二氧化硅致肺泡上皮细胞连接蛋白43定位的变化

目的 研究SiO2刺激肺泡巨噬细胞（PAM）上清液对肺泡上皮细胞连接蛋白43（Cx43)定位的影响，以深入探索SiO2抑制肺泡上皮细胞是隙连接通讯（GJIC）的作用水平。方法 不同剂量的SiO2刺激大鼠（SD）PAM培养上清液作用于正常貂肺泡上皮细胞株CCL－64细胞[在体积分数为2％的小牛血清培养基（RPMI 1640）中加体积分数为5％的不同剂量的SiO2刺激PAM的上清液]。采用间接免疫荧光的CCL－64细胞化学法和激光共聚集扫描显微镜（LCSM，Leica TCS SP)进行Cx43定位的测定。结果 正常培养的CCL－64相邻细胞连接处有明亮的斑片状标记， 分布、连接成线；SiO2刺激PAM培养上清液作用的CCL－64细胞连接处标记斑点逐渐减少，Cx43标记斑点出现在胞浆内，呈无特异性定位状态，但随着SiO2剂量的增加，细胞内大多数的Cx43标记斑点向细胞核聚集。结论 SiO2刺激PAM培养上清液可以改变肺泡上皮细胞Cx43的定位。推测SiO2抑制肺泡上皮细胞GJIC功能可能与Cx43的内移有关。     

石英粉尘对肺泡Ⅱ型上皮细胞脂质过氧化影响的研究

石英与健康大鼠的肺泡Ⅱ型上皮细胞共同培养４ｈ后，细胞存活率随石英剂量增加而下降，胞浆标志酶乳酸脱氢酶（ＬＤＨ）活性随石英剂量增加而增加，说明石英可导致Ⅱ型上皮细胞的损伤和破坏．扫描电镜可见细胞表面结构紊乱、细胞破损．Ⅱ型上皮细胞中丙二醛（ＭＤＡ）含量增加，还原型谷胱甘肽（ＧＳＨ）减少，谷胱甘肽过氧化物酶（ＧＳＨ－Ｐｘ）增加，表明石英可引发Ⅱ型上皮细胞膜脂质过氧化反应增强．推测石英引发膜脂质过氧化反应可能是造成Ⅱ型上皮细胞直接损伤的重要机制之一。     

矽肺发病机理研究

石英(SiO2）进入肺中首先被AM吞噬。石英损伤巨噬细胞，导致细胞的氧化代谢下降，并导致释放一些生物活性物质，对细胞有毒性作用，或能促进成纤维细胞增生与胶原台成，有的还可抑制腔原合成。通过获取支气管肺泡冲洗液(BALF)，可以得到肺泡巨噬细胞及其分泌物。本课题中进行，以下的几项检查。     

石英对大鼠肺上皮细胞和成纤维细胞的增殖抑制及致hprt基因突变的研究

目的探讨石英对大鼠肺泡II型上皮细胞和成纤维细胞的增殖抑制及致hprt基因突变的差异。方法采用(MTT噻唑蓝)比色法检测大鼠肺成纤维细胞和肺泡II型上皮细胞的增殖抑制毒性。以含6-巯基鸟嘌呤(6-TG)的培养基筛选突变细胞克隆,检测hprt基因突变频率。结果在相同的染毒条件下,肺泡II型上皮细胞对石英刺激比成纤维细胞更易受损伤,上皮细胞半数增殖抑制浓度(IC50)约为140μg/cm2,成纤维细胞的IC50约为282μg/cm2。在致hprt基因突变方面,石英粉尘对两种细胞都有致突变作用。在同样剂量染毒条件下,肺泡Ⅱ型上皮细胞的hprt突变频率为84.2×10-6～156.6×10-6,较成纤维细胞(67.6×10-6～114.3×10-6)更容易发生hprt基因突变,差异有显著性(P<0.05)。结论石英对大鼠肺成纤维细胞和肺泡II型上皮细胞的细胞毒性及对hprt基因致突变作用强度存在明显差异。肺泡II型上皮细胞对石英刺激的反应敏感性高于成纤维细胞。     

石英粉尘与香烟雾吸收液致大鼠氧化损伤的实验研究

目的 探讨石英粉尘（SiO2)与烟溶液（CSS）单独及联合作用致大鼠氧化损伤的影响。方法 雄性Wistar大鼠随机分为4组，检测血液中一氧化氮（NO）、丙二醛（MDA）的含量，超氧化物歧化酶（SOD），谷胱甘肽过氧化物酶（GSH-Px)的活性以及肺巨噬细胞（AM）的DNA损伤状况，结果 石英粉尘单独作用时，实验大鼠血液中NO、MDA含量以及DNA迁移长度明显增加，而SOD、GSH-Px的活性明显降低，且与生理盐水组相比差异均有显著性，烟溶液单独作用于大鼠后，NO含量及DNA迁移长度明显增加，而GSH-Px活性明显降低，SiO2与CSS联合作用时，可协同增加大鼠体内NO、MDA的含量及DNA的损伤程度，结论 SiO2与CSS联合作用时对机体内NO、MDA的增加和DNA损伤有协同作用。     

