To investigate the effects of osimertinib on hepatocellular carcinoma (HCC) and angiogenesis, and its combinatory effects with venetoclax in HCC. Viability was assessed by flow cytometry of Annexin V in multiple HCC cell lines after drug treatment. In vitro angiogenesis assay was performed using primary human liver tumor associated endothelial cell (HLTEC). HCC-bearing model was generated by subcutaneous implantation of Hep3B cells to investigate the efficacy of osimertinib alone and its combination with venetoclax. Osimertinib significantly induced apoptosis in a panel of HCC cell lines regardless of EGFR expression level. It inhibited capillary network formation and induced apoptosis in HLTEC. Using HCC xenograft mouse model, we further showed that osimertinib at non-toxic dose inhibited tumor growth by 50

目的 探讨亚低温治疗对重症病毒性脑炎患儿酸碱平衡及炎症细胞因子(T N F-α、IL-6、IL-10)、可溶性凋亡相关因子(s-Fas)水平的影响.方法 以2014年1月～2018年12月武汉儿童医院收治的93例重症病毒性脑炎患儿为研究对象,随机分为亚低温治疗组和常规对照组,亚低温治疗组在肛温32～35℃状态下给予常规治疗,治疗时间为3～10 d,对照组仅给予常规治疗.检测并比较两组治疗后不同时间点患儿动脉血PO2、PCO2、pH值,脑脊液及血清中乳酸以及细胞因子T N F-α、IL-6、IL-10、s-Fas水平变化.结果 ①亚低温治疗48、72 h及治疗后10 d,亚低温治疗组患儿动脉血PO2、PCO2均低于常规对照组(均P<0.05).两组比较,血pH值在各时段差异均无统计学意义(均P>0.05).②亚低温治疗48 h及72 h后,亚低温治疗组患儿的血清乳酸水平明显低于常规对照组(均P<0.05).亚低温组的患儿脑脊液乳酸水平在急性期及恢复期均低于常规对照组(均P<0.05).③血清T N F-α、IL-6水平在亚低温治疗后72 h及10 d均明显低于常规治疗组(均P<0.05);在亚低温治疗48、72 h后,亚低温治疗组血清IL-10水平高于常规治疗组(均P<0.05),血清s-Fas水平明显低于常规治疗组(均P<0.05).④亚低温治疗组患儿脑脊液TNF-α水平在恢复期明显低于常规治疗组(P<0.05),脑脊液IL-6水平在急性期及恢复期均明显低于常规治疗组(均P<0.01),脑脊液s-Fas水平在急性期明显低于对照组(P<0.01).结论 亚低温治疗在降低重症病毒性脑炎患儿动脉血PO2、PCO2的同时,也降低了脑脊液乳酸水平,且对动脉血pH值无明显影响.亚低温治疗可以降低重症病毒性脑炎患儿血清及脑脊液TNF-α、IL-6及s-Fas水平,抑制凋亡,并在治疗初期提高IL-10水平,抑制免疫反应.

Background: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. Methods: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. Findings: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts [0·6 (0·6-0·8)] but increases in neutrophil counts [4·7 (3·6-5·8)] than 27 mild cases [1.1 (0·8-1·4); 2·0 (1·5-2·9)]. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8+ T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-lymphocyte ratio (NLR) (AUC=0·93) and neutrophil-to-CD8+ T cell ratio (N8R) (AUC =0·94) were identified as powerful prognostic factors affecting the prognosis for severe COVID-19. Interpretation: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R and NLR may serve as a useful prognostic factor for early identification of severe COVID-19 cases. Funding: The National Natural Science Foundation of China, the National Science and Technology Major Project, the Health Commission of Hubei Province, Huazhong University of Science and Technology, and the Medical Faculty of the University of Duisburg-Essen and Stiftung Universitaetsmedizin, Hospital Essen, Germany.

