结核分枝杆菌感染小鼠的脾脏和肺脏组织荷菌量与病理变化

目的分析结核急性感染小鼠脾脏和肺脏的组织荷菌量以及病理的动态变化。方法以3×105CFU结核分枝杆菌标准株H37Rv尾静脉途径感染雌性C57BL/6J小鼠,测定感染后1 d、1周、2周、3周、4周、6周和8周肺脏及脾脏组织的荷菌量,脾脏和肺脏组织经HE染色分析病理变化。结果感染动物的脾脏荷菌量在前3周逐步提高,在4～8周脾脏荷菌量维持稳定,肺脏组织在2～8周组织荷菌量逐步升高。病理分析显示动物感染3周后肺脏和脾脏出现肉芽肿,在6～8周病理损伤加重。结论小鼠经尾静脉感染高剂量结核分枝杆菌在8周前表现为急性感染状态,适用于抗结核药物的体内药效学评价。     

A BAC transgenic Hes1-EGFP reporter reveals novel expression domains in mouse embryos

Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative state of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)^1Hri, to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates some previous observations on Hes1 expression and suggests new, hitherto unrecognised expression domains including expression in the definitive endoderm at early somite stages before gut tube closure and thus preceding organogenesis. This mouse line will be a valuable tool for studies addressing the role of Hes1 in a number of different research areas including organ specification, development and regeneration.     

Characteristics of long-term survivors of brain metastases from lung cancer

Background and aim

Long-term survival of lung cancer patients with brain metastases (BM) is very rare. Our aim is to report the characteristics of patients who survived for at least three years after a BM diagnosis.

Materials and methods

Nineteen lung cancer patients who had survived ≥3 years after a BM diagnosis were identified in our database. Seven (37%) had undergone whole-brain radiotherapy (WBRT) only, five (26%) BM surgery + WBRT, three (16%) BM surgery + WBRT + BM radiosurgery, and four (21%) no WBRT (one, surgery; one, radiosurgery; two, BM surgery + radiosurgery). Their characteristics were compared with historical data for 322 lung cancer patients with BM (control group, CG), who had received WBRT between 1986 and 1997.

Results

Median survival from BM in long survivors group was 73 months (in CG – 4 months). Characteristics comparison: median age 55 vs. 58 (CG), p = 0.16; female sex 68% vs. 28% (CG), p = 0.003; RTOG/RPA class 1 – 75% vs. 13% (CG), p = 0.00001; adenocarcinoma histology 84% vs. 24% (CG), p < 0.00001; control of primary tumor 95% vs. 27% (CG), p < 0.00001; extracranial metastases 0 vs. 26% (CG), p = 0.01; single BM 63% vs. 9% (CG), p = 0.00001; surgery of BM 53% vs. 14% (CG), p = 0.00001.

Conclusions

Beside prognostic factors already recognized as favorable in patients with BM, the adenocarcinoma histology and female sex were prevalent in long-term survivors of BM from lung cancer.     

高分辨MSCT薄层重组在常规胸部体检中的应用

目的：探讨多排螺旋CT（MSCT）高分辨薄层重组对体检中肺部疾病的诊断价值,以及MSCT薄层重组与常规CT扫描图像质量及诊断准确率对照。方法：搜集做64排常规CT扫描同时行薄层重组的2473例体检病例中筛选的97例患者进行回顾性分析。结果：97例患者在常规CT扫描中有80例可以显示病变,而在MSCT薄层重组中97例均有阳性发现;其中双肺弥漫性病变79例（两肺间质纤维化78例,肺内多发转移瘤1例）,肺结核3例,肺内孤立结节12例,早期中央型肺癌1例,先天性支气管闭锁1例以及纵隔肿瘤1例;MSCT薄层重组图像可清晰显示肺内小叶间隔线增厚、弥漫分布的囊性病变、肺内结节的细微征象。结论：MSCT薄层重组在胸部体检中的诊断阳性率、细节分辨率、图像质量及确诊率均优于常规CT,可作为肺内病变尤其是早期肺癌筛查方法之首选。     

