3 cases of fetal growth restriction with severe preeclampsia).Results:1 Compared with normal term pregnancy placenta:1) The MVDs in normal early pregnancies are lower, especially in deciduas; 2) There are no significant differences between term pregnancy and mild preeclampsia; 3) MVDs decrease dramatically in placentas and deciduas of severe preeclampsia than term pregnancies (P<0.05)2 The same result can be seen in the corresponding serum. The serum level of VEGF in normal early pregnancies is lower and it increases in normal term pregnancies; There are no significant differences between term pregnancy and mild preeclampsia, too. But the level decrease dramatically in serum of severe preeclampsia (P<0.05).The results monitored by Immunohistochemistry can be seen in the corresponding tissues:1. Netrin-1 expressed in cytoplasm of trophoblast and endothelial cell in placenta, and VEGF was mainly lay in parts of endothelial cells. Netrin-1 and VEGF both expressed in decidual stromal cells, decidual cell cytoplasm.2. Results are showed as following with the optical density of samples in each group:A. Compared with normal term pregnancy placenta, it were no significant difference in the levels of VEGF in chorionic villi or placentas in mild preeclampsia, or decidua in early pregnancies and mild preeclampsia. But VEGF in severe preeclampsia placenta and decidua were significantly decreased (P<0.05).B. Compared with normal term pregnancy placenta, it were no significant difference in the levels of Netrin-1 in chorionic villi of in early pregnancies or placentas in mild preeclampsia, or decidua in early pregnancies and mild preeclampsia. But Netrin-1 in severe preeclampsia placenta and decidua were significantly decreased (P<0.05).3 The result can be seen in the other groups by western blot:1. The expression level of Netrin-1 in the norm term pregnancy are the highest, and it decrease by turns of groups in fetal growth restriction, mild preeclampsia, severe preeclampsia, fetal growth restriction with mild preeclampsia and fetal growth restriction with severe preeclampsia. Statistical analysis confirmed that Netrin-1 in normal term prgnancy are the highest (P<0.05), but there are no significant differences in mild and severe preeclampsia groups. The expression level of Netrin-1 decrease drastically in the two groupss when they are combination with fetal growth restriction(P<0.01).2. The expression level of VEGF in the fetal growth restriction groups are the highest, and it decrease in severe preeclampsia, fetal growth restriction with mild preeclampsia and fetal growth restriction with severe preeclampsia. Statistical analysis confirmed that VEGF decreased dramatically in placentas of fetal growth restriction with mild preeclampsia and fetal growth restriction with severe preeclampsia. (P<0.05).Conclusion:1 Netrin-1, VEGF and other factors affected angiogenesis during pregnancy.2 Netrin-1 and VEGF played important roles in the pathogenesis of severe preeclampsia.3 Netrin-1 plays more important role than VEGF in the formation of placental vascular network, involves in pathogenesis of fetal growth restriction with.