Research On The Change Of Progesterone Receptor And TNF-??IL-1??NF-?B In The Deciduas Stromal Cell Which Causal Relationship With Spontaneous Abortion

The occurrence of unexplained recurrent spontaneous abortion (URSA) and its relevance with cytokines, immunity system, hormone and receptor regulation has attracted more and more attention. The series of factors has a close relationship with the endometrial convert decidual normall, the blastula implant, and fetation growth. During the early stages of pregnancy, progesterone play a significant role in a successful pregnancy. progesterone acts mostly through progesterone receptor (PR), a certain amount of PR is required in decidua to react to progesterone?The degradation in the amount of receptor and its function will affect the normal functions of the corresponding hormone. It's known that using progesterone or HCG alone does not result in an ideal outcome in the cases of habitual abortion. Therefore the reason for abortion with a sufficient supply of progesterone becomes a hot topic worldwide.The Inflammation factor is one of great importance, the mechanism of its cause to abortion is an important direction in research.many papers had already reported that inflammatory cytokine participate in the physiological and pathological process of abortion, preterm, and delivery. The investigation of Tumour necrosis factor(TNF)-?, interleukin(IL)-1?, nucleus factor (NF)-?B are of high frequency. Their correlation the course of cellular immune response, induction of cell-mediated inflammation, and cell proliferation, transcription activation, apoptosis. Lipopolysacchiride(LPS) is important compose of cell wall of the G negative bacteria, it can activate TNF-??IL-1??NF-?B expression in cells. We detected the expression of progesterone receptor(PR), Tumour necrosis factor(TNF)-?, interleukin(IL)-1?and nucleus factor (NF)-?B in deciduas of patients with spontaneous abortion, the result had distinct difference compare with normal pregnancy, we also did original cultivation of women decidual stromal cell(DSC), observed the changes in progesterone receptor and other cytokines when different concentration lipopolysacchiride(LPS) were added into DSC. It becomes more evident that inflammation is one important reason in inducing spontaneous abortion by decreasing progesterone receptor.Our study is divided into three sections. In part one, we study PR mRNA using RT-PCR; we also study PR,TNF-?,IL-1?,NF-?B expression by Western Blot in deciduas of patients with spontaneous abortion and normal pregnancy women. In part two, we built a model of human decidual, cultivated and identified the decidual stromal cell(DSC) from early normal pregnancy women. we added different concentrations of lipopolysacchiride(LPS) into DSC, simulated infection by G negative bacteria, observed the rate of inhibition and ultrastructure by the transmission electron microscopy(TEM). In part three, we added different concentrations of LPS into DSC, extracted DNA and used RT-PCR to study the difference in PR?TNF-??IL-1?NF-?B mRNA expression. Meanwhile we extracted protein, studied PR and NF-?B protein expression differences using western blot. Therefor we further confirmed that LPS caused an increase in inflammatory cytokines and PR reduction in the unexplained recurrent spontaneous abortion. Part oneStudy of the changing of PR during spontaneous abortion Objective:To observe the differences in the expression of PR?TNF-?IL-1??NF-?B in deciduas of patients with spontaneous abortion and normal pregnancy women.Methods:we collected decidua tissues of 30 cases of patients with natural abortion as the study group, and induced abortion as the control group. we extracted the DNA from the decidua tissues, tested the quantity of mRNA of PR using the reverse transcription-PCR method. And immunohistochemistry in site was used to observe the difference in the expression of PR?TNF-?IL-1??NF-?B protein in deciduas of patients and normal pregnancy. Analyse the relationship between PR and TNF-??IL-1??NF-?B, and the influence of PR on pregnancy.Results:1. The expression of PR mRNA in study group was significant lower than control group(P<0.05).2. Compared the study group with the control, the percentage of positive staining cases and the mean of staining score of PR were lower in study group than that of control group, and there was no significant difference in the percentage of positive staining cases(P>0.05). Yet there was a significant difference in the mean of staining score between study group and control group in the stromal cells of decidua(P<0.05);3. Compared with the control group, the percentage of positive staining cases and the mean of staining score of TNF-??IL-1?and NF-?B were higher in study group,and there was a significant difference between the study group and the control group in the stromal cells of decidua(P<0.05);4. The correlation analysis among TNF-?, IL-1?, NF-?B and PR showed a negative correlation between NF-?B and PR in the cells of decidua in study group (r=-0.628, P<0.05), and TNF-?and IL-1?has a positive correlation with NF-?B (r=0.410, P<0.05; r= 0.501, P<0.05).Conclusions:1. Compared with the normal pregnancy, the expression of PR mRNA was reduced in the spontaneous abortion;2. Compared with the normal pregnancy, the expression of PR protein was reduced in the spontaneous abortion;3. TNF-?, IL-1?, and NF-?B protein