OBJECTIVE: To investigate the effects of removing microglia from spinal cord on nerve repair and functional recovery after spinal cord injury (SCI) in mice. METHODS: Thirty-nine 6-week-old female C57BL/6 mice were randomly divided into control group ( n=12), SCI group ( n=12), and PLX3397+SCI group ( n=15). The PLX3397+SCI group received continuous feeding of PLX3397, a colony-stimulating factor 1 receptor inhibitor, while the other two groups were fed a standard diet. After 14 days, both the SCI group and the PLX3397+SCI group were tested for ionized calcium binding adapter molecule 1 (Iba1) to confirm that the PLX3397+SCI group had completely depleted the spinal cord microglia. The SCI model was then prepared by clamping the spinal cord in both the SCI group and the PLX3397+SCI group, while the control group underwent laminectomy. Preoperatively and at 1, 3, 7, 14, 21, and 28 days postoperatively, the Basso Mouse Scale (BMS) was used to assess the hind limb function of mice in each group. At 28 days, a footprint test was conducted to observe the gait of the mice. After SCI, spinal cord tissue from the injury site was taken, and Iba1 immunofluorescence staining was performed at 7 days to observe the aggregation and proliferation of microglia in the spinal cord. HE staining was used to observe the formation of glial scars at the injury site at 28 days; glial fibrillary acidic protein (GFAP) immunofluorescence staining was applied to astrocytes to assess the extent of the injured area; neuronal nuclei antigen (NeuN) immunofluorescence staining was used to evaluate neuronal survival. And 5-hydroxytryptamine (5-HT) immunofluorescence staining was performed to assess axonal survival at 60 days. RESULTS: All mice survived until the end of the experiment. Immunofluorescence staining revealed that the microglia in the spinal cord of the PLX3397+SCI group decreased by more than 95% compared to the control group after 14 days of continuous feeding with PLX3397 ( P<0.05). Compared to the control group, the BMS scores in the PLX3397+SCI group and the SCI group significantly decreased at different time points after SCI ( P<0.05). Moreover, the PLX3397+SCI group showed a further decrease in BMS scores compared to the SCI group, and exhibited a dragging gait. The differences between the two groups were significant at 14, 21, and 28 days ( P<0.05). HE staining at 28 days revealed that the SCI group had formed a well-defined and dense gliotic scar, while the PLX3397+SCI group also developed a gliotic scar, but with a more blurred and loose boundary. Immunofluorescence staining revealed that the number of microglia near the injury center at 7 days increased in the SCI group than in the control group, but the difference between groups was not significant ( P>0.05). In contrast, the PLX3397+SCI group showed a significant reduction in microglia compared to both the control and SCI groups ( P<0.05). At 28 days after SCI, the area of spinal cord injury in the PLX3397+SCI group was significantly larger than that in SCI group ( P<0.05); the surviving neurons significantly reduced compared with the control group and SCI group ( P<0.05). The axonal necrosis and retraction at 60 days after SCI were more obvious. CONCLUSION The removal of microglia in the spinal cord aggravate the tissue damage after SCI and affecte the recovery of motor function in mice, suggesting that microglia played a neuroprotective role in SCI.
Animals ; Spinal Cord Injuries/surgery* ; Microglia/pathology* ; Female ; Mice ; Mice, Inbred C57BL ; Nerve Regeneration/drug effects* ; Spinal Cord/pathology* ; Pyrroles/administration & dosage* ; Aminopyridines/administration & dosage* ; Recovery of Function ; Disease Models, Animal ; Calcium-Binding Proteins/metabolism* ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors* ; Microfilament Proteins/metabolism* ; Glial Fibrillary Acidic Protein/metabolism*

ObjectiveTo investigate the current status of cognition and attitude towards hospice care among medical staff in Liaocheng，analyze the related influencing factors，and to provide reference for further development of hospice care services. MethodsUsing the method of convenient sampling，404 medical staff from all levels of hospitals in Liaocheng were selected as the research subjects from January to June 2022 to conduct a questionnaire survey on the cognition and attitude towards hospice care.Statistical methods were used to analyze the related influencing factors. ResultsThe knowledge score of hospice care among medical staff in Liaocheng was （13.02 ± 4.10），with an average score rate of 65.10%.The score of attitude was （38.67 ± 5.64），with an average score rate of 64.50%.Age （41~50 years old），having received education and training，treated or cared for patients in the middle and late stages，and understanding ethics and morality，as well as cultural customs were significantly positively correlated with knowledge scores.Age （> 50 years old），professional title （deputy senior professional title），position （medical treatment），and experience in treating or caring for patients in the middle and late stages were positively related to attitude scores. ConclusionThe cognition and attitude towards hospice care among medical staff in Liaocheng were at a moderate level.Strengthening the construction of a standardized hospice care system is helpful to improve the cognition and attitude level towards hospice care among medical staff.

