| Preeclampsia is a common hypertensive disorder in pregnancy and is one of theimportant reasons for the mortality and morbidity of maternal and perinatal infant.The pathogenesis of preeclampsia remains unclear and mainly includes geneticdefects, placenta micro environmental damage and immune abnormalities, etc. Theimmunological and genetic mechanisms are playing a crucial role in PE pathogenesisand have drawn increasing attention.Normal pregnancy requires formation immune tolerance between the maternaland the fetus. The pregnant success depends on the the immune micro environmentalbalance and maintenance of immune tolerance. When there is imbalance, the immunetolerance is broken, the immune rejection response is induced and may lead to theoccurrence of preeclampsia. As main effector cells, T cells play important roles in theimmune response. CD4+CD25+Foxp3+T cell is a kind of T cell subset withregulatory function and specific phenotype.Treg cells are defined by the expression of forkhead box protein3(Foxp3), thegene of transcription factor maps to the short arm of X chromosome (Xp11.23). Tregcells have immunosuppressive function and play an important role in maintaining thestability of autoimmunity. Foxp3is a key switch molecule in the development andimmune regulation of Treg cells. The research confirmed that Foxp3gene mutationscould lead to CD4+CD25+Treg function change, which was associated with a varietyof autoimmune diseases, inflammation, and tumor development. It was demonstratedin mouse models that inactivation of Foxp3results in a deficiency of Tregs anddeficiency of Foxp3might impair the suppressive function of Tregs. Several previousstudies found that the Foxp3gene polymorphisms was associated with theautoimmune diseases, such as systemic lupus erythematosus (SLE), autoimmunethyroid diseases (AITDs), type I diabetes (TID), and allergic rhinitis. Therefore,evaluation of such polymorphisms may predict the susceptibility to certain diseases. Decreased numbers of circulating Tregs have been observed in women withpregnancy complications, including recurrent pregnancy loss and PE. In thedevelopment of preeclampsia, it is not clear whether Foxp3gene mutations affect thematernal-fetal immune function and placenta immune microenvironment. Theassociation between Foxp3polymorphisms and PE has not been reported in HanChinese so far. Hence, it is necessary to investigate the effect of Foxp3polymorphisms on the susceptibility of PE in different populations. Our hypothesis isthat Foxp3genetic heterogeneity determines the individual differences withpreeclampsia. It means that Foxp3gene polymorphism may decide the susceptibilityof preeclampsia.The study is helpful for us to explore the role of Foxp3genepolymorphism in preeclampsia, and provide new ideas for the research on thepathogenesis of preeclampsia.TGF-β1is mainly produced by T lymphocytes, and is an important cytokine andimmunomodulatory factor involved in regulating cell growth and differentiation andmaintaining self-tolerance and T-cell homeostasis. The reaearch showed that theamount of TGF-β1on fetal growth and development was nessersary. TGF-β1wassynthesized and secretoried in the placenta and played an important role on embryogrowth. It was a very complicated molecular mechanism that TGF-β1involved inoccurrence of preeclampsia.The abnormal expression of TGF-β1could limit totrophoblastic invasion the lining of the uterus and uterine placental blood flow,resulting in shallow implant of trophoblast cells and abnormally high impedance ofthe uterine placental vascular, formating the pathophysiological changes ofpreeclampsia, causing the incidence of preeclampsia.TGF-β1polymorphism mightaffect the level of serum TGF-β1, the latest research showed that TGF-β1genemutation was a risk factor for preeclampsia in Dutch and Korean, but it was notreported in China. There are the genetic variations in different geography andethnicity, it is unclear to wheather it is TGF-β1gene mutations may involve in theoccurrence of preeclampsia in China.IL-17is a pro-inflammatory cytokine from Th17cells, can induce expression of avariety of cytokines such as IL-6, IL-23, tumor necrosis factor-α, and activation of acute phase proteins and induces matrix metalloproteinases (metalloproteinases,MMPs) synthesis, and may be involved in the pathogenesis of some autoimmunediseases. It was believed that IL-17might involved in embryo implantation andtrophoblast