肺上皮细胞自噬体在结核分枝杆菌感染中保护作用的分子机制

目的：构建Atg5．真核表达载体并瞬时转染肺上皮细胞细胞株,探讨自噬在结核分枝杆菌感染上皮细胞中保护作用的分子机制。方法：设计针对Atg5的RNAi序列,化学合成后经过变性,退火连接到pSilencerTM3．1-H1hygro真核表达载体,经测序验证其正确性。脂质体法瞬时转染真核细胞A549,免疫印迹法检测瞬时转染的效果。用结核分枝杆菌分别感染正常和自噬表达低下的A549细胞．通过检测LDH来观察细胞的坏死情况。结果：成功的构建了pSilencerTM3．1-H1hygro真核表达载体并建瞬时转染了A549细胞株,成功抑制了细胞的自噬功能。Atg5-细胞对结核杆菌的抵抗能力下降。结论：在自噬表达低下的细胞中,细胞对结核分枝杆菌的抵抗能力有明显下降。在结核分枝杆菌感染上皮细胞的过程中,自噬是一种保护机制。     

水飞蓟宾诱导肺腺癌Anip973 细胞凋亡的分子机制研究

目的:探讨水飞蓟宾诱导肺腺癌Anip973细胞系细胞凋亡的分子机制。方法:采用MTT法、倒置显微镜和电子显微镜等形态学检测以及流式细胞仪(FCM)技术检测、DNALadder分析、凋亡分子PARP的表达检测细胞凋亡,同时进行凋亡相关蛋白Bax、Bcl-2、caspase-3和caspase-9表达活性分析。结果:(1)水飞蓟宾对人肺腺癌Anip973细胞系细胞的增殖有显著抑制作用;(2)水飞蓟宾作用Anip973细胞48h后,随着浓度的增加,倒置显微镜下可见细胞数目减少,胞体变小、变圆,到高浓度时出现较多的死亡细胞;(3)扫描电镜观察发现,随着水飞蓟宾作用浓度的增加,Anip973细胞中出现增多的凋亡细胞,凋亡细胞表现出典型的超微结构特征;(4)流式细胞仪检测的结果发现,随着药物作用时间的延长,Anip973细胞的G1期细胞比例增多,S期细胞明显减少,G2期细胞略有减少,并出现明显的凋亡峰。(5)水飞蓟宾作用后的Anip973细胞出现明显的DNALadder和PARP降解增加等凋亡特征;(6)水飞蓟宾作用后,Anip973细胞中的凋亡相关蛋白Bax表达增加、caspase-3和caspase-9酶活性增加,而Bcl-2表达降低。结论:水飞蓟宾在体外有抑制人肺腺癌细胞Anip973的增殖作用,并通过激活线粒体依赖的caspase凋亡通路,诱导其凋亡。     

无症状吸烟者的肺功能改变情况分析

目的:探讨烟龄≥15年,日吸烟量≥15支的无症状男性吸烟者的肺功能改变情况。方法:选择男性无症状吸烟者190人及非吸烟者180人,进行肺功能测定,并比较两组人群的肺功能改变情况。结果:吸烟组与非吸烟组比较,肺活量(VC)、用力肺活量(FVC)、第1秒用力呼气容积(FEV1)、Tiffeneau 1秒率(FEV1/VC)结果改变不明显,而Gaensler 1秒率(FEV1/FVC)、最大分钟通气量(MVV)、用力呼气50%肺活量的呼气流量(FEF50%)、用力呼气75%肺活量的呼气流量(FEF75%)、呼出25%～75%肺活量时的平均流量(FEF25%～75%)、肺一氧化碳弥散量(DLCO)结果均有显著降低,有统计学意义。结论:通过对无症状吸烟人群肺功能测定结果进行分析。发现有些吸烟者虽无临床症状,但已经出现了小气道及肺弥散功能的损伤,提醒吸烟者应早期戒烟,关爱自身健康,净化生存环境,提高生活质量。     

Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species

Pulmonary neuroepithelial bodies (NEBs) are extensively innervated organoid groups of neuroendocrine cells that lie in the epithelium of intrapulmonary airways. Our present understanding of the morphology of NEBs is comprehensive, but direct physiological studies have so far been challenging because the extremely diffuse distribution of NEBs makes them inaccessible in vivo and because a reliable in vitro model is lacking. Our aim has been to optimise an in vitro method based on vibratome slices of living lungs, a model that includes NEBs, the surrounding tissues and at least part of their complex innervation. This in vitro model offers satisfactory access to pulmonary NEBs, provided that they can be differentiated from other tissue elements. The model was first optimised for living rat lung slices. Neutral red staining, reported to stain rabbit NEBs, proved unsuccessful in rat slices. On the other hand, the styryl pyridinium dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), showed brightly fluorescent cell groups, reminiscent of NEBs, in the airway epithelium of living lung slices from rat. In addition, nerve fibres innervating the NEBs were labelled. The reliable and specific labelling of pulmonary NEBs by 4-Di-2-ASP was corroborated by immunostaining for protein gene-product 9.5. Live cell imaging and propidium iodide staining further established the acceptable viability of 4-Di-2-ASP-labelled NEB cells in lung slices, even over long periods. Importantly, the in vitro model and 4-Di-2-ASP staining procedure for pulmonary NEBs appeared to be equally reproducible in mouse, hamster and rabbit lungs. Diverse immunocytochemical procedures could be applied to the lung slices providing an opportunity to combine physiological and functional morphological studies. Such an integrated approach offers additional possibilities for elucidating the function(s) of pulmonary NEBs in health and disease. This work was supported by the following research grants: Fund for Scientific Research Flanders (G.0155.01 to D.A.), NOI-BOF (to D.A.) and BOF-RUCA Small Projects (KPO2 to D.A., I.B. and F.V.M.) from the University of Antwerp.     

In vivo cryochemotherapy of a human lung cancer model

Cryotherapy, an efficient technique to destroy tumour cells, is sometimes applied locally as a palliative treatment in lung cancers. It can be performed in combination with chemotherapy. Our aims were to determine in vivo: (1) the effects of cryochemotherapy in a human lung adenocarcinoma, (2) if it presents a benefit compared to the separate treatments and (3) if cryotherapy allows a tumour retention of the drug. Cells from the A549 cell line were xenografted into SCID mice. Tumours were treated by cryotherapy, chemotherapy (injection of Vinorelbine: Navelbine) or both and were studied morphologically at variable time points. Apoptosis was analysed by immunohistochemical staining of cleaved caspase-3 and by TUNEL. Intratumour Navelbine concentration was assessed by high performance liquid chromatography. Necrosis was important 2 h after cryochemotherapy (45% of the tumour surface) and at the later time points. Expression of cleaved caspase-3 was not significantly different from that of untreated tumours, except at the time point of 2 h where it was maximal (58%). Navelbine concentration was more important in tumours treated by chemotherapy than in tumours treated by cryochemotherapy, demonstrating that in our model, the benefit of the association observed 2h after treatment was not due to a concentration-dependent effect.     

Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung

Branching morphogenesis in the lung serves as a model for the complex patterning that is reiterated in multiple organs throughout development. Beta-catenin and Wnt signaling mediate critical functions in cell fate specification and differentiation, but specific functions during branching morphogenesis have remained unclear. Here, we show that Wnt/beta-catenin signaling regulates proximal-distal differentiation of airway epithelium. Inhibition of Wnt/beta-catenin signaling, either by expression of Dkk1 or by tissue-specific deletion of beta-catenin, results in disruption of distal airway development and expansion of proximal airways. Wnt/beta-catenin functions upstream of BMP4, FGF signaling, and N-myc. Moreover, we show that beta-catenin and LEF/TCF activate the promoters of BMP4 and N-myc. Thus, Wnt/beta-catenin signaling is a critical upstream regulator of proximal-distal patterning in the lung, in part, through regulation of N-myc, BMP4, and FGF signaling.