[摘 要] 目的：探究包被蛋白复合体β1亚基（COPB1）在食管鳞状细胞癌（ESCC）中的表达，及其对ESCC细胞恶性生物学行为的影响、作用机制及临床意义。方法：采用2014～2018年间在河北医科大学第四医院生物样本库中82例ESCC组织及癌旁组织，常规培养正常食管鳞状上皮细胞HEEC和食管癌细胞KYSE-150、KYSE-170、Eca109、TE1、KYSE-30、KYSE-450，用转染试剂将pcDNA3.1-vector（空载体）、pcDNA3.1-COPB1载体，si-NC和si-COPB1转染至KYSE-150、TE1细胞中，记为NC、COPB1-OE、si-NC和si-COPB1组。用数据库数据分析COPB1 mRNA在泛癌组织中的表达及其表达与免疫细胞浸润的关系，qPCR法检测ESCC组织和细胞中COPB1、PIK3CB、CD68、CD163、CD206、ARG1、IL-10 mRNA水平表达情况，WB法检测ESCC组织和各组细胞中的COPB1、PI3K、CD68、CD163、CD206、p-AKT蛋白表达，克隆形成实验和MTS实验检测各组细胞的增殖能力，划痕愈合实验和Transwell实验检测各组细胞的迁移和侵袭能力，免疫组织化学染色（IHC）法检测ESCC组织中COPB1和CD206蛋白表达。以人单核细胞白血病细胞（THP-1）构建巨噬细胞模型，用佛波酯（PMA）和IL-3和IL-4和ESCC细胞上清液诱导巨噬细胞转型，用qPCR和WB法检测CD68和CD206m RNA和蛋白的表达。结果：COPB1在泛癌组织和ESCC组织中均呈高表达且与淋巴结转移和TNM分期有关联（均P < 0.01），COPB1高表达的ESCC患者总生存期短（P < 0.05），COPB1是潜在的ESCC的诊断标志物。COPB1在KYSE-150和TE1细胞中也呈高表达（均P < 0.05），过表达或敲减COPB1可明显抑制或促进KYSE-150和TE1细胞的增殖能力、迁移和侵袭能力（均P < 0.05）。COPB1表达变化诱导的差异表达基因主要富集于PI3K/AKT通路（均P < 0.001）, COPB1可促进PI3K/AKT通路的活化（P < 0.05），COPB1高表达可导致M2型巨噬细胞浸润增加（P < 0.05），COPB1高表达促进TAM/M2极化（P < 0.05）。结论：COPB1在ESCC组织中呈高表达，其可激活PI3K/AKT通路及调控肿瘤免疫微环境促进 ESCC发生发展，COPB1有望成为ESCC诊断和预后的生物标志物及治疗靶点。

Objective:To construct the c-myc gene silenced hepatocytes, study the effect of c-myc gene silence on expression of oncogenes and apoptosis genes in hepatocytes treated with PM2.5.Methods:According to the c-myc gene mRNA sequence provided by GenBank, three interfering sequences were designed and synthesized, the recombinant lentiviral vector was transfected into L02 hepatocytes. The real-time quantitative PCR and western blotting were used to identify the effect of c-myc gene silencing. L02 cells and c-myc gene silenced cells were used as experimental subjects. The normal L02 cells and c-myc silenced cells were treated with 50 μg/ml PM 2.5 water soluble solution, 10 μM positive control Cr 6+ and a blank control, the treatment period was 24 h. The mRNA levels of oncogenes (c-myc, c-fos, k-ras, p53) and apoptotic genes (Caspase-3, Caspase-8, Caspase-9) were detected by real-time PCR. The protein levels of oncogenes and apoptotic genes were detected by western blotting. Results:The mRNA level and protein level of c-myc decreased by 81% and 70% in c-myc silenced cells when compared with the normal L02 hepatocytes, the above results indicate that c-myc gene silenced cells were successfully constructed. After c-myc silenced cells were treated with PM2.5 water soluble solution, The mRNA levels of c-myc, c-fos, and k-ras decreased by 84.1%, 45.4%, and 54.6% ( P<0.05) , p53 increased by 192.9% ( P<0.05) , and the expression of Caspase-3, Caspase-8, and Caspase-9 decreased by 24.4%, 36.1%, 60.9% ( P<0.05) . In the Cr 6+ positive control group, the expression of c-myc, c-fos, and k-ras decreased by 72.1%, 82.2%, and 54.0% ( P<0.05) , p53 increased by 250.0% ( P<0.05) , the expression of Caspase-3, Caspase-8, and Caspase-9 decreased by 34.6%, 36.0%, 68.9% ( P<0.05) , respectively, when compared with the normal L02 hepatocytes ( P<0.05) . Western blotting results showed that the protein levels of c-myc and c-fos increased, p53 decreased after PM 2.5 exposure; the protein levels of Caspase-3, Caspase-8, Caspase-9 increased after PM 2.5 exposure ( P<0.05) . When in comparison with the c-myc silenced group, the protein levels of c-myc and c-fos decreased, p53 protein increased in PM 2.5 exposed group ( P<0.05) . Conclusion:c-myc gene silenced cells were successfully constructed in this paper. PM 2.5 could promote the expression of oncogenes and apoptotic genes in L02 cells, and c-myc gene silencing can inhibit the expression of oncogenes and apoptotic genes after PM 2.5 treatment in L02 cells.