SiO2刺激肺泡上皮细胞间隙连接功能下调的研究

目的探索矽肺发病过程中,SiO2对肺泡上皮细胞增殖和间隙连接通讯功能(GJIC)的影响.方法不同剂量的SiO2刺激SD大鼠肺泡巨噬细胞(PAM)培养上清液,作用于正常貂肺泡上皮细胞株CCL-64细胞.用四唑盐(MTT)法测定CCL-64细胞增殖(以A570nm值表示);采用激光扫描共焦显微镜(LSCM,LeicaTCS SP)用荧光光漂白后再恢复(FRAP)技术测定CCL-64细胞GJIC功能[以荧光扩散率K(×10-3/s)]表示.结果体积分数为5%的SiO2刺激的PAM上清液能诱导CCL-64细胞增殖(F=9.679,P＜0.01),并抑制CCL-64细胞间GJIC功能(F=20.587,P＜0.01).在50～500 μg/mlSiO2范围内,随SiO2浓度增加,两者均呈良好的剂量依赖性关系(GJICr=-0.943,P＜0.05;增殖r=0.891,P＜O.05).结论SiO2刺激PAM培养上清液可以抑制肺泡上皮细胞的GJIC功能,并诱导细胞增殖.推论肺泡上皮细胞GJIC功能下调在SiO2介导的肺泡上皮组织损伤中有重要的作用.     

lncRNA MRAK050699对二氧化硅粉尘诱导大鼠肺泡Ⅱ型上皮细胞上皮-间质转换的影响

[背景]矽肺发病机制未明且没有特效治疗方法,长链非编码RNA(lncRNA)在矽肺纤维化进程中或可发挥作用.[目的]探讨lncRNA MRAK050699对SiO2粉尘诱导大鼠肺泡II型上皮细胞(RLE-6TN细胞)发生上皮-间质转换(EMT)过程的影响.[方法]建立大鼠肺泡巨噬细胞(NR8383细胞)与RLE-6TN...     

实验性大鼠矽肺纤维化中核转录因子Sp1的表达及作用

目的初步探讨核转录因子Sp1在实验性矽肺发生发展中的作用。方法复制SD大鼠矽肺模型,用免疫组织化学法检测体内SiO2刺激的肺巨噬细胞、肺泡Ⅱ型上皮细胞等肺纤维化效应细胞中核转录因子Sp1蛋白表达的定位和动态变化。结果大鼠实验性矽肺模型中,SiO2刺激组与空白对照组相比,肺巨噬细胞、肺泡Ⅱ型上皮细胞、肺间质细胞中转录因子Sp1蛋白表达上调,于染尘后第14天达高峰。结论SiO2能上调肺内多种细胞Sp1的表达,Sp1可能在矽肺的发生发展过程中起重要作用。     

凋亡蛋白及其配体系统介导的细胞凋亡与尘肺的关系

尘肺是生产过程中长期吸入含游离二氧化硅（SiO2）的粉尘而发生的以肺组织纤维化为主的疾病。近年来关于石英引起的细胞功能改变、崩解死亡和肺泡结构及其细胞受损破坏，进而导致肺组织纤维化病变的机制的研究不断深入并取得许多重要的进展。目前，对尘肺纤维化的研究已进入到细胞、细胞因子和信号转导等领域。     

职业医学

04 0 6 56　二氧化硅对成纤维细胞和肺上皮细胞的DNA损伤作用 /余　晨…∥中国职业医学 2 0 0 2 ,2 9( 3) 2 9～ 31研究小剂量二氧化硅 (SiO2 )粉尘对中国仓鼠肺成纤维细胞 (CHL)和貂肺上皮细胞 (CCL_6 4)的DNA损伤作用。采用彗星试验 ,按SiO2 粉尘不同的作用浓度 (终浓度     

Antibody producing cells in the spleens of mice treated with pathogenic mineral dust.

Experiments were carried out to assess the effect of intraperitoneal injection of the mineral dusts, titanium dioxide, quartz, or asbestos, on splenic lymphocyte antibody forming cells in immunised mice. Titanium dioxide and quartz caused similar, about one third, reductions in plaque forming cells; asbestos caused substantial reduction to about a quarter of the number found in control spleens. The inhibition of antibody forming cells in the spleen found with chrysotile was dose dependent and both chrysotile and crocidolite asbestos were similar in activity. Systemic immunomodulation after local deposition of mineral dust may be important to the development of disease.     

