涎腺导管内癌的病理诊断及新进展

涎腺导管内癌(intraductal carcinoma,IDC)是一种主要发生在腮腺,在导管内或囊内生长为主的罕见肿瘤,但也可伴浸润性生长,生物学行为相对惰性。具有独特的形态学、免疫表型及分子遗传学特征。主要包括4种亚型:闰管型、顶浆分泌型、闰管-顶浆分泌混合型和嗜酸型。闰管型多数存在NCOA4-RET融合,个别存在STRN-ALK融合;混合型存在TRIM27-RET融合,顶浆分泌型具有PIK3CA和HRAS突变或TP53缺失;嗜酸型具有TRIM33-RET融合或BRAF V600E突变。目前多数观点认为与涎腺导管癌不同,即使存在浸润,仍具有良好的预后,罕见复发和淋巴结转移,无远处转移。因此精准诊断对于临床治疗的选择极其重要。     

Clinical analysis of peritoneal dialysis in the treatment of rapidly progressive glomerulonephritis

Objective To observe the clinical characteristics and prognosis of patients with rapidly progressive glomerulonephritis (RPGN) caused by lupus nephritis, antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, or primary glomerulonephritis who were treated with peritoneal dialysis (PD) and then withdrew PD because of renal recovery. Methods Data of the above patients were retrospectively analyzed. The patients were diagnosed as RPGN and received PD therapy in Kidney Disease Center, the First Affiliated Hospital, College of Medicine, Zhejiang University from February 2009 to August 2018. The patients were divided into early withdrawal group (PD time≤183 days, n=24) and late withdrawal group (PD time＞183 day, n=24). The differences of clinical characteristics between the two groups were compared. The cumulative incidence of adverse events in both groups was analyzed using Kaplan-Meier curves. Cox proportional hazards model was used to analyze the risk factors influencing the prognosis of patients. Results Forty-eight RPGN patients were included. The median time of maintaining PD was 178(76, 378) days. Compared with the late withdrawal group, the patients in early withdrawal group had lower levels of urine volume, serum albumin and parathyroid hormone, and lower rates of gross hematuria and hypertension at the beginning of PD, and received higher rates of methylprednisolone impulse, combined immunosuppressive agents, and hemodialysis or continuous renal replacement therapy (all P＜0.05). At the time of PD withdrawal, the levels of serum creatinine, serum calcium, serum albumin and parathyroid hormone in the early withdrawal group were significantly lower than those in the late withdrawal group (all P＜0.05). The Kaplan-Meier curves showed that there was no significant difference in the cumulative survival of patients in both groups (log-rank test χ2=3.485, P=0.062). Cox regression analysis revealed serum creatinine≥209 μmol/L at the time of PD withdrawal was an independent risk factor for poor prognosis (HR=5.253，95%CI 1.757-15.702, P=0.003). Conclusions PD can be used for RPGN patients caused by lupus nephritis, ANCA-associated vasculitis and primary nephritis. Serum creatinine≥209 μmol/L at the time of PD withdrawal is an independent risk factor for poor prognosis.     

eIF4EBP3 was downregulated by methylation and acted as a tumor suppressor by targeting eIF4E/β-catenin in gastric cancer

Gastric Cancer - Epigenetic aberrations of tumor suppressor genes (TSGs), particularly DNA methylation, are frequently involved in the pathogenesis of gastric cancer (GC). Through a methylome...     

依维莫司联合全反式维甲酸逆转急性早幼粒细胞白血病细胞耐药的研究

目的探讨依维莫司联合全反式维甲酸(简称维甲酸)逆转急性早幼粒细胞白血病(APL)细胞株NB4-R1耐药的作用。方法应用CD11b染色流式细胞术及硝基四唑氮蓝(NBT)还原实验检测两药联合应用对细胞分化的影响, 流式细胞术检测细胞周期情况, Annexin V/PI双染色检测细胞凋亡情况, 蛋白质印迹法检测自噬相关蛋白微管结合蛋白轻链3(LC3)、Beclin 1及早幼粒白血病-维甲酸受体融合蛋白(PML-RARα)、磷酸化核糖体S6激酶(P-P70S6K)、磷酸化4E结合蛋白1(P-4E-BP1) 等表达水平。结果与维甲酸组比较, 联用组能诱导耐药细胞株NB4-R1细胞的分化, 并将细胞增殖阻止在G ₁期而对细胞凋亡无明显影响。100 nmol/L依维莫司组、1μmol/L维甲酸组、联用组、对照组NB4-R1细胞培养48 h后分化百分率分别为(2.29±0.57)%、(17.06±2.65)%、(54.47±4.91)%、(2.54±0.53)%; 处于G ₁期的细胞百分率分别为(35.20±11.97)%、(33.54±6.25)%、(53.70±8.73)%、(27.40±6.01)%; 四组细胞凋亡细胞百分率分别为(2.30±0.14)%、(2.25±0.21)%、(2.40±0.28)%、(1.95±0.07)%。与维甲酸组比较, 联用组mTOR信号通路下游的P70S6K、4E-BP1分子磷酸化水平下降, LC3-II和Beclin 1的表达上调, 且能部分降解融合蛋白PML-RARα。 结论依维莫司联合维甲酸能诱导NB4-R1细胞分化, 且能阻滞细胞周期而不致细胞凋亡, 其机制可能与依维莫司联合维甲酸抑制mTOR信号通路激活自噬作用从而降解PML-RARα蛋白有关。     

Helicobacter pylori induces epithelial-mesenchymal transition in gastric carcinogenesis via the AKT/GSK3β signaling pathway

