Objective To investigate the effects of granulocyte macrophage colony-stimulating factor (GM-CSF) combined with interleukin-12 (IL-12) genes on apoptosis of hepatoma cells.Methods The hepatoma cell lines were cultured in vitro and were divided into four groups: GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,negative control group (empty load group),respectively.The PIB-CMV3-GM-CSF and PIB-CMV3-IL-12 eukayotic expression vector was built,and 36 h after transfection,fluorescence microscope was used to detect the transfection effect;the expression level of IL-12,GM-CSF,p53,p38 and C-JUN mRNA were detected by RT-PCR,and Western blot was used to examine the expression level of IL-12,GM-CSF,p53,p38 and C-JUN protein.In addition,the flow cytometry was applied to detect cell apoptosis.Results Through fluorescence microscope,green fluorescence was observed in cells of GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,indicating that the plasmid has successfully transferred into cells.In addition,the expression of p53mRNA in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were 1.2±0.10,4.3±0.98,4.2±0.34,9.2±0.87,and the protein expression were 1.0±0.10,3.6±0.34,3.8±0.30,5.0±0.60.Compared with the empty load group,the expression level of p53 mRNA and protein were significantly increased in the three plasmid transfection groups (P<0.01).The expression of p53 mRNA and protein were significantly increased in co-transfection group than GM-CSF group and IL-12 group (P<0.01),while in the comparison with GM-CSF transfection group and IL-12 transfection group,the expression level of p53mRNA and protein in the co-transfection group could be improved to a higher degree(P<0.01).Meanwhile,p38 C-JUN mRNA expression levels in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were as follows: 7.5± 0.9,3.5±0.45,3.7±0.25,1.0±0.11,while p38protein expression levels were 10.1±1.03,6.1± 0.67,7.1 ± 0.61,1.0 ± 0.12,respectively,C-JUN mRNA expression levels were 11.2 ± 1.20,4.1 ± 0.19,3.3 ± 0.30,1.0 ± 0.01,separately,C-JUN protein expression levels were 2.25 ± 0.2,1.8 ± 0.13,1.4 ± 0.12,1.0 ± 0.09.P38, C-JUN mRNA and protein levels were significantly reduced in the three plasmid transfection groups compared with the empty load group (P<0.01).The expression of p38,C-JUN mRNA and protein were reduced to a lower degree in co-transfection group than in GM-CSF transfection group and IL-12 transfection group (P<0.01).Flow cytometer showed that the hepatoma cell apoptosis rate of the empty load group,GM-CSF transfection group,IL-12 transfection group,co-transfection group were (3.43±0.9)%,(5.87±1.02)%,(7.32±1.1)%,(17.47±2.11)%,the rates of the three plasmid transfection groups were significantly higher than that of the empty load group (P<0.01).And the apoptosis rate was significantly increased in the co-transfected group compared with other plasmid groups (P<0.01). Conclusion The combination of GM-CSF and IL-12 could significantly accelerate the apoptosis of hepatoma cells by up-regulating the expression of p53,and down-regulating the expression of p38 and C-JUN.

