目的   初步探讨生长因子受体结合蛋白14（GRB14）在肺腺癌患者预后中的具体作用机制。方法   通过TIMER数据库、UALCAN数据库及GEPIA数据库，探讨GRB14 mRNA在肺腺癌及正常肺组织中的表达。运用免疫组织化学通过组织芯片（75例肺腺癌患者和75例癌旁组织）检测其蛋白表达水平，收集国外肿瘤研究团队上传至TCGA数据库229例肺腺癌患者的临床数据，分析评估GRB14在肺腺癌患者的表达及其临床特征及生存预后之间的关系。应用TIMER数据库对GRB14肺腺癌患者进行免疫浸润分析。String数据库探讨GRB14与其他蛋白之间是否存在相互作用。结果  TIMER数据库分析显示，相比正常组织，GRB14 mRNA在多种实体肿瘤和肺腺癌组织中高表达（P＜0.05）。使用UALCAN数据库和GEPIA数据库以正常样本为对照组，肺腺癌患者的GRB14的表达均增加（P＜0.01）。免疫组织化学检测组织芯片结果显示，GRB14蛋白在肺腺癌的表达高于正常肺组织（肺腺癌6.07±1.01 vs 癌旁组织4.80±1.22；P＜0.01）。TCGA数据库分析显示，肺腺癌患者中GRB14高表达组和低表达组的中位总生存期分别为（41.59±5.20）月和（88.67±16.69）月；结合TCGA数据库绘制ROC曲线，发现GRB14的表达对肺腺癌患者具有一定的诊断价值。单因素回归分析结果显示，肿瘤分期（Ⅲ-Ⅳ）（P＜0.01）、肿瘤原发灶的情况（T3-4）（P＜0.01）、淋巴结转移（N1-3）（P＜0.01）和GRB14表达（P＜0.01）是影响肺腺癌中位总生存期的因素；Cox多因素回归分析显示，淋巴结转移（N1-3）（P＜0.05）和GRB14表达（P＜0.01）是影响肺腺癌中位总生存时间的因素。TIMER数据库分析显示，GRB14 mRNA 表达与巨噬细胞（r=-0.164，P＜0.01）、中性粒细胞（r=-0.175，P＜0.01）和树突状细胞（r=-0.148，P＜0.01）具有相关性。通过String数据库分析发现与GRB14相互作用的蛋白质包括EGFR、HRAS、FGFR1、INSR、CNGA1、COBLL1、LYPLAL1、TNKS2、TNKS、PRKCZ。结论  GRB14表达增加与肺腺癌患者预后不良相关。

       Objective  To assess the specific mechanism of growth factor receptor-bound protein 14（GRB14）in the prognosis of lung adenocarcinoma（LUAD）patients．Methods  The expression of GRB14 mRNA in LUAD and normal lung tissue was explored using TIMER database，UALCAN database，and GEPIA database．The expression of GRB14 protein was examined by immunohistochemistry using a tissue microarray．Then，the associations of GRB14 expression with clinicopathological features and clinical outcomes of LUAD were validated by analyzing TCGA database at the mRNA level and statistically evaluating the results．TIMER database was used to analyze immune infiltration of GRB14 in LUAD．Protein-protein interaction of GRB14 were analyzed using the String database．Results  Using the TIMER database，we found that GRB14 mRNA was highly expressed in various solid tumors and LUAD tissues compared to normal tissues（P＜0.05）．Comparing with the normal group，the expression of GRB14 was increased in LUAD（P＜0.01）via using UALCAN database and GEPIA database．The expression level of GRB14 protein in the LUAD tissues was significantly higher than that in the noncancerous LUAD tissues（LUAD［6.07±1.01］ vs benign，［4.80±1.22］；P＜0.01）in tissue microarray ．Median overall survival in the high and low GRB14 expression groups in LUAD was（41.59±5.2）and（88.67±16.69）months respectively．We plotted the ROC curves of 3-year survival rate and 5-year survival rate which again suggested that the model had good predictive performance．Univariate analysis revealed that individual cancer stages（Ⅲ-IV）（P＜0.01），tumor（T3-4）（P＜0.01），lymph node metastasis（N1-3）（P＜0.05）and GRB14 expression（P＜0.01）were risk factors affecting the median overall survival time of LUAD．According to Cox multiple regression analysis，we found that lymph node metastasis（N1-3）（P＜0.05）and GRB14 expression（P＜0.01）were  risk factors affecting the median overall survival time of LUAD．Using TIMER database analysis，the mRNA level of GRB14 was significantly correlated with macrophages（r=-0.164，P＜0.01），neutrophils（r=-0.175，P＜0.01）and dendritic cells（r=-0.148，P＜0.01）．Through analysis of the String database，it was found that proteins that interacted with GRB14 including EGFR，HRAS，FGFR1，INSR，CNGA1，COBLL1，LYPLAL1，TNKS2，TNKS，PRKCZ．Conclusions  The results of the present study suggest that GRB14 may efficiently predict poor survival in LUAD patients．