Objective:To study the effect of p38 mitogen-activated protein kinase (MAPK) gene silencing on expression of apoptotic genes and oncogenes in hepatocytes treated with PM 2.5. Methods:From June to September 2019, according to the p38MAPK gene mRNA sequence provided by GenBank, three interfering sequences were designed and synthesized, ligated into PLVX-shRNA2-puro after annealing, and the recombinant lentiviral vector was transfected into L02 hepatocytes. The p38MAPK silencing cells were identified by real-time fluorescent quantitative PCR and western blotting. The normal L02 cells and p38MAPK silencing cells were treated with 50 μg/mL PM 2.5 water soluble solution, 10 μmol/L positive control Cr 6+, and a blank control group was set up, the treatment time was 24 h. The mRNA levels of oncogenes (c-fos, c-myc, k-ras) , tumor suppressor gene (p53) and apoptotic genes (Caspase-3, Caspase-8, Caspase-9) were detected by real-time PCR. The protein levels of oncogenes and apoptotic genes were detected by Western blotting. Results:The expression levels of p38MAPK mRNA and protein in p38MAPK gene silencing cells were significantly lower than those in L02 hepatocytes ( P<0.05) , and the p38MAPK gene silencing cell line was successfully constructed. Compared with the blank control group, the expression levels of the oncogenes c-fos, c-myc, k-ras and the apoptosis genes Caspase-3, Caspase-8 and Caspase-9 increased, the expression level of tumor suppressor gene p53 decreased in the L02 hepatocyte group treated with PM 2.5 water soluble matter, and the differences were statistically significant ( P<0.05) . Compared with the L02 hepatocytes group treated with PM 2.5 water soluble matter, the expression levels of the oncogenes c-fos, c-myc, k-ras and apoptosis genes Caspase-3, Caspase-8 and Caspase-9 decreased, the expression level of tumor suppressor gene p53 increased in the p38MAPK gene silencing cells group treated with PM 2.5 water soluble matter, and the differences were statistically significant ( P<0.05) . Conclusion:PM 2.5 has effects on the expression of oncogenes, tumor suppressor genes and apoptotic genes in L02 hepatocytes, while p38MAPK gene silencing can inhibit the effects of PM 2.5 on L02 hepatocytes.

Objective:To construct the c-myc gene silenced hepatocytes, study the effect of c-myc gene silence on expression of oncogenes and apoptosis genes in hepatocytes treated with PM2.5.Methods:According to the c-myc gene mRNA sequence provided by GenBank, three interfering sequences were designed and synthesized, the recombinant lentiviral vector was transfected into L02 hepatocytes. The real-time quantitative PCR and western blotting were used to identify the effect of c-myc gene silencing. L02 cells and c-myc gene silenced cells were used as experimental subjects. The normal L02 cells and c-myc silenced cells were treated with 50 μg/ml PM 2.5 water soluble solution, 10 μM positive control Cr 6+ and a blank control, the treatment period was 24 h. The mRNA levels of oncogenes (c-myc, c-fos, k-ras, p53) and apoptotic genes (Caspase-3, Caspase-8, Caspase-9) were detected by real-time PCR. The protein levels of oncogenes and apoptotic genes were detected by western blotting. Results:The mRNA level and protein level of c-myc decreased by 81% and 70% in c-myc silenced cells when compared with the normal L02 hepatocytes, the above results indicate that c-myc gene silenced cells were successfully constructed. After c-myc silenced cells were treated with PM2.5 water soluble solution, The mRNA levels of c-myc, c-fos, and k-ras decreased by 84.1%, 45.4%, and 54.6% ( P<0.05) , p53 increased by 192.9% ( P<0.05) , and the expression of Caspase-3, Caspase-8, and Caspase-9 decreased by 24.4%, 36.1%, 60.9% ( P<0.05) . In the Cr 6+ positive control group, the expression of c-myc, c-fos, and k-ras decreased by 72.1%, 82.2%, and 54.0% ( P<0.05) , p53 increased by 250.0% ( P<0.05) , the expression of Caspase-3, Caspase-8, and Caspase-9 decreased by 34.6%, 36.0%, 68.9% ( P<0.05) , respectively, when compared with the normal L02 hepatocytes ( P<0.05) . Western blotting results showed that the protein levels of c-myc and c-fos increased, p53 decreased after PM 2.5 exposure; the protein levels of Caspase-3, Caspase-8, Caspase-9 increased after PM 2.5 exposure ( P<0.05) . When in comparison with the c-myc silenced group, the protein levels of c-myc and c-fos decreased, p53 protein increased in PM 2.5 exposed group ( P<0.05) . Conclusion:c-myc gene silenced cells were successfully constructed in this paper. PM 2.5 could promote the expression of oncogenes and apoptotic genes in L02 cells, and c-myc gene silencing can inhibit the expression of oncogenes and apoptotic genes after PM 2.5 treatment in L02 cells.