Antibody producing cells in the spleens of mice treated with pathogenic mineral dust

Experiments were carried out to assess the effect of intraperitoneal injection of the mineral dusts, titanium dioxide, quartz, or asbestos, on splenic lymphocyte antibody forming cells in immunised mice. Titanium dioxide and quartz caused similar, about one third, reductions in plaque forming cells; asbestos caused substantial reduction to about a quarter of the number found in control spleens. The inhibition of antibody forming cells in the spleen found with chrysotile was dose dependent and both chrysotile and crocidolite asbestos were similar in activity. Systemic immunomodulation after local deposition of mineral dust may be important to the development of disease.     

Exposure-response of silicosis mortality in Swedish iron ore miners

OBJECTIVES: To assess the exposure-response relationship between exposure to quartz and fatal silicosis. METHODS: The mortality from silicosis in 7729 miners was analyzed and compared to their estimated exposure to respirable quartz. The miners had been working as a miner for at least 1 year between 1923 and 1996. Their mortality between 1952 and 2001 was studied by using information from the national cause of death register. Both underlying and contributing causes of death were considered in the analysis. The exposure to quartz was estimated from job titles and using 3239 measurements of personal exposure to respirable quartz from 1965 to 1999. The mortality rates were adjusted to attained age and years of birth using a Poisson regression. RESULTS: The median cumulative exposure among the 7729 miners was 0.9 mg x years m(-3). There were 58 deaths from silicosis. Their median cumulative exposure was 4.8 mg x years m(-3). The crude mortality rate was 53 cases per 100,000 person-years with an exposure-response relationship. CONCLUSION: There seems to be an increased risk of fatal silicosis at exposure levels around 3 mg x years m(-3) for respirable quartz.     

Estimating the quartz exposure of South African gold miners

The possibility that dust and silica exposure estimates in epidemiological studies of South African gold miners have been underestimated has been postulated for some years. These exposure estimates were obtained by converting particle number concentrations measured with konimeters and thermal precipitators to respirable mass concentrations, primarily on theoretical considerations. A detailed review of the methodology has revealed that the theoretically based dust and silica estimates were probably underestimated. In the absence of systematic side-by-side thermal precipitator and modern respirable mass measurements in the South African gold mines, the true relationship between the respirable mass concentrations and the theoretically derived concentrations cannot be known. However, with many uncertainties, we estimate that the quartz exposures of South African miners derived from past theoretically based conversions from particle number to respirable mass underestimate the actual quartz exposures by a factor of about 2.     

On the defensive action of glutamate against the cytotoxicity and fibrogenicity of quartz dust.

The cytotoxic action of quartz (DQ12) particles on cultures of rat peritoneal macrophages, as estimated by the inhibition of the TTC-reductase activity, is considerably reduced by preincubation with glutamic acid and by adding sodium glutamate (15 mg/ml) to the drinking water of the rats donating the macrophages. This increase in macrophage resistance under the influence of glutamate is the most probable cause of the delay in the development of silicotic fibrosis shown in several experiments on rats intratracheally injected with quartz and then treated by prolonged administration of glutamate. This effect is probably connected with the influence of glutamate on the stability of the macrophage membranes, which can in its turn be explained by different mechanisms, including the influence on the synthesis and phosphorylation of adenosine nucleotides. Such an influence was shown in rats receiving glutamate by the change of the ATP/ADP ratio in macrophages, but not in erythrocytes. The resistance of rat erythrocytes to the haemolytic action of quartz is also not influenced by the action of glutamate neither in vitro nor in vivo. Such differences in the influences of glutamate on two types of cells, equally susceptible to quartz cytotoxicity but considerably differing in the character of energy metabolism, is an indirect proof of the role of the latter in the realisation of the anticytotoxic, and thereby antifibrogenic, effect of glutamate.     