Objective:To compare the assay and related substance detection methods of erythromycin lactobionate in the Chinese Pharmacopoeia 2020(ChP),USP 2023(USP),JP18(JP),BP2023(BP)and EP 11.0(EP),investigate the differences between the test results obtained from 7 batches of erythromycin lactobionate for injection samples by using ChP and BP methods and thus provide a reference for the improvement of specification of erythromycin lactobionate.Methods:The differences of test methods and limits under the items of assay and related substances of erythromycin lactobionate in the above five pharmacopoeias were listed and compared.The related substances and contents of erythromycin lactobionate for injection samples from different manufactures were tested with methods stated in ChP and BP,and then compared and analyzed.Results:The items of related substances were determined by high performance liquid chromotographic methods in the ChP,EP and BP.The test methods and limits in the EP and BP were the same,which were different from that in ChP.The related substances were not determined in the USP and JP.There are obvious differences between the chromatographic methods and limits for the items of related substances in the ChP and BP,i.e.,BP contains 7 specific impuri-ties,while ChP contains only 2 specific impurities.The limits for any other impurity and the total amount of impurities were lower in the BP than those in the ChP.The antibiotic microbiological assay was used as the test method for the item of assay in the ChP,USP and JP based on different bacterial strains,which was different from the chromatographic method used in the BP.Based on the different methods of the ChP and BP,the related substances and content determination results of 7 batches of erythromycin lactobionate for injection samples met the acceptance criteria.The detection efficiency of BP related substances inspection method for specific impurities and total impurities were significantly higher than that of the ChP method.Conclusion:The BP method is superi-or to the ChP method in the detection of erythromycin lactobionate and erythromycin lactobionate for injection related substances.In terms of content determination,the external standard method adopted by BP is feasible to replace the antibiotic microbiological assay adopted by the ChP.Related substances and content determination methods adopted by the BP can provide an important reference for the revision and improvement of erythromycin lactobionate standard in the ChP.

Objective:To investigate the role of transforming growth factor β1 (TGF-β1) and matrix metalloproteinase (MMP)-2 in pancreatic tissue repair and reconstruction in rats with acute pancreatitis and its potential mechanism.Methods:114 male SD rats were randomly divided into normal control group (CON group) and acute edematous pancreatitis model group (AEP group), acute necrotic pancreatitis model group (ANP group), ANP control group and ANP intervention group. The rat AEP model was constructed by subcutaneous injection of caerulein, and the rat ANP model was prepared by intraperitoneal injection of L-arginine. The ANP intervention group and ANP control group were prepared by intraperitoneal injection of TGF-β1 inhibitor SB431542 or DMSO 30 min before, 24 h and 48 h after pancreatitis induction, respectively. Hydroxyproline content in pancreatic tissue was determined by hydroxyproline kit. The expression of TGF-β1, phosphorylated Smad3 (p-Smad3), type Ⅲ collagen and MMP-2 in pancreatic tissue was detected by immunohistochemical method. The activity of MMP-2 was determined by gelatin enzyme spectrometry. The expression levels of MMP-2 and p-Smad3 proteins in pancreatic tissue were detected by Western blot.Results:The hydroxyproline content in CON group was (61.71±8.56)μg/mg protein. The hydroxyproline content in AEP group reached the peak (116.72±8.53)μg/mg on the 3rd day. The peak value of hydroxyproline content in ANP group was (174.93±11.75)μg/mg on day 5. The peak value in ANP group was significantly higher than that in AEP group, and the peak value of hydroxyproline content in AEP group was significantly higher than that in CON