小鼠Ⅱ型肺泡上皮细胞类器官三维培养体系的建立

本研究旨在建立小鼠Ⅱ型肺泡上皮细胞(type 2 alveolar epithelial cells, AT2)类器官三维(three-dimensional, 3D)培养体系的方法。采用酶消化与磁珠分选分离和纯化ICR小鼠肺AT2细胞,proSPC免疫荧光染色鉴定AT2细胞纯度;2维(two-dimensional, 2D)培养8 d,5-乙炔基-2’脱氧尿嘧啶核苷(5-ethynyl-2’-deoxyuridine, EdU)掺入和荧光染色法观察AT2细胞的增殖与分化;将AT2细胞与小鼠肺成纤维细胞(mouse lung fibroblasts, Mlg) 3D共培养,光学显微镜下观察类器官生长情况,收集生长13d的类器官,2%多聚甲醛固定后行HE染色,荧光染色观察类器官内部形态结构。结果显示,提取AT2细胞纯度超过95%;在体外培养1～8 d期间,AT2细胞EdU荧光染色阴性,未见增殖;AT2细胞形状逐渐趋向扁平鳞状、细胞表面积显著增大;在体外培养3 d后,有部分AT2细胞特异性标志物proSPC阳性细胞开始出现I型肺泡上皮细胞(type 1 alveolar epithelia...     

肺泡Ⅱ型上皮细胞转分化

哺乳动物肺泡上皮细胞主要由肺泡Ⅱ型上皮细胞（AECⅡ）和肺泡Ⅰ型上皮细胞（AECⅠ）组成。在肺发育和肺损伤修复过程中,AECⅡ可转分化为AECⅠ,体外原代培养的AECⅡ有这种转分化的特性。现对AECⅡ转分化的标志、影响及调控因素及其在肺损伤中的作用进行综述。     

大鼠肺泡Ⅱ型上皮细胞的原代分离

目的建立大鼠肺泡Ⅱ型上皮细胞(alveolar epithelial cells typeⅡ,AECⅡ)分离、纯化、原代培养及鉴定的方法。方法用4.2U/ml的弹性蛋白酶通过气管插管注入肺泡内,消化分离AECⅡ。把细胞悬液接种到包被有大鼠IgG的塑料平皿中纯化细胞。用电镜、碱性磷酸酶显色法、改良巴氏染色法、单宁酸染色法、免疫组化染色法鉴定AECⅡ。结果细胞纯度达到90%以上,倒置显微镜下可见细胞呈岛屿状生长。电镜下可见细胞内有大量板层小体,包膜上有绒毛结构。碱性磷酸酶染色法(BCIP/NBT)可见胞浆内有蓝色颗粒。改良巴氏染色法、单宁酸染色法可见胞浆内有黑色颗粒。抗大鼠肺泡表面活性蛋白A(surfactant protein A,SP-A)免疫组化染色呈阳性反应。结论弹性蛋白酶作用温和,不损伤胞膜,分离所得细胞活力好;IgG免疫粘附纯化法操作简单,纯化效率高。电镜、BCIP/NBT、巴氏染色、单宁酸染色及免疫组化染色等鉴定方法稳定可靠,特异性高。     

丹皮酚减轻晚期糖基化终末产物诱导的人Ⅱ型肺泡上皮细胞损伤

目的探究丹皮酚(Paeonol,Pae)对晚期糖基化终末产物(advanced glycationend end products,AGEs)诱导的人Ⅱ型肺泡上皮细胞(HEPApiC)损伤的影响及机制。方法用Pae预处理HEPApiC 1 h,然后用AGE-BSA刺激48 h。采用MTT法检测细胞活力;采用TUNEL法和Annexin V-FITC/PI染色法检测细胞凋亡;分别采用DCFH-DA测定法和SOD活性检测试剂盒检测细胞内ROS生成和SOD活性;采用Western blot检测细胞中cleaved-caspase3、Bax和细胞质细胞色素C(Cyt C)水平。结果 Pae能抑制AGEs诱导的HEPApiC活力降低、凋亡增加,ROS生成增加和SOD活性降低。另外,在HEPApiC中,Pae能抑制AGEs诱导的Cleaved-caspase3、Bax和细胞质Cyt C水平升高。结论 Pae通过抑制AGEs诱导的氧化应激和线粒体依赖性凋亡途径来抑制HEPApiC的凋亡。     