细胞周期蛋白D1在石英致人细胞恶性转化中的作用

目的探讨细胞周期蛋白D1在石英致人胚肺成纤维细胞(HELF)恶性转化过程中所起的作用。方法采用基因重组与基因导入的方法将表达正义和反义细胞周期蛋白D1 RNA的pXJ41-细胞周期蛋白D1导入石英恶性转化HELF细胞中。采用原位杂交和免疫组化的方法检测细胞中细胞周期蛋白D1基因表达情况,分析细胞周期蛋白D1导入前后细胞生长速度、倍增时间、细胞周期分布、软琼脂克隆形成能力的改变。结果石英诱导HELF细胞恶性转化过程中细胞周期蛋白D1基因过表达,反义细胞周期蛋白D1 RNA可抑制石英恶性转化细胞的生长增殖。与石英恶性转化细胞相比,反义pXJ41-细胞周期蛋白D1转染细胞培养至第8天时,生长速率下降58．69％,倍增时间从21．0h延长到31．4h,G1期细胞比例从45．1％增加到52．7％,S期细胞比例由40．3％下降到33．1％,克隆形成率显著下降,克隆明显变小。结论细胞周期蛋白D1的异常表达与石英恶性转化细胞有密切的关系,高水平表达的细胞周期蛋白D1对维持恶性转化细胞的恶性性状起重要的作用。     

Quartz hemolysis as related to its surface functionalities

Quartz has been observed to be a potent hemolytic agent in all red cell systems studied thus far. Its effect appears to be one of quartz surface—cell interaction: the active site on the mineral and its mechanism have long been considered the silanol group and its hydrogendonor character. The surface of the mineral quartz is hydrated in the presence of water to form such silanol groups, which are in part ionized. This imparts two functionalities, both of which are available for bonding. Binding of the proton-accepting polymer, polyvinyl-pyridine-N-oxide, to the hydrogen site on the quartz surface is readily achieved and the hemolytic effect of the mineral is blocked. However, metal salts (strong Lewis acids), at the same pH, also bind on the quartz surface and blunt hemolysis, even in the presence of unsatisfied hydrogen sites. Both the polymer compound and cationic metals may be bound to the surface simultaneously with no resultant interference in their respective bonding mechanisms. The surface of quartz is clearly bifunctional, and the ratio of sites is dependent on the degree of ionization of the silanol groups. Both bonding mechanisms are observed to be separate functionalities and must be taken into account to explain membrane activity.     

Peroxidative action of quartz in relation to membrane lysis

The possibility that the hydroxylative and peroxidative properties of quartz could be responsible for its ability to lyse various membranes has been investigated.Experiments with autoxidation of linoleate, haemolysis of red blood cells, lipid peroxidation in macrophages, and in vivo effects of antioxidants on collagen produced by quartz injected subcutaneously, have shown that peroxidation of membrane lipids is not the primary mechanism whereby quartz attacks membranes.     

Extracellular matrix components in bronchoalveolar lavage fluid in quartz exposed rats

To investigate the long term effects of quartz, bronchoalveolar lavage (BAL) and analysis of lung silica were performed in rats (n = 20) one, four, and 12 months after exposure to intratracheally instilled crystalline silica. Total and relative concentrations of silica in the lungs were highest one month after exposure. At this time BAL fluid concentrations of total cells, macrophages, and lymphocytes increased five to 10-fold compared with saline instilled controls (n = 19). The number of polymorphonuclear cells (PMNs) increased about 200-fold. The increased number of PMNs persisted during the year. Furthermore, albumin and fibronectin concentrations increased continually during the year, about two to fivefold the values of controls. Hyaluronan, by contrast, increased during the four month period (about eightfold) but decreased after one year to the one month concentration. Phospholipids in BAL fluid, raised already after one month, remained high at one year. The findings suggest progressive damage of the alveolar and interstitial tissues. Moreover, the increases in components of the extracellular matrix capable of building fibrotic networks are in agreement with the microscopical findings of fibrosis. Because only total cells, macrophages, and albumin concentrations correlated weakly with the silica contents of the lung, it is unlikely that the relation between quartz burden and the reaction in the lung is simple.     

Extracellular matrix components in bronchoalveolar lavage fluid in quartz exposed rats.

To investigate the long term effects of quartz, bronchoalveolar lavage (BAL) and analysis of lung silica were performed in rats (n = 20) one, four, and 12 months after exposure to intratracheally instilled crystalline silica. Total and relative concentrations of silica in the lungs were highest one month after exposure. At this time BAL fluid concentrations of total cells, macrophages, and lymphocytes increased five to 10-fold compared with saline instilled controls (n = 19). The number of polymorphonuclear cells (PMNs) increased about 200-fold. The increased number of PMNs persisted during the year. Furthermore, albumin and fibronectin concentrations increased continually during the year, about two to fivefold the values of controls. Hyaluronan, by contrast, increased during the four month period (about eightfold) but decreased after one year to the one month concentration. Phospholipids in BAL fluid, raised already after one month, remained high at one year. The findings suggest progressive damage of the alveolar and interstitial tissues. Moreover, the increases in components of the extracellular matrix capable of building fibrotic networks are in agreement with the microscopical findings of fibrosis. Because only total cells, macrophages, and albumin concentrations correlated weakly with the silica contents of the lung, it is unlikely that the relation between quartz burden and the reaction in the lung is simple.