Objective: To investigate the clinicopathological characteristics of cardial mixed adenoneuroendocrine carcinoma (MANEC) and analyze its prognostic factors. Methods: The retrospective and descriptive study was conducted. The clinicopathological data of 34 patients with primary cardial MANEC who were admitted to the Changzhou No.2 People′s Hospital of Nangjing Medical University from January 2008 to January 2018 were collected. There were 20 males and 14 females, aged from 39 to 81 years, with an average age of 66 years. All the 34 patients underwent resection of cardia cancer and postoperative pathological examination. Observation indicators: (1) surgery and treatment; (2) clinical manifestations and pathological conditions; (3) pathological examination of surgical resection specimens; (4) follow-up and survival; (5) analysis of prognostic factors. Follow-up using outpatient examination and telephone interview was conducted to detect the survival of patients and tumor recurrence and metastasis up to December 2018. Measurement data with normal distribution were represented as Mean±SD. Measurement data with skewed distribution were expressed as M (range). Count data were expressed as absolute numbers or percentages. Kaplan-Merier method was used to calculate the survival time and rate and draw the survival curve, and log-rank test was used for the survival analysis. Univariate and multivariate analyses were performed using the COX proportional risk model. Results: (1) Surgery and treatment: all the patients underwent surgery successfully and postoperative systemic chemotherapy based on cisplatin + etoposide. (2) Clinical manifestations and pathological conditions: patients had epigastric discomfort, abdominal pain and abdominal distension as the first symptoms. Of 34 patients, number of males, cases with age ≥60 years, cases with esophageal involvement, cases with stable microsatellite, cases with higher CA19-9, cases with elevated cancer embryonic antigen, cases of tumor pathologic TNM stage Ⅲ-Ⅳ, cases with tumor diameter ≥5 cm, cases with vascular tumor emboli, cases with positive lymph node metastasis, cases with nerve invasion were 20, 29, 31, 28, 4, 3, 29, 30, 27, 30, 29, respectively. (3) Pathological examination of surgical excision specimens: the masses of patients were mainly ulcer-type, with the diameter of 3.0-8.4 cm. Of the 34 patients, 1 had tumor infiltrated into submucosa, 5 infiltrated into muscle layer, 18 infiltrated into serosal layer, and 10 infiltrated into extra-serous fibrous adipose tissues. Microscopy examination showed that all tumors were composed of two components including adenocarcinoma and neuroendocrine carcinoma, and the two components accounted for more than 30%. Among adenocarcinoma components of the 34 patients, 14 were poorly differentiated tubular adenocarcinoma, 6 were mucinous adenocarcinoma, 6 were moderately differentiated tubular adenocarcinoma, 5 were low-adhesion carcinoma, 1 was highly differentiated tubular adenocarcinoma, and 2 were papillary adenocarcinoma. Among the neuroendocrine carcinoma components of the 34 patients, 10 were small cell type and 24 were large cell type. Of the 34 patients, 10 had adenocarcinoma and neuroendocrine carcinoma closely adjacent but not confused, and 24 had adenocarcinoma and neuroendocrine carcinoma cross-mixed. Immunohistochemistry examination of 34 patients showed that the components of neuroendocrine carcinoma were positive for synaptophysin, pheochromoin A and nerve cell adhesion molecule. The components of adenocarcinoma were positive for broad-spectrum cytokeratin, cytokeratin 8/18 and cytokeratin 7. (4) Follow-up and survival: 34 patients were followed up for 8.0-68.0 months, with a median time of 53.7 months. The 34 patients had survived for 21-49 months, with a median time of 35 months. The 1-, 3-, 5-year survival rates were 93.31%, 53.60%, and 20.62%. (5) Ananlysis of prognostic factors: results of univariate analysis showed that CA19-9, tumor diameter, intravascular tumor thrombus, tumor pathological TNM stage, lymph node metastasis, microsatellite detection, and histological classification were the related factors affecting the prognosis of patients with cardial MANEC (risk ratio =1.724, 0.327, 1.401, 1.612, 1.542, 1.876, 0.945, 95% confidence interval: 1.226-3.467, 0.218-0.776, 1.171-4.432, 0.694-4.054, 0.987-3.776, 1.217-4.341, 0.614-2.115, P<0.05). Results of multivariate analysis showed that the tumor pathological TNM stage Ⅲ-Ⅳ, positive lymph node metastasis, stable microsatellite, neuroendocrine carcinoma as the main histological classification were independent risk factors affecting the prognosis of patients with cardial MANEC (odds ratio=1.667, 1.441, 1.306, 3.501, 95% confidence interval: 1.013-4.915, 1.035-5.746, 1.006-6.213, 2.076-8.528, P<0.05). Conclusions Cardial MANEC is composed of two components including adenocarcinoma and neuroendocrine carcinoma, and the two components account for more than 30%. The tumors in the neuroendocrine cacinoma area present as solid nest-like pattern, rosettes-shaped or organ-like pattern, with high nuclear-to-plasma ratio and fine chromatin, and it is easy to see mitotic figures. Adenocarcinoma components are tubular adenocarcinoma, mucinous adenocarcinoma, papillary adenocarcinoma with various differentiation. The adenocarcinoma and neuroendocrine carcinoma components can be cross-mixed, and also can be closely adjacent but not confused. Tumor pathological TNM stage Ⅲ-Ⅳ, positive lymph node metastasis, stable microsatellite, neuroendocrine carcinoma as the main histological classification are independent risk factors affecting the prognosis of patients with cardial MANEC.