Helicobacter pylori (H. pylori) is a main risk factor for gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is involved in the development and progression of H. pylori-associated GC. However, the exact molecular mechanism of this process remains unclear. The AKT/GSK3β signaling pathway has been demonstrated to promote EMT in several types of cancer. The present study investigated whether H. pylori infection induced EMT, and promoted the development and metastasis of cancer in the normal gastric mucosa, and whether this process was dependent on AKT activation. The expression levels of the EMT-associated proteins, including E-cadherin and N-cadherin, were determined in 165 gastric mucosal samples of different disease stages by immunohistochemical analysis. The expression levels of E-cadherin, N-cadherin, AKT, phosphorylated (p-)AKT (Ser473), GSK3β and p-GSK3β (Ser9) were further determined in H. pylori-infected Mongolian gerbil gastric tissues and cells co-cultured with H. pylori by immunohistochemical analysis and western blotting. The results indicated that the expression levels of the epithelial marker E-cadherin were decreased, whereas the expression levels of the mesenchymal marker N-cadherin were increased during gastric carcinogenesis. Their expression levels were associated with H. pylori infection. Furthermore, H. pylori infection resulted in downregulation of E-cadherin expression and upregulation of N-cadherin expression in Mongolian gerbils and GES-1 cells. In addition, an investigation of the associated mechanism of action revealed that p-AKT (Ser473) and p-GSK3β (Ser9) were activated in GES-1 cells following co-culture with H. pylori. Furthermore, following pretreatment of the cells with the AKT inhibitor VIII, the expression levels of E-cadherin, N-cadherin, p-AKT and p-GSK3β did not show significant differences between GES-1 cells that were co-cultured with or without H. pylori. The levels of p-AKT and p-GSK3β were increased in H. pylori-infected Mongolian gerbils. In conclusion, the present study demonstrated that H. pylori infection activated AKT and resulted in the phosphorylation and inactivation of GSK3β, which in turn promoted early stage EMT. These effects were AKT-dependent. This mechanism may serve as a prerequisite for GC development.     

NLRC3 alleviates hypoxia/reoxygenation induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6

BackgroundNOD-like receptor family CARD domain containing 3 (NLRC3) plays an important role in both innate and adaptive immunity. This study was to explore the function and related mechanisms of NLRC3 in a hypoxia/reoxygenation (H/R)-induced inflammatory response in RAW264.7 cells.MethodsLiver ischemia-reperfusion (I/R) model in mice and H/R model in RAW264.7 cells were constructed. Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3, TRAF6, p–p65, p65, IκB–α, and the K63-linked ubiquitination level of TRAF6 in cells. The immunofluorescence assay was performed to evaluate the nuclear level of the NF–κB (p65). ELISA was conducted to measure the content of IL–1β in serum and cell supernatant. The interaction between NLRC3 and TRAF6 in cells was analyzed by the Co-IP assay.ResultsThe NLRC3 protein level in liver tissue was decreased with the prolongation of reperfusion time (P < 0.05). The expression of NLRC3 and IκB–α protein in RAW264.7 was decreased gradually, while the expression of p–p65 and TRAF6 proteins and K63-linked ubiquitination of TRAF6 were increased gradually with the prolongation of reoxgenation time (P < 0.05). The Co-IP assay revealed that NLRC3 and TRAF6 can bind to each other directly. However, NLRC3 had no effect on the expression of TRAF6 protein. The ubiquitination test results showed that the K63-linked ubiquitination level of TRAF6 in H/R + Lv–NLRC3 group was significantly lower than that in the H/R + negative control (NC) group (P < 0.05). Moreover, the activation of NF–κB in H/R + Lv–NLRC3 group was inhibited compared with that in the H/R + NC group, and the level of the inflammatory factor IL–1β in the cell culture supernatant was also decreased accordingly (P < 0.05).ConclusionsNLRC3 might alleviate H/R-induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.     

肾小管上皮细胞表型转化与糖尿病肾病

肾小管病变及肾间质纤维化在糖尿病肾病中的重要作用逐渐被认识,其中肾小管上皮细胞向间充质细胞转化(EMT)可能是其发挥作用的关键环节,与肾间质纤维化的程度相平行。在糖尿病肾病中引发EMT的因素主要有高血糖、晚期糖基化终末产物、蛋白尿、血管紧张素Ⅱ、结缔组织生长因子等。     

雷帕霉素对内皮祖细胞数量与功能的影响

目的观察雷帕霉素对外周血内皮祖细胞（EPCs）数量与功能的影响。方法采用密度梯度离心法从外周血获得单个核细胞，将其接种在人纤维连接蛋白包被的培养板上，培养7天后收集贴壁细胞，加入不同浓度（1.0、2．0、5．0ug／m^3）雷帕霉素分别培养6、12、24、48h。激光共聚焦显微镜鉴定，FITC-UEA-I和Dil-acLDL双染色阳性细胞为正在分化的EPCs，流式细胞仪检测其表面标志以进一步鉴定EPCs，并在倒置荧光显微镜下计数。然后分别采用MTT比色法、改良的Boyden小室、黏附能力测定实验和体外血管生成试剂盒来观察EPCs的增殖能力、迁移能力、黏附能力和体外血管形成能力。结果EPCs数量随雷帕霉素浓度与作用时问增加而减少，其增殖能力、迁移能力、黏附能力和体外血管形成能力亦随雷帕霉素浓度与作用时间增加而降低。结论雷帕霉素降低EPCs的数量、增殖能力、迁移能力、黏附能力和体外血管形成能力，并呈浓度和时间依赖性。