invasion process and suggested that Th17might be play a role duringpregnancy. Studies showed that IL-17concentrations in serum were significantlyincreased in patients with atherosclerosis, angina and myocardial infarction and othercardiovascular diseases. The latest research found that the IL-17and other Th17family cytokines were closely related to preterm, premature rupture of membranesand intrauterine infection, but changes of IL-17expression level in patients withpreeclampsia remain unclear.What are the roles of Treg cells and Foxp3, TGF-β1and IL-17molecules in thepathogenesis of preeclampsia? whether Foxp3and TGF-β1gene polymorphism lociare risk factors for preeclampsia, whether the genes of Foxp3and TGF-β1are thesusceptibility genes of preeclampsia, are worthy of the further research and explore, inorder to provide theoretical basis and experimental basis for the prediction andprevention of preeclampsia. Part Ⅰ The Levels of CD4+CD25+Treg Cells and TGF-β1in Preeclampsia PatientsObjective To detect CD4+CD25+Treg cells and TGF-β1expression levels in patientwith preeclampsia and explore their roles in the pathophysiological process ofpreeclampsia and provide experimental basis for immune pathogenesis ofpreeclampsia.Methods68cases of patients with preeclampsia(33mild preeclampsia and35severe preeclampsia) and30cases of normal pregnant women were Chose fromOctober2011to December2012in guangzhou severe maternal treatment center andguangzhou medical university affiliated third hospital. Preeclampsia diagnosticcriteria was in accordance with national textbooks《Obstetrics and Gynecology》7thEdition. An average age of the patients was29.8±5.4years, average gestational weekwas34.8±3.6weeks. The medical complications and gestational diabetes wereexcluded out in all cases, there were no differences in age, production times andpregnancy body mass index between normal control group and preeclampsia, allpatients were untreated, no blood transfusion and immunosuppressant therapy. Tregcells were detected by Flow cytometry, TGF-β1levels in serum were tested by ELISA.The experimental datum were expressed as mean±standard deviation (X±s) andanalyzed by SPSS13.0statistical software, a single factor analysis of variance andspearson linear correlation analysis method were used to deal with experimentaldatum of the groups, inspection standard for a=0.05.Results CD4+CD25+Treg cell ratio in peripheral blood of mild preeclampsia andsevere preeclampsia were6.34%±0.98%and4.31%±0.85%respectively, the ratioswere significantly lower than those in normal pregnant women (12.38%±2.63%,P<0.05). TGF-β1levels in peripheral blood of mild preeclampsia and severepreeclampsia were112.63±24.43ng/mL and134.72±34.86ng/mL respectively, thelevels were significantly higher than that in control group（87.67±21.24ng/mL）,P <0.05, and TGF-β1levels rised to accompany with the increase of the illness severity.Conclusion Treg cell ratio in peripheral blood of patients with preeclampsia wassignificantly decreased, TGF-β1levels increased significantly, Treg cells and TGF-β1may play an important role in the pathogenesis of preeclampsia. Part Ⅱ The Effects of FOXP3, TGF-β1and IL-17in the Pathogenesis of PreeclampsiaObjective To detect the TGF-β1, Foxp3and IL-17expression levels in the placentatissue with preeclampsia and explore the role of the molecules in the pathogenesis ofpreeclampsia, provide experimental basis for probing new treatment strategies ofpreeclampsia.Methods The object of study, the inclusion criteria and exclusion criteria were sameas part Ⅰ. Allthe conner was told that the purpose of collecting the placenta andsigned informed consent, after approved by ethics committee of guangzhou medicaluniversity affiliated third hospital, the clinical data and specimens from all cases werecollected. The placenta tissue were selected immediately after operation from thecentral zone in the placenta maternal side, all specimens were thoroughly washed withnormal saline and fixed in10%neutral formalin solution24hours at least, embeddedin paraffin and4μm by serial sections. TGF-β1, Foxp3and IL-17expression levelswere assay by immunohistochemical method. Positive cells were as followingrequirements: microscopic tissue structure was clear, positive particles was correctlylocated, staining was significantly higher than background. Positive results criteriawere that the cytoplasm or tissue were stained brown, the result was determined