expression increased in the spontaneous abortion;4. a negative correlation between NF-?B and PR in the spontaneous abortion is expressed;Part TwoThe study of LPS cause of damage in Decidual In VitroObjective: To cultivate and identify the original decidual stromal cell(DSC), build a successful model of human decidual in vitro, add different concentrations of lipopolysacchiride(LPS), simulate the infection of Gram-negative bacteria during pregnancy, and at last study the inhibition of LPS to the DSC cultivated in vitro.Methods:We obtained fresh decidua at about 6-8 weeks of pregnancy, treated the tissues through enzyme digestion and Percoll density centrifugation culture total deciduas cells and purified the cells through nature proliferation. We tested the expression of PRL in the decidual stromal cell(DSC) using immunecytochemistry stain and immunofluorescence confirmation the DSC, and thus built a model of human DSC in vitro. Then we added different concentrations of lipopolysacchiride(LPS) into the model, setting the final concentrations as 25ng/ml,50ng/ml,100ng/ml, 200ng/ml. After cultivation of 24h,48h,72h, we test the inhibition ratio of the infected cells with MTT method, observe structural changes in the cells through light microscope and transmission electron microscopy(TEM).Results:We built a successful model of human decidual, different components of decidual cells were visible and most of them were decidual stromal cell. The cells were fusiform or in irregular star shapes, with the nucleus big and round at the centre of cell. Used immunecytochemistry stain and immunofluorescence method have proven 90% of the cells to be PRL positive, which shows that DSC was highly purified, and that their secretion functions remain. After the addition of different concentrations of LPS, we tested the inhibition ratio of the cells by the MTT method and found that in 24 hours, compared to the control group, the different concentrations of LPS had no obvious effect on the inhibition of the cells. P was over 0.05 and not statistically significant. Yet the ratio of inhibition showed a significant difference in 48h and 72h. The trial group with high concentration of LPS (200 ng/ml) showed an inhibition ratio of 16.88%, significantly higher than that of a low concentration group (25 ng/ml) by 3.96%. Under the light microscope, the effect of the low concentration of LPS to the cells were almost invisible, while that of high concentration LPS showed a slight change in DSC. Under the TEM, the synapses of the cells in the control group were clearly visible, with little microvilli, evenly distributed chromatin, and complete nucleolus and nuclear membrane; while in the trial group the size of the cells shrank, surface projection increased, the amount of vacuole in cytoplasm increased, and the mitochondria swelled, along with other signs of cell apoptosis. And with the increase in the concentration of LPS, the cytoplasmic organelle damage became more evident: cell condensation, cytoplasmic edema, microvilli overgrowth, nucleus condensation, and nuclear membrane rupture all appear.Conclusions:1. We have successfully cultivated original decidual stromal cell.2. With different concentrations of LPS acted on cultured DSC for 48h and 72h, the inhibition ratio significantly increased, and the concentration contributed to the ratio. 3. Different concentrations of LPS can cause the DSC to go through ultrastructural change, and with the increase in LPS concentration, the cytoplasmic organelle damage gradually aggravates.Part ThreeStudy On the Influence of LPS on PR(?TNF-?IL-1??NF-?B)of The Decidual Stromal CellObjective:To investigate the difference in the expression of the PR and cytokines of cultivated DSC infected by LPS.Methods:We took third generation DSC, added different concentrations of LPS, setting the final concentrations as below: control group(0ng/ml), study group 1 (25ng/ml), study group 2 (50ng/ml), study group 3 (100ng/ml), study group 4 (200ng/ml).48 hours later we used the RT-PCR to test the mRNA of PR?TNF-?IL-1??NF-?B; and then the Western Blot to test the protein of PR?NF-?B influenced by LPS.Results:1. Different concentrations of LPS induced mRNA of PR to show an obvious trend of degression. Compared with control group, PR mRNA of study group1,2,3 have decreased, but not distinctively (P>0.05); a distinct reduction was shown in PR mRNA of study group 4 compare with control group (P<0.05), as well as with study group 1,2,3.2. Different concentrations of LPS induced the mRNA of TNF-??IL-1?and NF-?B to show a distinct increase in abundance(P<0.05). A slight increase is seen between the study groups, but nothing distinct(P>0.05).3. The PR protein expression of DSC cells in study group was obviously lower than that of control (P<0.05), and with the rising of LPS concentration, the PR protein expression decreases gradually; The NF-?B protein expression of DSC cells in study group was obviously higher than that of control (P<0.05) and with the rising of LPS concentration, the NF-?B protein expression gradually increases.Conclusions:1. Different concentrations of LPS induced PR mRNA to show an obvious degression.2. Different concentrations of LPS induce TNF-?IL-1?and NF-?B mRNA to all show a distinct trend of increase.3. With the rising of the LPS concentration, the PR protein of DSC cells gradually decreases while NF-?B protein expression increases.