Objective:To study the effect of p38 mitogen-activated protein kinase (MAPK) gene silencing on expression of apoptotic genes and oncogenes in hepatocytes treated with PM 2.5. Methods:From June to September 2019, according to the p38MAPK gene mRNA sequence provided by GenBank, three interfering sequences were designed and synthesized, ligated into PLVX-shRNA2-puro after annealing, and the recombinant lentiviral vector was transfected into L02 hepatocytes. The p38MAPK silencing cells were identified by real-time fluorescent quantitative PCR and western blotting. The normal L02 cells and p38MAPK silencing cells were treated with 50 μg/mL PM 2.5 water soluble solution, 10 μmol/L positive control Cr 6+, and a blank control group was set up, the treatment time was 24 h. The mRNA levels of oncogenes (c-fos, c-myc, k-ras) , tumor suppressor gene (p53) and apoptotic genes (Caspase-3, Caspase-8, Caspase-9) were detected by real-time PCR. The protein levels of oncogenes and apoptotic genes were detected by Western blotting. Results:The expression levels of p38MAPK mRNA and protein in p38MAPK gene silencing cells were significantly lower than those in L02 hepatocytes ( P<0.05) , and the p38MAPK gene silencing cell line was successfully constructed. Compared with the blank control group, the expression levels of the oncogenes c-fos, c-myc, k-ras and the apoptosis genes Caspase-3, Caspase-8 and Caspase-9 increased, the expression level of tumor suppressor gene p53 decreased in the L02 hepatocyte group treated with PM 2.5 water soluble matter, and the differences were statistically significant ( P<0.05) . Compared with the L02 hepatocytes group treated with PM 2.5 water soluble matter, the expression levels of the oncogenes c-fos, c-myc, k-ras and apoptosis genes Caspase-3, Caspase-8 and Caspase-9 decreased, the expression level of tumor suppressor gene p53 increased in the p38MAPK gene silencing cells group treated with PM 2.5 water soluble matter, and the differences were statistically significant ( P<0.05) . Conclusion:PM 2.5 has effects on the expression of oncogenes, tumor suppressor genes and apoptotic genes in L02 hepatocytes, while p38MAPK gene silencing can inhibit the effects of PM 2.5 on L02 hepatocytes.

Objective To explore the pathological characteristics and prognostic influencing factors of T1 invasive ductal breast carcinoma with calcification.Methods The clinicopathological and follow-up data in 172 patients with initially treated operable T1 invasive ductal breast cancer in this hospital from June 2012 to June 2013 were analyzed restrospectively.The patients were divided into the calcification group and non-calcification group based on the breast X-ray image features.The differences of pathological characteristics between two groups,related factors,and relationship between the calcification expression with patient survival were analyzed.Results The pathological types,lymph node metastasis,Her-2 overexpression,TNM stage and Ki-67 had statistically significant difference between the calcification group and non-calcification group(P＜0.05).The multivariate analysis showed that the cases type,lymph node metastasis and Ki-67 were the related risk factors affecting the calcification expression(P＜0.05).The 3-year disease-free survival rate in the the calcifi cation group and non-calcification group were 87.30％ and 95.06％ respectively.The lymph node status and calcification were the independent predictive risk factors affecting the disease-free survival time of invasive ductal breast carcinoma(P＜0.05).Conclusion Calcification is visible X-ray risk factor of T1 invasive ductal breast carcinoma prognosis.