bysemi-quantitative integration method, based on the proportion of positive cells perslice and colored shades scoring. The experimental datum were analyzed by SPSS13.0statistical software, a single factor analysis of variance and spearson linear correlationanalysis method were used to deal with experimental datum of the groups, inspectionstandard for a=0.05.Results TGF-β1positive expression rates in placenta of mild preeclampsia and severepreeclampsia were75.76%and85.7%respectively, the positive rates weresignificantly higher than those in normal pregnant women (36.67%, P <0.01). Foxp3positive expression rates in placenta of mild preeclampsia and severe preeclampsiawere51.52%and28.57%respectively, the rates were significantly lower than that inthe control group (86.67%, P<0.05). IL-17positive expression rates in placenta of mild preeclampsia and severe preeclampsia were81.82%and91.42%respectively,the rates were significantly higher than that in the control group (40%), P<0.05.IL-17and TGF-β1expression were negatively correlated with the weight of theplacenta and fetus, Foxp3expression were positively correlated with the weight of theplacenta and fetus.Conclusions1. The results that the levels of Foxp3decreased and levels of IL-17increased inplacenta tissue of preeclampsia suggested that the unbalance of Th17/Treg ratio mayplay a role in pathogenesis of preeclampsia, which can lead to immunosuppressionfunction abate, inflammation, cause imbalance of maternal-fetal immune tolerance,and affect the growth of the placenta and fetus.2. TGF-β1expression level in placenta of preeclampsia significantly increased andwas significantly positively correlated with the degree of illness, TGF-β1may mainlybe produced by the placenta trophocyte, abnormally high expression of TGF-β1maymainly play the roles of proinflammation and affection vascular endothelial cells, leadto the blood pressure increased and participate in the pathological process ofpreeclampsia. Part Ⅲ The Role of TGF-β1Gene Polymorphismin the Preeclampsia SusceptibilityObjective To detect TGF-β1gene locus polymorphism and explore the relationshipbetween TGF-β1gene polymorphism and hereditary susceptibility in the preeclampsia,to clarify TGF-β1molecular mechanism in the preeclampsia.Methods The source of the research object, the inclusion criteria, exclusion criteriaand collection time were same as part Ⅰ.156cases of patients with preeclampsia and252cases of normal pregnant women were chose, an average age of the patients was28.9±6.1years, average gestational time was35.7±4.3weeks, the average gestationaltime of normal pregnancy control group was38.6±2.9weeks. All conner was toldthat the purpose of collecting the blood and signed informed consent, after approvedby ethics committee of guangzhou medical university affiliated third hospital. theperipheral blood specimens were collected and the leukocytes were separated, thegenomic DNA was extracted by phenol-chloroform method, the primers weredesigned, TGF-β1-509C/T and+869T/C gene loci polymorphism of the preeclampsiagroup and normal control group were measured by PCR-RFLP and sequence analysis,TGF-β1levels of the preeclampsia group and control group in serum were measuredby ELISA, the experimental datum were analyzed by SPSS13.0.Results The genotypes of TGF-β1+869T/C were CC, CT and TT, the theirfrequencies in preeclampsia group were33.33%,62.18%and4.49%respectively, thegenotype frequencies of the normal control group were13.09%,48.41%and13.09%respectively, CC and CT genotype frequencies in preeclampsia group weresignificantly higher than that in control group, while the TT genotype frequency ofwas significantly lower than that in control group, P <0.01. C allele frequency(64.42%) in preeclampsia group was significantly higher than that in control group(37.30%), T allele frequency was significantly lower than that in the normal controlgroup, P <0.01. CC, CT and TT genotypes were related to TGF-β1levels in serum.CC, CT and TT genotype frequencies of TGF-β1-509in preeclampsia group were11.54%,52.57%and35.89%respectively, the genotype frequencies in the normalgroup were13.89％、46.82％39.29％respectively, there were no significant difference between preeclampsia group and control group, P>0.05. CC,CT and TTgenotype were not related to TGF-β1levels in serum.TGF-β1+869CT/-509CT, TGF-β1+869CC/-509CT and TGF-β1+869CC/-509TT alliance genotype frequencies in preeclampsia group were significantly