不同城市PM2.5致小鼠肝脏纤维化的效应差异研究

为阐明PM_2.5诱发肝脏纤维化的潜在机制以及导致这种不良效应的主要组分,本研究以采自我国太原、北京、杭州和广州市的PM_2.5暴露10月龄C57BL/6雌性小鼠4周后,利用组织切片染色,荧光定量PCR (qRT-PCR)以及Western blot技术检测小鼠肝脏纤维化的发生,并采用皮尔森相关系数法分析不同城市PM_2.5暴露组小鼠肝脏纤维化相关基因表达水平与各城市典型化学组分的线性相关关系.结果发现,与对照组相比,太原组和杭州组小鼠肝脏脏器系数显著降低;太原组小鼠肝脏胶原沉积面积显著增加,肝脏纤维化相关基因(Col1a1、Col3a1、TGF-β和MMP2)的转录水平显著升高,并且也仅有太原组PM_2.5暴露诱导了小鼠肝脏Col1a1蛋白的显著增高,而其他城市PM_2.5暴露组并未见上述纤维化相关因子的显著变化.相关性分析结果显示,Cr、Mn、Mo、Cs、Pb、Bi、U和Fe等金属组分与Col1a1和Col3a1的mRNA表达呈显著正相关,除NA、AC及BaP外其余15种多环芳烃均与Col1a1表达显著相关,18种PAHs之和与Col3a1表达显著相关.上述结果表明,PM_2.5暴露可导致小鼠肝脏纤维化的发生,其中太原市PM_2.5诱导肝脏纤维化发生最为显著,TGF-β及其通路中相关信号分子在介导PM_2.5诱发肝脏纤维化发生中发挥了重要作用,Cr、Mn、Mo、Cs、Pb、Bi、U、Fe及PAHs很可能是细颗粒物暴露导致小鼠肝脏纤维化的关键毒性组分.     

NLRP3 inflammasome activation determines the fibrogenic potential of PM2.5 air pollution particles in the lung

Airborne fine particulate matter(PM_2.5) is known to cause respiratory inflammation such as chronic obstructive pulmonary disease and lung fibrosis. NLRP3 inflammasome activation has been implicated in these diseases; however, due to the complexity in PM_2.5compositions, it is difficult to differentiate the roles of the components in triggering this pathway. We collected eight real-life PM_2.5samples for a comparative analysis of their effects on NLRP3 inflammasome ...     

Duplex,triplex and quadruplex PCR for the preimplantation genetic diagnosis (PGD) of cystic fibrosis (CF), an exhaustive approach

Most of cystic fibrosis (CF) pre-implantation genetic diagnosis (PGD) cases described to date are limited to the detection of ΔF508. Beside this predominant mutation, over 1000 mutations have been identified, rendering the development of a mutation-based PGD protocol impracticable. This is the reason why we, as well as the others, have developed PGD strategies on the basis of the identification of the pathogenic haplotype instead of the mutation(s). In a previous article, we reported the conditions for the co-amplification of two intragenic polymorphic markers and the F508 locus. Here we describe an improved protocol allowing the additional amplification of two new intragenic markers, intron 1 CA repeat (I1CA) and IVS17bTA. This new protocol should, theoretically, allow us to provide a diagnosis to all couples requiring PGD for CF. Using single lymphoblasts, we have tested four different PCR configurations, including one duplex, two triplexes and one quadruplex PCR. All of them gave results compatible with a clinical application. The number of single lymphoblasts tested in each series varied from 89 to 155. PCR efficiency ranged from 95.4 to 100%. A complete haplotype was achieved for 83.2 to 90.7% of the tested cells, with an allele drop out (ADO) rate comprised between 6.0 and 11.6%. We present here three cases that we performed either with the former test (one case using the triplex PCR combining F508, IVS8CA and IVS17bCA) or with the new one (one case using the triplex combining F508, I1CA and IVS17bTA and one case using a quadruplex test). We obtained two single pregnancies. Copyright © 2004 John Wiley & Sons, Ltd.     