Objective To investigate effect of mild hypothermia on nearon-soecific enolase (NSE)and S100B in serum and cerebrospinal fluid (CSF) of children with severe viral encephalitis.Methods Eighty-three children with severe viral encephalitis were randomly divided into 2 groups:mild hypothermia group and regular therapy group.Mild hypothermia group received cooling(32 35 ℃C)for 3--10 days within 24 h.Control group received ordinary treatment.The clinical symptoms and encephalogram were observed before and after therapy.The contents of NSE and S100B in the serum and CSF were detected by ELISA.Results ① Compared with regular therapy group,symptom persisting time,Glasgow Coma Scale (GCS) and hospitalization time of mild hypothermia group were decreased significantly (P＜0.05 or P＜0.01).② There were no statistical differences in NSE and S100B protein serum levels at 0 h and 24 h post mild hypothermia treatment(P＞0.05).But NSE and S100B protein serum levels were significantly lower in the mild hypothermia group than in the regular therapy group at 48 h,72 h post mild hypothermia treatment(P＜0.05 or P＜0.01).③ After 3-10 days of mild hypothermia treatment,children's CSF S100B protein and NSE levels of the mild hypothermia group were significantly decreased,compared with the regular therapy group(P ＜0.05 or P＜0.01).Conclusion The treatment of mild hypothermia can reduce serum and CSF S100B protein and NSE levels,and improve the clinical symptoms of children with severe viral encephalitis.

Objective To examine the effect of silencing tumor necrosis factor(TNF)receptor-associated factor-6(TRAF6) on the inflammatory responses in mice with lipopolysaccharide (LPS)/D-galactosamine (GalN)-induced acute liver failure (ALF).Methods The small interfering RNA(siRNA)sequence that could most effectively target TRAF6 was screened out in vitro. The plasmid pTRAF6-shRNA was delivered into BALB/c mice by hydrodynamics-based gene transfection(HGT)twice at an interval of 24 h.Twenty-four h after the second HGT,LPS/D-GalN was intraperitoneally administrated to BALB/c mice to establish the endotoxin-induced inflammatory model of acute liver failure.Animals were divided into four groups:normal con-trol group,model control group,blank plasmid/LPS group and RNAi/LPS group.Blood samples were collected at 4,8 and 16 h after LPS/D-GalN injection.HE staining was used to observe the pathological changes of liver tissues.Meanwhile,TRAF6,in-terleukin-6(IL-6)and cyclooxygenease(COX)-2 mRNA levels were detected by real-time PCR.Inflammatory cytokines[TNF-α, IL-1β,transforming growth factor (TGF)-β1 and IL-6 ]were measured by ELISA.Western blot analysis was used to detect TRAF6 and NF-κB p65 expression in the nuclear extracts of hepatocytes.Results Our results showed that TRAF6 mRNA and protein expressions were remarkably reduced by 60.13% and 52.08%,respectively,in the mouse liver(P<0.01).ELISA indi-cated that the secretions of TNF-α,IL-1βand TGF-β1 were markedly increased after LPS/D-GalN inj ection,with TNF-α,IL-1βpeaking at 8 h,and TGF-β1 at 1 6 h.Furthermore,the levels of TNF-α,IL-1βand TGF-β1 were lower in RNAi/LPS group than in model control group at different detection time points.Real-time PCR showed that IL-6 ,COX-2 and TRAF6 mRNA levels were obviously lower in RNAi/LPS group than in model control group(P<0.01).Moreover,total NF-κB p65 protein levels&nbsp;were effectively increased in RNAi/LPS group as compared with normal control group(P<0.05).However,NF-κB p65 levels in the nuclear extracts in RNAi/LPS group were significantly lower those in model control group(P<0.05).Conclusion Ap-plication of pTRAF6-shRNA can successfully alleviate LPS/D-GalN-induced acute liver inj ury in vivo by inhibiting NF-κB ac-tivities and decreasing the expression of inflammatory cytokines and inflammatory mediators.