目的 探讨磷酸二酯酶4抑制剂对人肺泡巨噬细胞(AM)吞噬非生物性颗粒及革兰阳性菌、阴性菌能力的影响。方法 使用Ficolll-Hypaque密度梯度法将外周血单核细胞分离的静脉血,在含有2 ng/m GM-CSF的培养液中经12天诱导培养成AM替代细胞模型—单核细胞源性巨噬细胞(MDM)。用酶标仪检测MDM经磷酸二酯酶4抑制剂Rolipram预处理过夜(16~18 h)后吞噬荧光标记的非生物颗粒Beads和热灭活的流感嗜血杆菌(H.influenzae)、金黄色葡萄球菌(S.aureus)量的改变,另使用MTT法检测细胞活性。结果 成功建立的MDM细胞模型对Beads和细菌的吞噬呈时效关。Rolipram在实验浓度(10^~8~10^-5 M)下对MDM吞噬Beads、H.influenzae和S.aureus能力无明显促进或抑制作用,也不影响MDM的活性。结论 磷酸二酯酶4抑制剂不会因升高巨噬细胞内cAMP水平而影响其吞噬非生物颗粒和细菌的能力。

Objective To investigate the influence of phosphodiesterases 4 inhibitor on the phagocytosis of non-biological particles and gram-positive bacteria, gram-negative bacteria by human alveolar macrophages. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood from 12 healthy volunteers using Ficoll-Hypaque density gradients. Monocytes were incubated with media containing 2 ng/ml GM-CSF for 12d to allow full differentiation into macrophage (MDM), a functionally equivalent model of human AM. MDM were pretreated with Rolipram overnight (16-18h),phagocytosis of fluorescent labeled beads and H.influenzae,S.aureus by MDM was measured using a fluostar optima fluorimeter. Cell viability was assay with MTT. Results MDM phagocytosis of beads and bacteria was a time-dependant process. Rolipram in the concentration of 10^-8-10^-5M didn't inhibit or promote phagocytosis of beads and bacteria by MDM, and didn't affect the cell viability. Conclusion Phosphodiesterases 4 inhibitor would not affect the human macrophage phagocytic capacity of non-biological particles and bacteria associated with enhanced intracellular cAMP level.

目的 确定CD4⁺CD25⁺Treg调节性T细胞在重症肺炎克雷伯菌肺炎中的表达以及意义,探讨CD4⁺CD25⁺Treg在重症肺炎克雷伯菌肺炎的免疫抑制中的调控作用。方法 通过气管内滴注肺炎克雷伯菌菌液建立重症肺炎模型。采用流式细胞仪检测CD4⁺CD25⁺Treg细胞及酶联免疫吸附法(ELISA)等方法检测各种细胞因子。结果 重症肺炎克雷伯菌肺炎大鼠的脾脏和肺中CD4⁺CD25⁺Treg的数量增加。使用了CD25抗体(PC61)去除机体内源性的CD4⁺CD25⁺Treg,分别去除脾脏和肺的94%和90%的CD4⁺CD25⁺Treg。CD25抗体组在建模4 h,12 h及24 h后,肺部MPO及血清IL-1,IL-6,MIP-2较对照组高(P<0.05),肺和BLA比对照组高(P<0.05),CD25抗体组大鼠生存率比对照组低(P<0.05)。结论 内源的CD4⁺CD25⁺Treg对大鼠抑制重症肺炎克雷伯菌肺炎的过度免疫损害反应起到保护作用。

Objective To confirm the expression and meaning of the T regular cell in the severe Klebsiella pneumonia, and to evaluate the regular and control affect in the immunologic suppression of the severe Klebsiella pneumonia. Methods To build the severe pneumonia model by intratracheally inoculated with Klebsiella pneumoniae bacteria. To check sorts of inflammation factors by the methods of ELISA and flow cytometry. Results The quantity of the CD4⁺CD25⁺Treg in the splenic and lungs of the mice with severe Klebsiella pneumonia were increased. Anti-CD25Ab(PC61) was used to remove endogenousCD4⁺CD25⁺Treg. Anti-CD25 treatment remove 90% of CD4⁺CD25⁺Treg cells. The cytokine production(IL-1β,IL-6,MIP-2)in the anti-CD25-treated group were significantly increased. And it also increased significantly in the airway neutrophil infiltration, while the survival rate had been decreased. Conclusion Endogenous CD4⁺CD25⁺Treg can provide obvious protection effect to the restraining the over immunity damage of the severe Klebsiella pneumonia for the mice.