higherthan that in normal pregnancy group, P <0.05. The TGF-β1+869TT/-509CC, TGF-β1+869TT/-509CT and TGF-β1+869TT/-509TT alliance genotype frequencies inpreeclampsia group were significantly lower than that in normal pregnancy group,P <0.01.Conclusion1. CC and CT genotype frequencies of TGF-β1+869T/C in preeclampsia gourpwas significantly higher, TT genotype frequency was significantly decreased, C allelesignificantly increased and the T allele was significantly decreased, TGF-β1+869T/Cpolymorphism may be a risk factor for preeclampsia occurance in Guangdong Hanpopulation of China.2. There were no significant differences of TGF-β1-509C/T genotype and allelefrequencies between preeclampsia group and control group, TGF-β1-509C/T may beindependent risk factor for the preeclampsia, but as combined with TGF-β1+869T/C,alliance genotype can significantly increase the risk of preeclampsia and induce theoccurrence of preeclampsia. Part Ⅳ The Role of Foxp3Gene Polymorphismin Preeclampsia SusceptibilityObjective To detect Foxp3gene loci polymorphism and analyze the relationshipbetween Foxp3gene polymorphism and hereditary susceptibility in the preeclampsia,to elucidate Foxp3pathogenesis in the preeclampsia.Methods The source of the research object, the inclusion criteria, exclusion criteriaand collection time were same as part Ⅲ.The peripheral blood specimens werecollected and leukocytes were separated, the genomic DNA was extracted by phenol-chloroform method, the primers were designed and synthesized, Foxp3-6054(del/ATT, rs5902434) gene locus,-3279(A/C, rs376158) locus and-924(A/G, rs2232365)locus polymorphism were measured by PCR-ARMS (amplification refractorymutation system) and sequence analysis in the preeclampsia group and normalcontrol group, the genotype and allele frequencies of Foxp3-6054,-3279and-924were analyzed statistically by SPSS13.0.Results TT genotype frequency of Foxp3-6054was significantly decreased in patientswith preeclampsia, P<0.01, TT relative risk values (RR) and95%confidence intervals(95%CI) were0.734and0.562～0.960respectively. The genotype frequencies ofFoxp3-6054were analyzed by Logistic regression analysis. The results showed thatalliance genotype AT+AA significantly increased the risk of preeclampsia, relativerisk RR was1.206,95%CI was1.035-1.405.GG, GA and AA genotypic frequencies of Foxp3-924in preeclampsia group were0.4019,0.4579and0.1402respectively, G and A allele frequencies were0.6308and0.3691respetively, there were no significant differences in genotypes and alleles ofFoxp3-924between preeclampsia and control group, P>0.05. AA, CA, CC genotypicfrequencies of Foxp-3279in preeclampsia group were0.0128,0.2564and0.7308respectively. C and A allele frequencies were0.8589and0.1411respetively, therewere no significant differences in genotypes and alleles of Foxp3-3279betweenpreeclampsia group and control group, P>0.05.Foxp3-6054AA/-3279CC alliance genotypic frequencies in preeclampsia group was significantly higher than that in the normal control group, P <0.01. Foxp3-6054TT/-3279CC alliance genotype in preeclampsia group was significantly lower than thenormal control group, P <0.01. There are no significant differences of Foxp3-6054/-924and Foxp3-3279/-924alliance genotypes betwwen preeclampsia group andcontrol group, P>0.05.Foxp3-6054AA/TGF-β1+869CC、 Foxp3-6054AT/TGF-β1+869CC、Foxp3-6054AT/TGF-β1+869CT and Foxp3-6054TT/TGF-β1+869CC alliancegenotypes in preeclampsia significantly higher than those in control group, P <0.05.Conclusion1. The TT genotype frequencies of the Foxp3-6054in the preeclampsia wassignificantly decreased, TT frequency decline may be a risk factor of preeclampsia.Foxp3-6054locus mutation may be participate in the occurance of preeclampsia.2. There were no significant differences of Foxp3-924and Foxp3-3279genepolymorphism between preeclampsia group and control group, two locus may beindependent risk factors.3. There are significant differences of Foxp3-6054AA/-3279CC andFoxp3-6054TT/-3279CC alliance genotypes between preeclampsia group and controlgroup, these alliance genotypes may be susceptibility genotypes for preeclampsia.4. Interaction between TGF-β1and Foxp3different loci may form a high-riskgenotypes inducing onset of preeclampsia. The research suggests that alliancegenotypes may be preeclampsia susceptible factor and preeclampsia may be caused bythe multiple genes coaction. |
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