Edta anticoagulant tube

Approximately 5 ml venous blood samples were collected from the study participants in EDTA-anticoagulant tubes (BD Biosciences) and centrifuged at 1,520 × g for 5 min at 4°C. The plasma samples were transferred into RNase/DNase-free tubes and frozen at −80°C for miRNA extraction. For conventional tumor marker determination, the plasma samples were separated and kept at −20°C until assayed.

A total of 12 healthy volunteers with good periodontal health (37.5±6.3 years) with no known periodontal pocket formation or attachment loss, no alveolar bone resorption and no systemic disease were selected from the Second Affiliated Hospital of Jinzhou Medical University from September 2017 to December 2017. Additional exclusion criteria were presence of gingivitis, periodontal disease, orthodontic treatment or a history of long-term use of antibiotics or other drugs. All subjects participating in the experiment were subjected to periodontal examination by the same dentist. The subjects clearly understood the purpose of the experiment, agreed to participate and provided written informed consent. The collection of specimens was approved by the Ethics Committee of the Second Affiliated Hospital of Jinzhou Medical University (Jinzhou, China). A total of 10 ml venous blood was collected from each volunteer and stored in EDTA anticoagulant tubes (BD Biosciences, San Jose, CA, USA). PBMCs were isolated by the Ficoll-Paque density gradient centrifugation method (650 × g; 18°C for 20 min) and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). All cells were grown in an incubator containing 5% CO₂ at 37°C in 100% humidity.