胎鼠肺泡Ⅱ型上皮细胞分离、纯化与鉴定

肺泡Ⅱ型上皮细胞（type Ⅱ alveolar epithlial cell,AEC Ⅱ）一直是肺疾病领域的研究热点和难点.AEC Ⅱ是肺泡上皮细胞的＂祖细胞＂,与肺组织生理功能密切相关.获取胎鼠AEC Ⅱ为该领域提供了良好的实验模型和基础.迄今为止,尚没有一套完善的有关AEC Ⅱ分离、纯化与鉴定的方法.文章就目前国内外AEC Ⅱ分离、纯化、鉴定的实验室方法进行综述与介绍,为肺疾病领域的研究奠定实验基础.     

经气管灌注血管紧张素Ⅱ导致凋亡性急性肺损伤(英文)

为研究外源性血管紧张素Ⅱ（angiotensinⅡ,ANG）在急性肺损伤和肺泡上皮细胞凋亡中的作用,经气管分别给雄性Wistar大鼠（175-200g）灌注ANG、ANG加caspase抑制剂ZVAD—fmk、ANG加ANG受体1阻断剂losartan和仅灌注磷酸盐缓冲溶液（PBS）。6或20h后在体灌洗动物肺脏,测定灌洗液中血红蛋白（hemoglobin,Hb）和荧光物质（BODIPY）标定的白蛋白含量（在灌洗前15min静脉注入BODIPY-白蛋白）。TUNEL测定显示,灌注ANG6h后,支气管和肺泡上皮细胞内标定的DNA片断显著增2H（P〈0．05）;ANG所致的DNA片断增加可被同时灌注ZVAD—fmk或losartan阻断。灌注ANG后免疫标定caspase3阳性细胞数量显著增多（P〈0．01）,ZVAD—fmk或losartan同样显著减少caspase3阳性细胞的数量。灌注ANG显著增加肺泡灌洗液中荧光标定的白蛋白（P〈0．01）和Hb的含量（P〈0．05）;ZVAD—fmk或losartan亦显著抑制荧光白蛋白和Hb含量的变化。结果表明,肺泡上皮细胞在体暴露于外源性ANG足以引起ANG受体1介导的上皮细胞凋亡和肺泡屏障损伤。     

香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

肺部微生物组成与特发性肺纤维化生存率相关性研究

目的 运用16S rRNA基因测序技术分析特发性肺纤维化（idiopathic pulmonary fibrosis,IPF）患者支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）的微生物组成,研究肺部微生物组成与IPF预后相关性。方法 选择2016年3月—2018年3月在本院诊治的IPF患者39例,作为IPF组,以体检健康成年人为对照组,40例。收集患者临床资料、生存时间,检测治疗前肺泡灌洗液中微生物组成。结果 IPF组Shannon指数明显高于对照组（P=0.024）。IPF组患者奈瑟氏球菌、金黄色葡萄球菌、溶血葡萄球菌、肺炎链球菌检出率明显高于对照组（P=0.005,0.003,0.003,0.000）。39例IPF患者随访时间为2~60个月,随访结束时患者死亡20例,存活19例,生存率为48.7%,平均生存时间39.8个月,奈瑟球菌、金黄色葡萄球菌、溶血葡萄球菌、肺炎链球菌检出与IPF患者的预后成反比。结论 IPF患者肺内微生物多样性增加,奈瑟球菌、金黄色葡萄球菌、溶血葡萄球菌、肺炎链球菌检出与IPF患者生存率呈负相关,可能成为改善IPF的潜在靶点。     

