Subjects And Methods
Patients
2.1.1Peripheral blood collection
The research objects obtained from PE patients who were admitted from January 2014 to December 2017 to the obstetrics of public hospitals in Qingdao, Jinan, Binzhou and other regions. We used R i386 3.4.3 software to select 1138 cases as the case group and 1342 cases as the control group from PE sample bank, to explore the possible relationship between the SNP site Val166Met in BDNF and the occurrence of PE. At the same time, screening 200 pairs pregnant women with PE and their fetuses and 208 pairs healthy pregnant women and their fetus from pregnant women who admitted to the obstetrics department of Qingdao University Affiliated Hospital from January 2018 to June 2018. Collecting their peripheral blood to proceed the study of maternal-fetus paring.
Case group follow the diagnosis criteria of PE that were SBP≥140mmHg, DBP≥90mmHg, proteinuria ≥300mg/24h appearing after 20 gestation weeks15. Besides, cases group excluded following situations: gestational diabetes mellitus, thyroid disease, connective tissue diseases, hepatic and renal dysfunction and other complications and required the age of PE women over 25. The enrollment criteria of healthy control are as follows: (1) age≥25 (2) gestation weeks ≥30 (3) normal blood pressure during pregnancy and no history of hypertension (4) normal result of blood and urine tests and normal hepatorenal function during the pregnancy (5) There is no other complications and some pathological conditions like placenta implantation, placenta previa, placental abruption, premature rupture of membranes and so on. This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University and obtained the written informed consent from all participants.
2.1.2 Placenta collection
We screened the 21 PE patients and 21 healthy pregnant women who admitted to the Affiliated Hospital of Qingdao University from June 2017 to June 2018 to evaluate the expression level of BDNF. Placental tissues were obtained from normal or PE pregnancies immediately after cesarean section. Square segments (estimate as 1×1×1 cm) were removed from maternal side and avoided parts of blood clots and infarction. The tissues were rinsed with saline, quickly snap-frozen in liquid nitrogen, and stored at -80 °C for further processing.
  1. Taqman real-time fluorescence quantitative PCR
  2. Use whole blood genomic DNA extraction kit to extract the peripheral blood DNA, then detect the DNA concentration and purity by ultraviolet spectrophotometer. Use designed probes to perform the fluorescence quantitative polymerase chain reaction (PCR). The PCR amplification volume is a total of 7ul, including 1 ul template DNA, 0.05 ul 20× Taqman probe stock solution, 4.2 ul autoclaved deionized water, 1.75ul 2×MIX. Amplification process using TIB-8600 fluorescence quantitative PCR instrument: denaturation at 95℃ for 10min, annealing at 95℃ for 15s, extension at 60℃ for 1min, 45 cycles, reading results after extension at 60℃. Performing the sequencing test to verify the accuracy of Taqman probe real-time PCR results.