group. The hydroxyproline content at day 3, 5 and 7 in the ANP intervention group was (108.07±10.48)μg/mg, (137.14±8.66)μg/mg and (112.35±13.16)μg/mg, respectively, and that at day 3, 5 and 7 in the ANP control group was (132.35±14.2)μg/mg, (175.43±13.75)μg/mg and (137.92±12.65)μg/mg, respectively. TGF-β1 immunohistochemical peak score in control group, AEP group and ANP group was (0.12±0.03), (1.96±0.21) and (3.00±0.28), respectively. p-Smad3 immunohistochemical peak score was (0.15±0.05), (2.05±0.20), and (3.05±0.24), while type Ⅲ collagen immunohistochemical peak score was (0.11±0.04), (1.56±0.15), and (3.10±0.17). MMP-2 immunohistochemical peak score was (0.05±0.03), (1.45±0.20), and (2.45±0.15), respectively. The immunohistochemical peak scores of TGF-β1, p-Smad3, type Ⅲ collagen and MMP-2 in ANP group were significantly higher than those in AEP group. The immunohistochemical peak scores of TGF-β1, p-Smad3, type Ⅲ collagen and MMP-2 in pancreatic tissue of ANP intervention group and ANP control group were (2.36±0.21), (2.25±0.22), (2.47±0.19), (2.00±0.10) and (3.02±0.21), (3.01±0.19), (3.05±0.24), (2.43±0.11), respectively, which in ANP intervention group was significantly lower than those in the ANP control group. The peak value of MMP-2 activity in pancreatic tissue of CON group, AEP group and ANP group was (10.85±1.73), (85.78±7.16) and (115.43±8.7), respectively, which in ANP group was significantly higher than that in AEP group, and in AEP group was significantly higher than that in CON group. In ANP intervention group and ANP control group 3 and 5 days after molding, the expression levels of MMP-2 protein in pancreas were 0.20±0.01, 1.19±0.02, 0.52±0.01, 1.54±0.05, respectively; p-Smad3 protein expression levels were 0.30±0.04, 0.66±0.11, 1.95±0.05, 1.30±0.01, respectively; and MMP-2 and p-Smad3 in ANP intervention group was significantly lower than those the ANP control group. All the differences among the groups above were statistically significant (all P value <0.001). Conclusions:TGF-β1 and MMP-2 play an important role in tissue remodeling and extracellular matrix deposition after acute pancreatitis inflammation.

Objective: To compare the clinical effects of endoscopic thyroidectomy using a modified gasless transsubclavian approach and the traditional neck approach for unilateral papillary thyroid carcinoma (cN0). Methods: The clinical data of 135 patients with cN0 papillary thyroid carcinoma who underwent unilateral thyroidectomy in the Department of Thyroid Surgery, the First Hospital of Jilin University from October 2020 to November 2022 were retrospectively analyzed. There were 37 males and 98 females, aging (43.2±8.8) years (range: 21 to 59 years). There were 51 cases using the modified gasless transsubclavian approach (TS group) and 84 cases using the traditional neck approach (TN group). Comparative analyses were performed between the operative results of the 2 groups by t-test, Wilcoxon rank sum test, and χ² test. Results: All endoscopic operations were successfully completed without conversion to the traditional neck approach. Compared to the TN group, the TS group had a longer operation time (M(IQR)) (73.5 (22.5) minutes vs. 90.0 (30.0) minutes, Z=-5.831, P<0.01), more postoperative drainage (60 (25) ml vs. 95 (45) ml, Z=-6.275, P<0.01), higher hospitalization costs (22 687 (3 488) yuan vs. 26 652 (2 431) yuan, Z=-6.944, P<0.01), and a higher rate of parathyroid autotransplantation (15.5% (13/84) vs. 60.8% (31/51), χ²=29.651, P<0.01). There was no significant difference in the total exposure rate of the central compartment, postoperative hospitalization time, the number of dissected lymph nodes, the number of metastatic lymph nodes, C-reactive protein ratio before and after operation, and preoperative and postoperative parathyroid hormone (all P>0.05). Conclusions: Endoscopic thyroidectomy using the modified gasless transsubclavian approach is safe for cN0 papillary thyroid carcinoma, with longer operating time, more postoperative drainage, higher hospitalization costs, and moredifficulty in preserving the inferior parathyroid gland in situ compared to traditional open surgery.