AIM:To establish a new model for predicting survival in acute-on-chronic liver failure (ACLF) patients treated with an artificial liver support system.METHODS:One hundred and eighty-one ACLF patients who were admitted to the hospital from January 1, 2012 to December 31, 2014 and were treated with an artificial liver support system were enrolled in this retrospective study, including a derivation cohort (n = 113) and a validation cohort (n = 68). Laboratory parameters at baseline were analyzed and correlated with clinical outcome. In addition to standard medical therapy, ACLF patients underwent plasma exchange (PE) or plasma bilirubin adsorption (PBA) combined with plasma exchange. For the derivation cohort, Kaplan-Meier methods were used to estimate survival curves, and Cox regression was used in survival analysis to generate a prognostic model. The performance of the new model was tested in the validation cohort using a receiver-operator curve.RESULTS:The mean overall survival for the derivation cohort was 441 d (95%CI: 379-504 d), and the 90- and 270-d survival probabilities were 70.3% and 58.3%, respectively. The mean survival times of patients treated with PBA plus PE and patients treated with PE were 531 d (95%CI: 455-605 d) and 343 d (95%CI: 254-432 d), respectively, which were significantly different (P = 0.012). When variables with bivariate significance were selected for inclusion into the multivariate Cox regression model, number of complications, age, scores of the model for end-stage liver disease (MELD) and type of artificial liver support system were defined as independent risk factors for survival in ACLF patients. This new prognostic model could accurately discriminate the outcome of patients with different scores in this cohort (P < 0.001). The model also had the ability to assign a predicted survival probability for individual patients. In the validation cohort, the new model remained better than the MELD.CONCLUSION:A novel model was constructed to predict prognosis and accurately discriminate survival in ACLF patients treated with an artificial liver support system.

目的：分析丙型肝炎病毒（hepatitis C virus, HCV）1b 型非结构蛋白3（non-structure protein 3, NS3）丝氨酸蛋白酶区序列的变异规律及影响意义。方法使用基因型别特异性引物,通过巢式 PCR 的方法扩增119例慢性 HCV 1b 型患者血清中 HCV NS3区。对 PCR 产物进行测序后获得 NS3区核苷酸及氨基酸序列。分析119例 HCV 1b 型 NS3区序列的同源性及种系进化,分析 NS3丝氨酸蛋白酶区的变异情况及重要功能位点的突变情况。结果湖北地区 HCV 1b 型与 HCV 1b 型标准株及亚洲地区序列同源性较高,核苷酸序列同源性为85.94％及87.68％,氨基酸序列同源性可达96.83％及92.39％,而与欧美地区1b 型同源性较低,核苷酸序列同源性均为85.57％,氨基酸序列同源性分别为92.17％及93.38％。进化树分析提示湖北地区序列与中国其他地区序列亲缘性较接近,而与日本、东南亚地区及欧美地区的1b 型亲缘性较远。 NS3丝氨酸蛋白酶区185个氨基酸内有34个位点有氨基酸突变,但其重要的功能区包括催化酶底物特异性结合位点以及 Zn2﹢结合位点在所有序列中均高度保守,无一例发生突变。结论对湖北地区 HCV 1b型 NS3丝氨酸蛋白酶区的变异规律及生物学意义的研究进一步丰富和完善了对 HCV 1b 型基因组变异情况的认识。为了解 HCV1b 型在中国地区的进化过程提供一定理论基础。

