Regulation Of Monocyte Chemoattractant Protein-1 By Lipopolysaccharide In First Trimester Decidual Cells

From the immunological angle, pregnancy can be perceived as a successful semiallograft and there is a delicate maternal-fetal balance,which enable the fetus to develop normally. The establishment and maintenance of pregnancy is decided by both decidual regional and systemic immunologic condition of the mother. On one hand, the mother has to establish immunologic tolerance to permit fetus to implant and develop. On the other hand, decidual immunologic system ought to activate adequately to eliminate pathogens lest infection would damage the balance between mother and fetus, which will contribute to the rejection of mother and result in such pathological pregnancy as abortion, fetal malformation, FGR, preeclmpsia and eclampsia. Many immunologic cells and cytokines take part in the balance between mother and fetus. It is reported that monocyte chemoattractant protein-1 (MCP-1) has bilateral function during immunologic process, anti-inflammation as well as mediating tolerance. Decidual stromal cells (DSC) of first trimester express MCP-1 and its receptor -CCR2 at a high level and several inflammatory factors can promote DSC to express MCP-1, which indicate that MCP-1 plays an important role in the maintenance of immunologic banlance during infection. So we supposed that infection would affect the expression of MCP-1 on DSC. There hasn't been a culture technique of DSC that is generally acknowledged. To confirm the reliability of our result, we also investigate the cuture condition of DSC in vitro.Objective: To observe the regulation of MCP-1 by LPS in first-trimester decidual stromal cells of different passages under the conditions of the micro-environment simulation of early pregnancy (17-β-Estradiol and Progesterone) in vitro.Methods:According to conventional methods, We isolated first trimester decidua(6-8 weeks) of normal pregnant woman, cultivated and passaged with or without the presence of 17-β-Estradiol and Progesterone. We used immunocytochemical staining (Cytokeratin 7 and vimentin) to identify the purity of decidual stromal cells and FCM to detect TLR4 and CCR2 expression on the cell surface. When isolated cells grown to confluence (about 80%),DSC of passage 1 and passage 3 are respectively divided into such groups as control group or LPS stimulation group. In other cases, isolated cells were cultured in complete medium with or without 17-β-Estradiol and Progesterone. After 3 passages, they were divided into control group or LPS stimulation group respectively. Cultured for 24h in the environment of 37℃,5%CO2, supernatant and total RNA were collected. Expression of MCP-1 at transcription level was detected by semi-quantitative RT-PCR method and real-time quantitative PCR, while the secretion of MCP-1 in supernant was determined by ELISA.Results:1,Results about the technique of DSC in vitro:①Compared with cells of passage 1, cytoplasmic granulations in cells of passage 3were diminished and protrution of cytomembrane declined when observed under optic microscope.②It was showed by immunocytochemical staining that compared with cells of passage 1,DSC purity of cells of passage 3 was improved(P<0.05).③FCM results indicated that TLR4 and CCR2 expression level on DSC surface of passage 3 is higher than that of passage 1 DSC.2,Results about the effect of LPS in first trimester DSC①The results of RT-PCR,real-time quantitative PCR and ELISA showed that no matter at transcription level or at protein level, cells of passage 1 express MCP-1 at a high level before the stimulation of LPS. However, after treatment of LPS, changes of expression level has no significant difference (P□0.05). As to cells of passage 3, the expression of MCP-1 was significantly up-regulated in LPS treatment group, compared with control group (P＜0.05). It is interesting that expression level of MCP-1 was much lower in control group of passage 3 than that of passage 1(P<0.05), but higher in LPS treatment group (P<0.05).②RT-PCR,real-time quantitative PCR and ELISA showed that both at transcription level and at translation level, compared with DSC without any treatment (LPS or 17-β-Estradiol and Progesterone), the expression of MCP-1 was significantly up-regulated in other three groups:LPS stimulated group,17-β-Estradiol and progesterone treated group, LPS plus 17-β-Estradiol and progesterone treated group. Compared with 17-β-Estradiol+progesterone treated group or LPS treated group, the expression level of MCP-117-β-Estradiol and progesterone plus LPS combined treatment group was significantly up-regulated (P＜0.05).Conclusions:1,The purity of DSC in cells we isolated can be improved through passage, however, during the process of passage, the expression of TLR4 and CCR2 on the cytomembrane declined, which maybe affect the function of DSC.2,Through combination with its membrane receptor-TLR4, LPS can directly or indirectly up-regulate expression of MCP-1 in DSC at both transcription level and translation level, thus decidual regional immunological was disturbed.3,Meanwhile, during the first trimester, MCP-1 expression of DSC was up-regulated, which suggested MCP-1 plays an essential role in the occurrence and maintenance of normal pregnancy.4,Moreover, combination of 17-β-Estradiol and progesterone could augment the effect of LPS on DSC, which is indicative of the importance of MCP-1 during the inflammatory process related to decidual immunological system.