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.