Objective:To investigate the effects of NOD-like receptor protein 3(NLRP3) inflammasome activation on the proliferation, migration and extracellular matrix desposition of activated pancreatic stellate cells(PSCs).Methods:The rat PSCs were isolated, cultured and identified, and were divided into control group or LPS group based on the pretreatment with LPS (10 μg/ml for 24 hours) or without. The expression of NLRP3 inflammasome associated molecules in PSCs culture medium was detected by ELISA. The PSCs with NLRP3 inhibition were constructed by shRNA carrying lentivirus infection and were divided into LPS+ negative control group and LPS+ lentivirus group based on whether the cells were treated with LPS and infected by lentivirus or not. The alteration in cell proliferation and migration were detected by CCK-8 kit and transwell chamber method. The expression of extracellular matrix α-SMA and collagen in PSCs was detected by immunofluorescence staining and the expression of TGF-β mRNA was analyzed by RT-qPCR.Results:The cytoplasm of PSCs which were cultured for 24 hours was rich in bright annular lipid droplets, and the cells expressed desmin. After 7 days of culture, the cell became larger in size, the lipid droplets basically disappeared, and the cells were activated and expressed α-SMA. The expression of caspase-1, IL-1β and IL-18 in the supernatant of PSCs culture medium in LPS group were significantly higher than those in control group (1.55±0.04 vs 0.65±0.03), (2.02±0.04 vs 1.05±0.05) and (1.70±0.05 vs 0.97±0.03), respectively. After inhibiting by lentivirus infection, the expression of NLRP3 in the lentivirus group (0.25±0.04) was significantly lower than that in negative control group (0.68±0.05). In control group, LPS group, LPS+ negative control group and LPS+ lentivirus group, the A490 values was 0.61±0.02, 1.15±0.06, 0.96±0.05, and 0.56±0.01, respectively; the migrating PSCs number was (64.12±4.58), (121.67±8.02), (111.67±4.67) and (69.67±8.08)/HF, respectively; the relative expression of α-SMA was 0.78±0.05, 4.12±0.04, 3.81±0.06 and 0.88±0.05, respectively; the relative expression of collagen was 0.65±0.03, 3.43±0.02, 2.67±0.02 and 0.48±0.03, respectively; and the expression of TGF-β mRNA was 0.22±0.03, 0.89±0.01, 0.86±0.03 and 0.43±0.02, respectively. The A490 value, the migrating cells number, the expression of α-SMA, collagen and the expression of TGF-β mRNA in LPS group and LPS+ negative control group was significantly higher than those in control group and LPS+ lentivirus group, and all the differences were statistically significant (all P value <0.05). Conclusions:NLRP3 inflammasome activation may accelerate the extracellular matrix deposition and pancreatic fibrogenesis by promoting PSCs proliferation and migration ability via regulating the biological functions.

Objective:To investigate the application value of CT and MRI imageomics based on machine learning method in the diagnosis of pancreatic cancer.Methods:The clinical data of 62 patients with surgically resected and pathologically confirmed pancreatic cancer, who underwent enhanced CT scan, MRI plain or enhanced scan in Shanghai General Hospital between January 2014 and December 2021 were collected. According to the chronological order of surgery, 49 patients from January 2014 to December 2020 were enrolled in the training set and 13 patients from January 2021 to December 2021 were enrolled in the validation set. 3D-slicer 4.8.1 software was used to draw the region of interest in each layer of CT and MRI images for cancerous and paracancerous tissue segment. Image features were extracted by Python and the optimal feature set from the training set data was obtained by using Lasso regression model. The machine learning decision tree model was constructed. The receiver operating characteristic curve(ROC) curve was drawn, and the area under the curve (AUC) was calculated to evaluate the value of these three kinds of imageomics models in the diagnosis of pancreatic cancer.Results:The 1 767 CT features and 1 674 MRI features were obtained from enhanced CT scan, MRI plain scan and enhanced MRI scan, respectively. For the differential diagnosis model of cancerous tissue and paracancerous tissue, the enhanced CT scan data model obtained the optimal feature set involving 6 