AFP: alpha-fetoprotein; BCLC: Barcelona Clinic Liver Cancer (BCLC) staging system; HCC: hepatocellular carcinoma; GMPs-TACE, transarterial chemoembolization with gelatin sponge microparticles. CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease.
2.2 The standardized GSMs-TACE
The enrolled patients were treated with GSMs-TACE. The right femoral artery was punctured by Seldinger method and RH hepatic duct was introduced. Celiac arteriography and common hepatic arteriography were performed routinely. All the blood supply arteries of the tumors were identified according to the location, size and staining integrity of the tumors, combined with imaging data, and the auxiliary angiograms of the diaphragm artery, superior mesenteric artery, right renal artery, intercostal artery, left gastric artery, and internal thoracic arteries to determine whether there was a collateral blood supply artery to the tumor. Microparticles of different sizes (150μm, 350-560 μm and 560-710 μm) were selected during the procedure according to the tumor size and degree of staining. For patients with large tumors (diameter greater than 5 cm) and abundant nutrient arteries, GSMs of 350 or 560 μm in diameter were chosen; for patients with small tumors (diameter less than 5 cm) with no abundant blood supply arteries, GSMs of 150 μm in diameter were selected as the embolic agent. The appropriate dose of epirubicin (30-50 mg) was decided according to the tumor volume, and the dose of GSMs embolic agent (50-200 mg) was selected according to the tumor volume and degree of staining. Epirubicin was diluted with 50-100 mL of saline and then mixed with embolic agents of GSMs evenly. When the GSMs achieved a uniform and sparse suspension in epirubicin dilution, the mixture was used to slowly embolize the feeding artery of the tumor until the tumor staining disappeared completely, and the embolization usually lasted for 20-30 min.
2.3 Collection and treatment of blood sample
All patients signed the informed consent. Functions of liver and kidney, blood test and AFP were routinely examined 1 day before intervention and 4, 10, 20 and 30 days after surgery. Upper abdominal CT plain scan was performed 4 days after operation, and enhanced CT or MRI was performed 30 days after operation. 2 mL forearm venous blood was collected from all patients with HCC 1 day before operation, 10 days and 30 days after operation using blood collection tube treated with heparin sodium anticoagulant. Flow cytometry was used to determine Treg cell proportion in peripheral blood. 2 ml of forearm venous blood was also collected from the control group for the determination of Treg cell counts in peripheral blood, and the data were collected for statistical analysis.
2.4 Blood preparation and determination of Treg cell proportion by flow cytometry
(1) Venipuncture blood was collected using anticoagulant tube;
(2) 100 mL whole blood was added to the bottom of the dry powder reagent tube which has antibodies of CD25/CD4/CD127/CD3 in the bottom;
(3) the tube was vortexed for 0.5-1 s to mix well and incubated for 15 min at room temperature with light avoided;
(4) 500 μL of erythrocyte lysate was added to the tube. The tube was then incubated for 15 min at room temperature with light avoided;
(5) 2 ml PBS solution was added to each tube, and then oscillate the tube. Then the mixture was centrifuged at room temperature for 5 min with a centrifugal force of 300 g;
(6) the supernatant was removed, and then step 5 was repeated for one more time.
(7) the supernatant was removed, and then 500 mL PBS solution was added to each tube. The sample was then used for flow cytometry within 1 h;
(8) the gating strategy of flow cytometry was set as follows: Treg: cell population of ①CD4+ and CD3+; ②CD25+and CD127+ low.
2.5 Statistical analysis
The statistical analysis was conducted by SPSS software (provided by IBM, version 20.0). Results were expressed as mean ± SD. The ttest was used to compare the data of different groups. The test level was α=0.05, and P < 0.05 was considered statistically significant.