Prenatal diagnosis of cystic fibrosis using a monoclonal antibody specific for intestinal alkaline phosphatase

A monoclonal antibody (AAP-1), specific for the intestinal isoenzyme of alkaline phosphatase (ALP), has been used to develop an immunoassay for amniotic fluid samples. Values in the immunoassay correlated closely with those obtained by direct determination of phenylalanine-inhibitable ALP. A panel of 124 control second-trimester amniotic fluids and 21 fluids with a 1 in 4 risk of a cystic fibrosis fetus were examined in the immunoassay. Eight of 10 affected cases had values below an arbitrary cut-off of one third median, while all the non-affected cases were above this level. Almost identical results were obtained by enzymatic determination of phenylalanine-inhibitable ALP. However, in both systems the false positive rate (control fluids with values below one third median), was unacceptably high. It is pointed out that at present the most effective system for the prenatal diagnosis of cystic fibrosis is achieved by measuring the ratio of intestinal to total ALP in amniotic fluid supernatants. This is probably best effected by enzymatic assay in the presence of phenylalanine and homoarginine inhibition.     

Preconception and preimplantation diagnosis for cystic fibrosis

Preimplantation diagnosis provides couples at high genetic risk the possibility of avoiding genetic disease without the need for prenatal diagnosis and selective abortion of the affected pregnancy. Following extensive background work on the reliability of genetic diagnosis in a single cell, we offered on a research basis preimplantation diagnosis to five couples at risk for offspring with the delta-F508 mutation (the major mutation causing cystic fibrosis). There was no detrimental effect from polar body removal on either fertilization or preimplantation development. Genetic analysis, undertaken in 22 polar bodies and 15 corresponding blastomeres, identified 21 embryos of which ten were transferred.     

Amniotic fluid microvillar enzyme activities in the early detection of fetal abnormalities

Measurement of the microvillar enzymes, γ-glutamyltranspeptidase (GGTP), aminopeptidase M (APM) and alkaline phosphatase (ALP), in amniotic fluid supernatant has been proposed as a method for the early prenatal diagnosis of cystic fibrosis. The activities of these enzymes in a series of other fetal abnormalities have now been examined. GGTP activities were below the 5th percentile in 28 out of 54 cases of trisomy 21 and 9 of 14 cases of trisomy 18, while APM values were below this cut-off in 26 of 54 cases of trisomy 21 and 8 of 14 cases of trisomy 18. Abnormal ALP isoenzyme ratios were found in 6 of 54 cases of trisomy 21 and 4 of 14 cases of trisomy 18. If prenatal cytogenetic studies are routinely carried out on amniotic fluid cells, the occasional confounding effect of abnormal microvillar enzymes associated with fetal trisomies rather than with cystic fibrosis should be avoided.     

Prenatal detection of cystic fibrosis; comparative study of maltase and alkaline phosphatase activities in amniotic fluid

The potential value of microvillar enzymes in the prenatal diagnosis of cystic fibrosis (CF) has previously been demonstrated and is corroborated in the present comparative study. Maltase and alkaline phosphatase (ALP) activities were studied in the amniotic fluids of 57 pregnancies with a 1 in 4 risk for CF or with a known CF outcome and in 489 controls. A simple assay for maltase activity (MU-maltase) with the fluorogenic substate 4-methylumbelliferyl α-glucoside, offers great technical advantages and an at least equal detection rate of CF, when compared to the previously used test with maltose as substrate. Intestinal ALP was estimated either as phenylalanine inhibitable activity (PI-ALP) or as the proportions of residual activity in the presence of the inhibitors phenylalanine or homoarginine. MU-maltase and PI-ALP appeared the most successful methods: both tests were able to detect 14 of the 16 (88 per cent) pregnancies with fetal CF. Each of the two tests alone also allowed a correct prediction in 24 of the 25 pregnancies at risk but with normal outcome; however all 25 cases could be correctly predicted by a combined evaluation. It is suggested that more than one intestinal enzyme activity should be evaluated to allow optimal results in the prenatal monitoring of pregnancies at high risk for CF.     

Hyperechoic fetal bowel: The perinatal consequences

A number of publications have reported an association between the finding of hyperechoic fetal bowel on prenatal sonogram and disorders such as aneuploidy and cystic fibrosis. To define more precisely the significance of this finding, we systematically reviewed the published material on the subject. Based on a total of 357 reported cases, we documented a high prevalence of cystic fibrosis (25·6 per cent) and chromosome abnormality (12·4 per cent) associated with increased bowel echogenicity in the fetus. High rates of intrauterine growth retardation (14·9 per cent), fetal demise (9·0 per cent), and prematurity (15·3 per cent) were also found. The data were obtained from a population at high a priori risk for aneuploidy and included fetuses at 1 in 4 risk for cystic fibrosis reported in two studies. This increased the bias towards an adverse outcome. The rate of complications when a hyperechoic abdomen is noted in a low-risk fetal population has so far not been delineated. Although the high frequency of complications found is of concern and warrants investigation, extrapolation of these risk figures to a fetal population at low a priori risk may not be appropriate.