与成纤维细胞共培养的肺泡Ⅱ型上皮细胞的生物学特性

建立胎鼠肺泡Ⅱ型上皮细胞（AECⅡ）与肺成纤维细胞（LF）共培养模型,观察与LF共培养下AECⅡ的生物学特性。倒置相差显微镜观察AECⅡ形态和基本生长情况：RT-PCR和流式细胞术分别检测肺泡表面活性蛋白-C（SP-C）、水通道蛋白5（AQPS）mRNA及蛋白质表达;流式细胞术检测细胞周期及Ki67表达。结果显示,与LF共培养时,AECⅡ能较好地保留其细胞形态。SP-C mRNA及其蛋白质表达明显增加,而AQP5mRNA及其蛋白质表达则明显减少;LF促进AECⅡ增殖,使G2／M、S期细胞及表达Ki67^＋细胞的比率明显增多。结果提示,AECⅡ与LF共培养时,能更好地保留其细胞形态、分化及增殖特性。     

肺泡II型上皮细胞转分化

哺乳动物肺泡上皮细胞主要由肺泡II型上皮细胞(AECII)和肺泡I型上皮细胞(AECI)组成。在肺发育和肺损伤修复过程中,AECII可转分化为AECI,体外原代培养的AECII有这种转分化的特性。现对AECII转分化的标志、影响及调控因素及其在肺损伤中的作用进行综述。     

Major histocompatibility complex and alveolar epithelial apoptosis in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by fibroblast expansion, and tissue remodeling. It is considered a multifactorial disease but the possible involved genes are largely unknown. Interestingly, studies regarding the possible role of major histocompatibility complex (MHC) are scanty and show contradictory results. In this study, we evaluated the polymorphisms of the MHC, locus HLA-B, -DRB1, and -DQB1 in a cohort of 75 IPF patients and 95 controls by using PCR and hybridization with sequence-specific oligonucleotide probes. In addition, we examined the effect of bronchoalveolar lavage (BAL) from IPF patients with different MHC haplotypes on alveolar epithelial growth rate by WST-1 cell viability assay and on epithelial apoptosis by flow cytometry and by cleaved caspase-3 in cell homogenates. Three haplotypes were significantly increased in IPF: (1) HLA-B*15-DRB1*0101-DQB1*0501 (OR=10.72, CI=1.43–459.6; pC=0.011); (2) HLA-B*52-DRB1*1402-DQB1*0301 (OR=4.42, CI=1.21–24.1; pC=0.024); and (3) HLA-B*35-DRB1*0407-DQB1*0302 (OR=4.73, CI=1.53–19.5; pC=0.005). BAL from patients with the later haplotype significantly reduced epithelial growth rate (∼30%) and caused epithelial cell apoptosis assayed by cleaved caspase-3 (351.7±16.5 pg/10⁶ cells versus 264±24 from controls, and 274±36.8 and 256.5±10.7 from the other haplotypes; P<0.05), and DNA breaks labeling by flow cytometry (23.7±6.9% versus 3.1±0.7% from controls, and 6.5±0.6% and 7.6±1.2% from the other two haplotypes; P<0.01). These findings suggest that some MHC polymorphisms confer susceptibility to IPF, which might be related with the induction of epithelial cell apoptosis, a critical process in the development of the disease.     

Effects of type II alveolar epithelial cells on T cell mitogenic responses to concanavalin A and purified protein derivatives

To investigate the role of type II alveolar epithelial cells during the T cell-dependent host immune response to Mycobacterium tuberculosis (MTB), effects of MTB-infected A-549 human type II alveolar epithelial cells (A-549 cells) on T cell mitogenesis in response to concanavalin A (Con A) and purified protein derivatives (PPD) were studied. Human peripheral blood mononuclear cells (PBMCs) were cocultivated with uninfected or MTB-infected A-549 cells and Con A-and PPD-induced T cell mitogeneses were examined, and the following findings were obtained. T cell mitogenesis was inhibited by uninfected as well as MTB-infected A-549 cells, even when a dual-chamber culture system was used to prevent direct cell contact between PBMCs and A-549 cells. Therefore, it appears that A-549 cells suppress T cell mitogenesis by producing some unknown humoral suppressor factors.     

Establishment of immortalized alveolar type II epithelial cell lines from adult rats

Summary We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37° C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham’s F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number. However, at later passages the 6T cells became polyploid, while the 6TN genotype remained stable. The RLE-6T and 6TN cells were not tumorigenic in nude mice. The cell isolation methods reported and the novel cell lines produced represent potentially useful tools to study the role of pulmonary epithelial cells in neoplastic and nonneoplastic lung disease.     

Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells,fibroblasts, alveolar type II cells,airway epithelial cells and lymphocytes

Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC‐specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8⁺ T cells from bronchial lymph nodes (T‐BL) and CD8⁺ T cells from lungs (T‐LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up‐regulated and 70% down‐regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over‐expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types.     

Hemoglobin is expressed in alveolar epithelial type II cells

Hemoglobin is the main oxygen carrying heme protein in erythrocytes. In an effort to study the differential gene expression of alveolar epithelial type I and type II cells using DNA microarray technique, we found that the mRNAs of hemoglobin alpha- and beta-chains were expressed in type II cells, but not in type I cells. The microarray data were confirmed by RT-PCR. The mRNA expression of both chains decreased when type II cells trans-differentiated into type I-like cells. Immunocyto/histochemistry revealed that hemoglobin protein was specifically localized in type II cells of a lung cell mixture and rat lung tissue. The endogenous synthesis of hemoglobin in alveolar epithelial cells suggests that hemoglobin may have unidentified functions other than oxygen transport in the lung.     

Properties of freshly isolated type II alveolar epithelial cells

The biochemical characteristics of type II alveolar epithelial cells dissociated from adult rabbit lung by instillation of low concentrations of an elastase trypsin mixture are reported. Cells studied immediately (within 4 h) after isolation were found to incorporate the radioactively labelled precursors [U-¹⁴C]glucose, [methyl-³H]choline and [³H]palmitate into cellular phosphatidylcholine at rates 2–10-fold higher than previously reported for cells not subject to short-term cell culture. Secretion of phosphatidylcholine was stimulated by beta-adrenergic agonists. Measurement of specific activities of enzymes of phospholipid biosynthesis in subcellular fractions of isolated lung cells showed a significant enrichment of acyl coenzyme A-lysophosphatidylcholine acyltransferase, an enzyme believed to be involved in pulmonary surfactant phosphatidylcholine remodeling, in the endoplasmic reticulum of type II cells. These observations support the utility of freshly isolated type II cells as a model system for the study of the functions of the alveolar epithelium.     

Isolation of alveolar type II cells by centrifugal elutriation

Summary Centrifugal elutriation (counterflow centrifugation) was used to develop a reproducible method for obtaining a nearly pure population of isolated alveolar type II cells. Lung was dissociated into individual cells with recrystallized trypsin, and the type II cells were partially purified by centrifugation on a discontinuous density gradient. The alveolar type II cells were finally purified by centrifugal elutriation. Cells were collected from the elutriator rotor by stepwise increases in flow rates. Cells obtained at flow rates of 7 and 14 ml per min were lymphocytes, other small cells, a few type II cells and cell debris; cells collected at flow rates of 18 and 22 ml per min were mainly type II cells; and cells collected at flow rates of 28, 34 and 43 ml per min were macrophages, some type II cells, other lung cells and cell aggregates. At flow rates of 18 and 22 ml per min, 1.9±1.0×10⁶ cells per rat lung (mean±S.D.,n=30) were recovered of which 86±6% were type II cells. At these flow rates, 94% of the cells excluded the vital dye erythrosin B from their cytoplasm. They consumed oxygen at a rate of 101±21 nmol per hr·10⁶ cells (mean±S.D.,n=4), and their oxygen consumption increased only 10% after 10mm sodium succinate was added. The cells incorporated [¹⁴C]leucine into protein and lipid for 4 hr. Electron micrographs of the cells collected at flow rates of 18 and 22 ml per min show a high percentage of morphologically intact alveolar type II cells. We conclude that centrifugal elutriation is a reproducible method for obtaining nearly pure, metabolically active alveolar type II cells. Postdoctoral trainee supported by Grants HL-05251 and HL-07192 from the National Heart, Lung and Blood Institute. This work was supported by U.S. Public Health Service Grants Program-Project HL-06285 and Pediatric Pulmonary SCOR HL-19185, and by a grant-in-aid from the American Heart Association (77-1098).