16 pregnant women with singleton pregnancies from 28 to 36+6 weeks from the first affiliated hospital of Chongqing medical university were enrolled and divided into PL group and TL group with informed written consent. PL and TL were defined according to the guidelines of the American College of Obstetricians and Gynecologists (ACOG). Pregnant women who suffered preterm labor with infection and pregnancy complications such as pregnancy hypertension, diabetes, placental abruption or chronic diseases were excluded. The basic characteristic of these patients was presented in Table 1. Human FMs were collected within 30 minutes after delivery. These samples were stored as required for Weston blot, real-time quantitative PCR (qPCR) and Immunohistochemistry. Ethics approval was gained from the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (2019-137).
Human fetal membranes could secret chemokines recruiting T cells. Therefore, human amniotic cell lineage was used as the targeting cells in the present study. Human amniotic cell lineage—WISH (Wistar Institute Susan Hayflick) cell line was purchased from the commercialized company. They were cultivated in MEM (Gibco, USA) with 10% fetal bovine serum (PAN, Germany), 1% nonessential Amino Acid Solution, 100µg/ml streptomycin and 100 U/ml penicillin (Beyotime Biotechnology, Shanghai, China). Cells were kept in a wetted 5% CO2 atmosphere at 37 oC and the medium was changed every two days.
Extraction of total RNA and qPCR test
Total RNA was extracted by RNAiso Plus (Takara Bio Inc., Tokyo, Japan) and quantified by measuring the ratio of A 260nm/A 280nm. Total RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara Bio Inc., Tokyo, Japan). Thereafter, generated cDNA was subjected to qPCR analysis using SYBR Premix Ex Taq II kit (MCE, Shanghai, China) with specific primers (Table 2). Amplification and detection of mRNA was performed using Thermal Cycler Dice Real Time System (TP800; Takara Bio, Shiga, Japan). Relative quantity of target gene expression to β-actin gene was measured by comparative Ct method as described previously.
Total protein was harvested from the tissue by RIPA lysis buffer (ZSGB-BIO, Beijing, China) containing PMSF (99:1). Equal amounts of protein (40 μg) were electrophoresed on 10% SDS-polyacrylamide gels (Invitrogen) and blotted onto PVDF membranes. The membranes were incubated overnight with primary antibodies (IL-27Rα(1:1000; Affinity Biosciences, USA); Janus kinase 2 (JAK2), P-JAK2(Tyr1007, (1:1000; Affinity Biosciences, USA)), signal transducer and activator of transcription 1 (STAT1), p-STAT (Tyr701, (1:1000; Affinity Biosciences, USA)), STAT3, p-STAT3 (Tyr705,(1:1000; Affinity Biosciences, USA)) after blocked in 5% nonfat milk for 2 h; Then, the membranes were incubated with an HRP-conjugated anti-IgG secondary antibody, followed by detection with an ECL chemiluminescent detection system. The blots were imaged and quantified using ImageJ software, and the results were reported as primary antibody/β-actin ratio.
Hematoxylin and eosin (H& E) staining and immunohistochemistry (IHC)
Fetal membrane tissues were formalin fixed, and paraffin-embedded sections were stained with hematoxylin and eosin. IHC was used to localize IL-27Rα+ and T-bet+ cells within the fetal membranes. Paraffin sections of human FMs were dewaxed and rehydrated, followed by microwave antigen retrieval. Then, these sections were blocked with 3% H2O2 followed by 10% normal goat serum. Following that, these tissue sections were incubated with primary antibodies overnight at 4 centigrade (IL-27Rα, (1:200, santa, China); T-bet, (1:300, protein tech, China)). After thorough washes, they were incubated with biotinylated goat anti-mouse/Rabbit IgG. Positive antibody binding was detected with diaminobenzidine, followed by staining with hemotoxylin (Sigma).
Supernatant of cells culture were harvested and stored at -80°C until analyzed by Elisa Assay. Frozen human FMs were thawed on ice and solubilized in ice-cold PBS containing protease inhibitors PMSF. Samples were centrifuged at 5,000 g for 10 min at 4°C, and supernatant was stored at -80°C until analyzed by Elisa Assay. The total protein of human FMs homogenates was determined using Bradford’s reagent (Bio-Rad Protein Assay; Bio-Rad) according to the manufacturer’s instructions. Both the supernatant of cells culture and FMs tissue homogenate were measured by a human CCL2, CXCL9, CXCL10, and CXCL11 Elisa kit (Jiubang, China). All the experiments were conducted according to the manufacturer’s instructions. The absorbance of the supernatant was assessed at 450nm in a Multi-skan GO plate reader (Thermo Fisher Scientific, Waltham, MA, USA).
Statistical analysis was performed by prism soft-ware. Student’s t test or Mann–Whitney U test were used to assess continuous variables according to its distribution. Chi-square test were used to assess categorical variables. A p value< 0.05 was considered statistically significant.