目的 研究乙型肝炎病毒X蛋白(HBx)调节细胞内钙离子可能的分子机制,揭示乙型肝炎病毒(HBV)诱导肝癌的可能途径.方法 培养人胚肾细胞(HEK293),取第2代HEK293细胞共转染HBx基因、钙释放激活钙通道蛋白1(Orai1)基因或者间质相互作用因子1(STIM1)基因,免疫共沉淀(co-IP)实验观察细胞内蛋白结合情况,并使用谷胱甘肽巯基转移酶(GST)pull-down实验研究HBx与Orai1作用位点,使用钙离子测量/成像系统检测HBx蛋白对细胞内钙离子的影响.结果 转染后细胞生长状态良好,co-IP实验结果显示HBx蛋白在细胞内可以与Orai1蛋白结合,GST pull-down实验显示HBx蛋白可以与Orai1蛋白C末端结合,钙离子测量/成像系统检测显示HBx蛋白可升高活细胞钙内流.结论 HBx蛋白通过与细胞膜钙离子通道Oria1结合蛋白的C末端结合,增加细胞钙内流,扰乱细胞内钙离子平衡,从而影响细胞增殖等活性.

Objective To investigate the subcellular localization of p21 in hepatocellular carcinoma( HCC) tissues and its clinical significance. Methods 115 cases of HCC tissues based on liver cirrhosis were performed with immunohistochemical staining and subcellular localization for p21 was observed. The correlation between subcellular localization of p21 and patients' clinicopathological parameters was analyzed. Results The positive rate of p21 in HCC tissues was same with that in paracancerous tissues( 35. 7% vs 38. 3%,P > 0. 01),and significantly higer than that in liver tissues of cirrhosis( 10. 4%,P < 0. 01). p21 was mainly localized in nucleus in cirrhosis and paracancerous tissues( 91. 7% vs 93. 2%,P > 0. 01),but the positive rate of p21 in cytoplasm was the highest in HCC tissues( 75. 6% P < 0. 01). There was a significant correlation between the localization of p21 in cytoplasm and the tumor differentiation or metastasis,and the localization of p21 was not related with the age,HBV infection,size of the tumor; but with the differentiation and metastasis of the tumor. Conclusions p21 may change its subcellular localization in the process of transformation from cirrhosis to tumor. Higher the degree of malignancy of the tumor cells or earlier the tumor metastasis is,larger the preportion of cytoplasmic shift has. And the distribution of this subcellular localization has no association with the age of onset of tumors,size of the tumor or hepatitis B virus infection.

先天性肝内胆管囊性扩张症即Caroli病,是一种先天性肝内胆管多发节段性囊性扩张病,由法国学者Jaeque Caroli 于1958年首先描述、报道而得名.此病为一种少见病,发病率小于1/10万,本院于2012年3月收治1例,现报道如下.一、病例资料患者女性,13岁,因发现肝硬化伴反复畏寒、发热1年余入院.2010年12月,患者于受凉后出现畏寒、发热,自服感冒药后逐渐出现烦躁、意识模糊,呕咖啡色胃内容物1次,量少,入当地市级医院诊治,CT诊断为肝硬化,但原因不明,予对症处理后好转出院.2011至2012年,患者反复多次出现畏寒、发热,不伴腹痛、恶心、呕吐,在当地诊所予头孢类药物后体温可恢复.2012年3月2日,患者进食高蛋白食物后出现发热、尿黄、意识障碍再次入当地医院,抗感染、醒脑处理后暂好转,遂前来本院要求明确诊断. 关键词：胆管疾病;肝硬化;先天性肝内胆管囊性扩张症