features, the MRI plain scan model obtained the optimal feature set involving 16 features, and the enhanced MRI scan model obtained the optimal feature set involving 15 features. The diagnostic model based on enhanced CT scan had an AUC of 0.98 in the training set and 1 in the verification group. The AUC of the MRI plain scan and enhanced MRI scan models in both the training set and the validation set was 1. The specificity and sensitivity of machine learning decision tree model based on the three kinds of imageomics models in the diagnosis of cancerous tissue and paracancerous tissue were 100%. For the differential diagnosis model of splenic artery wrapping, the enhanced CT scan model didn′t obtain the optimal features and had no diagnostic efficacy. The MRI plain scan model and enhanced MRI scan model obtained the optimal feature set involving 5 and 4 features, respectively. The AUC of the MRI plain scan model in the training set and the validation set were 0.862 and 0.750, respectively, with diagnostic sensitivity of 93.8% and 50.0%, and specificity of 78.6% and 100%, respectively. The AUC of the enhanced MRI scan model in the training set and the validation set were 0.950 and 0.861, respectively, with diagnostic sensitivity of 90.0% and 93.6%, and specificity of 100% and 78.6%, respectively.Conclusions:Based on the radiomics of CT enhanced, MRI plain scan and enhanced MRI scan, the machine learning diagnostic model has an accuracy of more than 90% in differentiating pancreatic cancer from paracancerous tissue. For the differentiation of splenic artery wrapping in pancreatic cancer, the diagnostic model based on enhanced MRI scan haS the best diagnostic efficiency.

Objective:To evaluate the role of Toll-like receptor 4 (TLR4)/nuclear transcription factor κB (NF-κB) signaling pathway in long-term cognitive impairment induced by multiple exposures to sevoflurane anesthesia in neonatal rats.Methods:Seventy-five SPF healthy newborn Sprague-Dawley rats of either sex, aged 6 days, weighing 12-20 g, were divided into 3 groups ( n=25 each) using a random number table method: control group (group C), multiple exposures to sevoflurane anesthesia group (group S) and TLR4 inhibitor plus multiple exposures to sevoflurane anesthesia group (group I+ S). The rats in group S and group I inhaled 3% sevoflurane for 2 h at 6, 7 and 8 days after birth. TLR4 inhibitor TAK-242 10 mg/kg was intraperitoneally injected before each exposure to sevoflurane in group I, and the equal volume of normal saline was given instead in the other two groups. The spontaneous activity was evaluated by open field test on day 29 after birth, and the cognitive function was assessed by Morris water maze test on days 30-34 after birth. After the behavioral test, the blood samples from the abdominal aorta were collected, and then the rats were sacrificed under deep anesthesia to isolate the hippocampal tissues for measurement of the levels of S100β and neuron-specific enolase (NSE) in serum and hippocampal interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α) (by enzyme-linked immunosorbent assay), expression of TLR4, NF-κB p65 and phosphorylated NF-κB p65 (p-NF-κB p65) (by Western blot) and for microscopic examination of the pathological changes of hippocampal CA1 region after HE staining. Results:Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the TLR4 expression was up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was increased, the levels of serum S100β protein and NSE and hippocampal IL-1β, IL-6 and TNF-α were increased ( P<0.05), and the pathological changes in the hippocampal CA1 region were aggravated in group S. Compared with group S, the escape latency was significantly shortened, the number of crossing the original platform was increased, TLR4 expression was down-regulated, the ratio of p-NF-κB p65/NF-κB p65 was decreased, the levels of S100β and NSE in serum and hippocampal IL-1β, IL-6 and TNF-α were decreased ( P<0.05), and the pathological changes in hippocampal CA1 area were significantly attenuated in group P. Conclusions:The mechanism by which multiple exposures to sevoflurane anesthesia induces long-term cognitive impairment is related to activation of TLR4/NF-κB signaling pathway and increase in hippocampal inflammatory responses in neonatal rats.