Objective To investigate the risk factors influencing early and late recurrences after resection of primary clear cell carcinoma of the liver (PCCCL).Methods 214 PCCCL patients treated by curative resection from January 1996 to March 2006 were retrospectively analyzed.Recurrences were classified into early (≤1 year) and late (＞1 year) recurrences.Results 99 patients developed recurrences,with early recurrence in 28 patients and late recurrence in 71 patients.The 3-and 5-year overall survival (OS) rates for recurrent PCCCL were significantly worse than those with no recurrence (68.7％ and 46.2％ vs 72.2％ and 64.3％,P=0.003).The 1-,3-and 5-year OS rates for late recurrence were 100％,80.3％ and 54.6％,which were significantly better than those with early recurrence (85.7％,39.3％ and 25.0％,P=0.001).On multivariate analysis,aminoleucine transferase (ALT) level and vascular invasion were independent risk factors for early recurrence,while age was the only significant risk factor for late recurrence.Conclusions The time to recurrence was the main determinant for prognosis of recurrent PCCCL,Clarifying the different risk factors for early and late recurrences will help postoperative follow-up,early detection of recurrence,and hopefully will improve survival.

Objective To investigate the clinicopathologic characteristics and prognostic factors of primary clear cell carcinoma of the liver(PCCCL). Methods A total of 214 PCCCL patients treated by curative resection from January 1996 to March 2006 were retrospectively analyzed. Results The 1-,3-,and 5-year overall survival (OS) rates for PCCCL patients were significantly better than those of non-clear cell hepatocellular carcinoma ( NHCC ) patients ( 90.2％,70.6％,and 55.9％ vs 82.8％,62.7％ and 47.7％,P =0.001 ).Tumor size was significantly smaller in PCCCL group than in NHCC group ( x2 =4.37,P =0.04 ).Tumors of PCCCL group had a lower incidence of vascular invasion ( x2 =9.42,P =0.002) and a better differentiation than those of NHCC group ( x2 =4.30,P =0.04).Serum a-fetoprotein (AFP) level,tumor size,liver cirrhosis,and vascular invasion were independent risk factors impacting OS and disease-free survival (DFS) of PCCCL. Conclusions PCCCL is an uncommon subtype of HCC and has different clinicopathologic characteristics from NHCC. Complete surgical resection is the optimal treatment for PCCCL and its prognosis is much better than that of NHCC.

Objective To study the risk factors of post-hepatectomy hepatic decompensation (PHD) in patients with hepatocellular carcinoma.MethodWe reviewed 562 patients with Child-Pugh A classification,who underwent partial hepatectomy for hepatocellular carcinoma at Zhongshan Hospital,Fudan University between July 1st 2007 to December 31st 2007,to study the risk factors of hepatic decompensation.ResultsPreoperative high total bilirubin (TB) and low prealbumin (PA) were independent risk factors of PHD by logistic multivariate analysis ROC analysis revealed the cut-offs of preoperative PA predicting PHD were 0.14 g/L (sensitivity 41.4％; specificity 83.1％).The incidence of PHD was 16.0％ when TB≥20.4 μmol/L and PA＜0.14 g/L(OR=7.276,P=0.002).ConclusionThe Child-Pugh A patients recovered well when the preoperative liver function was as follows:TB＜20.4 μmol/L and PA≥0.14 g/L.

Objective To investigate the role and mechanism of TMPRSS4 in radiation induced metastasis of hepatocellular carcinoma (HCC).Methods Metastatic model of human HCC was established by orthotopic implantation of histologically intact human HCC tissue into the liver of nude mice.Mice bearing xenografts in liver were killed after radiation and the residual tumors were resected and reimplanted into the liver of normal nude mice.At the end of sixth week,the mice were killed and the histopathological features,tumor volume,intrahepatic and lung metastasis were evaluated.Expression of epithelial-mesenchymal transition (EMT) related genes including N-cadherin,Vimentin,SIP1 and TMPRSS4 were measured by Western blotting and RT-PCR.Results The tumor volume and frequency of lung metastasis of control group was 2.25±0.52 cm3 and 66.7％,respectively.Compared to control group,tumor diameter (1.61±0.51 cm3,P＜0.05) and lung metastasis (12.5％,P＜0.05) were significantly inhibited 2 days after radiation.Whereas,30 days after radiation,tumor growth recovered (2.60±0.61 cm3,P＞0.05) and lung metastasis was enhanced (100％,P＜0.05).There were no intrahepatic metastasis in the control group and in the group of reimplantation of HCC 2 days after radiation,while the tumors from those 30 days after radiation showed enhanced intrahepatic metastasis (18 ± 8.05,P＜ 0.01 ),with overexpression of SIP1,N-cadherin,Vimentin and TMPRSS4,and reduced expression of E-cadherin.Conclusion The metastasis potential of residual HCC after radiation was first inhibited and then promoted.Overexpression of TMPRSS4 plays a critical role in radiation induced long-term metastasis of HCC by facilitating EMT.