AIM: To investigate the impact of small hairpin RNA (shRNA)-mediated silencing of the tumor necrosis factor receptor-associated factor 6 (TRAF6) gene on the lipopolysaccharide (LPS)/toll-like receptor (TLR) 4 signaling pathway in vitro. METHODS: ShRNA sequences targeting the TRAF6 gene were designed, synthesized and used to construct eukaryotic expression plasmids. After transfection of the recombinant plasmids into RAW264.7 cells, cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay. Inflammatory cellular models were established by LPS stimulation. Levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) in the supernatants, mRNA expression of TRAF6, interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), protein expression of TRAF6, and translocation of NF-κB were assayed by ELISA, real-time quantitative PCR and Western blot, respectively. RESULTS: The mRNA and protein expression of TRAF6 was lower in cells transfected with TRAF6-shRNA1 or TRAF6-shRNA2 (79.17% and 68.74%, respectively) compared to other groups. Therefore, cells transfected with pGCsi-TRAF6-shRNA1 or 2 were used for subsequent experiments. TRAF6 knockdown significantly inhibited the proliferation of RAW264.7 cells within 72 h after transfection, reduced the production of pro-inflammatory cytokines and mediators including TNF-α, IL-1β, IL-6 and COX-2, and inhibited NF-κB nuclear translocation. Moreover, TRAF6 knockdown could suppress the release of TGF-β1 at the protein level. CONCLUSION: TRAF6 knockdown can, to some extent, inhibit early inflammatory response stimulated by LPS. TRAF6 may become a potential therapeutic target for many inflammation-related diseases.

AIM: To investigate the impact of small hairpin RNA (shRNA)-mediated silencing of the tumor necrosis factor receptor-associated factor 6 (TRAF6) gene on the lipopolysaccharide (LPS)/ toll-like receptor (TLR) 4 signaling pathway in vitro. METHODS: ShRNA sequences targeting the TRAF6 gene were designed, synthesized and used to construct eukaryotic expression plasmids. After transfection of the recombinant plasmids into RAW264.7 cells, cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay. Inflammatory cellular models were established by LPS stimulation. Levels of tumor necrosis factor-α (TNF-α), interleukin- 1β (IL-1β) and transforming growth factor-β1 (TGF-β1) in the supernatants, mRNA expression of TRAF6, interleukin-6 (IL-6) and cyclooxygenase- 2 (COX-2), protein expression of TRAF6, and translocation of NF-κB were assayed by ELISA, real-time quantitative PCR and Western blot, respectively. RESULTS: The mRNA and protein expression of TRAF6 was lower in cells transfected with TRAF6-shRNA1 or TRAF6-shRNA2 (79.17% and 68.74%, respectively) compared to other groups. Therefore, cells transfected with pGCsi-TRAF6- shRNA1 or 2 were used for subsequent experiments. TRAF6 knockdown significantly inhibited the proliferation of RAW264.7 cells within 72 h after transfection, reduced the production of pro-inflammatory cytokines and mediators including TNF-α, IL-1β, IL-6 and COX-2, and inhibited NF-κB nuclear translocation. Moreover, TRAF6 knockdown could suppress the release of TGF-β1 at the protein level. CONCLUSION: TRAF6 knockdown can, to some extent, inhibit early inflammatory response stimulated by LPS. TRAF6 may become a potential therapeutic target for many inflammationrelated diseases.

This study examined the effect of subcellular localization of P21 on the proliferation and apoptosis of HepG2 cells.The coding genes of the wild and the mutant P21 were amplified by mega primer PCR from the plasmid pCEP-WAF1 which contains human P21 cDNA in the nuclear localizational signal（NLS）sequence,and then inserted into the eukaryotic expression vector pDsRed1-C1.The recombinants were transfected into HepG2 cells.The transcription and expression of P21 were determined by RT-PCR and fluorescence microscopy.The cell proliferation was measured by MTT,and the cell cycle and apoptosis of HepG2 cells by flow cytometry.The results of restriction analysis,DNA sequencing and fluorescence microscopy confirmed the construction of the wild and the mutant P21 in the eukaryotic expression plasmid.The plasmid containing the mutant P21 was found to accelerate cell proliferation and the wild P21 plasmid to inhibit cell proliferation.Cell cycle analysis showed that the cell ratio of G0/G1 in the wild type group was significantly increased as compared with that in the mutant type group,and cell apoptosis analysis revealed that the apoptosis rate in the wild type group was much higher than that in the mutant type group.It was concluded that the subcellular localization of P21 may contribute to the development of hepatic cancer.