Objective:To evaluate the role of nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in edaravone-induced attenuation of long-term cognitive impairment caused by long-time sedation with propofol in the neonatal rats.Methods:Eighty SPF healthy newborn Sprague-Dawley rats of both sexes, aged 7 days, weighing 15-20 g, were divided into 4 groups ( n=20 each) using a random number table method: control group (group C), propofol group (group P), edaravone+ propofol group (group EP) and Nrf2 inhibitor ML385+ edaravone+ propofol group (group MEP). Propofol 75 mg/kg was intraperitoneally injected once a day for 7 consecutive days in P group, EP group and MEP group, respectively, while the equal volume of medium/long chain fat emulsion injection was intraperitoneally injected in C group. Edaravone 3 mg/kg was intraperitoneally injected at 30 min before each propofol injection in EP and MEP groups, and ML385 15 mg/kg was intraperitoneally injected simultaneously in group MEP. The spontaneous activity was evaluated by the open field test on day 29 after birth, and the cognitive function was assessed by Morris water maze test on days 30-34 after birth. The rats were sacrificed after the end of water maze test, and brains were removed and hippocampal tissues were obtained for determination of reactive oxygen species (ROS) levels (by flow cytometry), superoxide dismutase (SOD) and malondialdehyde (MDA) levels (by enzyme-linked immunosorbent assay) and expression of Nrf2 and HO-1 (by Western blot) and for microscopic examination of the pathological changes in the hippocampal CA1 area (using HE staining). Results:There was no significant difference in the speed, distance and time of stay at the center of the open field among the four groups ( P>0.05). Compared with C group, the escape latency was significantly prolonged, the number of crossing the original platform quadrant was reduced, the levels of MDA and ROS were increased, the activity of SOD was decreased, the expression of Nrf2 and HO-1 was down-regulated ( P<0.05), and the pathological injury was observed in the hippocampal CA1 region in group P. Compared with P group, the escape latency was significantly shortened, the number of crossing the original platform quadrant was increased, the levels of MDA and ROS in the hippocampus were decreased, the activity of SOD was increased, the expression of Nrf2 and HO-1 was up-regulated ( P<0.05), and the pathological injury in the hippocampal CA1 region was significantly alleviated in EP group. Compared with EP group, the escape latency was significantly prolonged, the number of crossing the original platform quadrant was reduced, the levels of MDA and ROS were increased, the activity of SOD was decreased, the expression of Nrf2 and HO-1 was down-regulated ( P<0.05), and the pathological injury was aggravated in the hippocampal CA1 region in MEP group. Conclusions:The mechanism by which edaravone attenuates long-term cognitive impairment caused by long-time sedation with propofol is related to activation of Nrf2/HO-1 signaling pathway and inhibition of oxidative stress in the neonatal rats.

Objective:To investigate the association between the expression of long non-coding RNA genes and the HULC rs7763881 polymorphism, recurrence, and metastasis after radical resection in patients with hepatocellular carcinoma (HCC).Methods:Paraffin tissue samples were selected from 426 cases diagnosed with HCC between January 2004 to January 2012. The expression of different genotypes of HULC gene locus rs7763881 in paraffin tissues was detected by PCR, and the association between different genotype expressions and clinical case characteristics of HCC [gender, age, TNM stage, alpha-fetoprotein, tumor maximum diameter (cm), vascular invasion, tumor capsule, tumor grade] was analyzed. Cox proportional risk regression model was used to analyze the correlation between different genotypes and clinicopathological features, prognosis, and recurrence. Survival analysis between different genotypes was performed using the Kaplan–Meier method for a parallel log-rank test.Results:There were 27 (6.3%) cases in the whole group who lost to follow-up. A total of 399 (93.7%) specimens were included in the study, and 105 (26.3%), 211 (52.9%) and 83 (20.8%) were included in the rs77638881 AA, AC, and CC genotypes, respectively. Kaplan-Meier curve showed that the postoperative overall survival and recurrence-free survival rate were significantly higher in patients with the AA than AC/CC genotype ( P < 0.05). Univariate analysis showed that the AC/CC genotype was closely related to tumor vascular invasion and recurrence or metastasis of HCC ( P < 0.05). Cox multivariate analysis results showed that patients with the AA genotype were taken as references, and the results showed that the risk of recurrence and metastasis in patients with the CA/CC genotype increased to varying degrees, with statistical significance ( P < 0.05). Conclusion:The rs7763881 polymorphic loci located on the HULC gene are closely related to HCC recurrence and metastasis after radical resection. Thus, it may be an indicator for evaluating HCC recurrence and metastasis.