Objective To construct the eukaryotic expression vector of pDsRed1-C1-p21NLS-gene and study its function.Methods The mutation with nuclear localization signal of p21 was obtained by site-directed mutagenesis.p21 NLS-was directly cloned into eukaryotic expressive plasmid pDsRed1-C1 and transfected into HepG2 Cells by liposome-mediated DNA transfection.The p21 NLS-gene expression,intracellular localization of the p21 and HBV antigens were detected by RT-PCR,fluorescence microscope and ELISA,respectively.Results The expression of pDsRed1-C1-p21NLS-was mainly localized in the cytoplasm compared to pDsRed1-C1-p21WT which was localized in the nucleus.The expression of pDsRed1-C1-p21NLS-improved the replication of HBV in HepG2.2.15.Conclusion The different intracellular localization of p21 has various impacts on replication of HBV in HepG2.2.15 cells.

Objective To compare the transfection efficiency and expression intensity of a green fluorescent protein (GFP) reporter gene in the liver of mice by using three non-viral transfection methods,i.e.,hydrodynamic injection,portal vein injection and peritoneal injection. Methods The naked GFP plasmids or liposome encapsulated plasmids were injected via the tail vein or portal vein or abdominal cavity of different mice with homogeneity. The blood and the liver were harvested 48 h after injection,and the contents of alanine aminotransferase (ALT) and total bilirubin (TB) were detected in serum. Also,expression intensity of GFP was evaluated by fluorescence microscopy. Results Of the three transfection methods employed,hydrodynamic gene delivery and portal vein injection conferred a stronger expression of GFP with a similar transfection efficiency (P0.05). However,after encapsulation by liposomes,the expression level of GFP was significantly higher than that of naked plasmid DNA. Conclusion High level gene expression in mouse liver can be achieved by hydrodynamic injection or portal vein injection of liposome encapsulated plasmid DNA,and hepatic delivery of the foreign gene can be accomplished by hydrodynamics-based injection.

AIM To compare the therapeutic effects of compound glycyrrhizin injection by hepatic arterial infusion and by intraperitoneal injection on rats with acute hepatic failure.METHODS D-galactosamine was injected into the abdominal cavity of rats to establish an experimental model of acute liver failure.Rats of acute liver failure were treated with compound glycyrrhizin injection by hepatic arterial infusion and by intraperitoneal injection.The level of plasma ALT,AST,TBil and the pathological morphologies of hepatic tissues were examined.RESULTS Treatment of hepatic arterial infusion could obviously ameliorate the hepatic function and pathological morphologies of hepatic tissues of the rats.Comparing with intraperitoneal injection,there were statistical significances(P0.01).CONCLUSION The improvement effects on liver function of experimental rats with acute hepatic failure by hepatic arterial infusion is better than by intraperitoneal injection.

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide（LPS）/toll-like receptor（TLR）-4 signaling pathway in vitro.Four plasmids（pGCsi-TRAF6-shRNA1,2,3,4） containing different shRNA sequences were designed and synthesized.The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay.Inflammatory cellular models were established by LPS stimulation.Levels of TNF-α,IL-1β and TGF-β1 in the supernatants,mRNA expressions of TRAF6,IL-6 and COX-2,protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA,real-time quantitative PCR and Western blotting,respectively.The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h.The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1,2 groups than in the TRAF6-shRNA3,4 groups.Therefore,pGCsi-TRAF6-shRNA1,2 were selected for the subsequent experiments.Our results still showed that pGCsi-TRAF6-shRNA1,2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-α,IL-1β,IL-6 and COX-2,and inhibit NF-κB nuclear translocation.Moreover,pGCsi-TRAF6-shRNA1,2 could suppress the release of TGF-β1 at the protein level.It was concluded that the recombinant plasmid pTRAF6-shRNA can,to some extent,inhibit inflammatory response stimulated by LPS at the initial phase.TRAF6 may become the potential therapeutic target of many inflammation-related diseases.