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<p><b>Background</b>Idiopathic pulmonary fibrosis (IPF) is an age-related and progressive interstitial lung disease. Up to 20% of cases of IPF cluster in families, genetic factors contribute significantly to the pathogenesis of the disease. This study aimed to explore the association between rare genetic variants and IPF in Chinese Han families.</p><p><b>Methods</b>A Han family, comprising three IPF patients and five unaffected their first-degree relatives, and 100 ethnically matched control individuals from North China were enrolled in this study. Peripheral blood was collected, and genomic DNA was extracted. To elucidate if rare genetic variants are associated with the familial IPF, we performed whole-exome sequencing of affected members from a Chinese Han IPF family. Candidate rare variants were then confirmed by Sanger sequencing.</p><p><b>Results</b>We identified a potentially damaging rare variant-a heterozygous mutation c.2146G>A in exon 6 of the gene encoding for telomerase reverse transcriptase (TERT), which results in an amino acid substitution (p.Ala716Thr). We confirmed the missense mutation by Sanger sequencing in all the affected family members but did not detect this mutation in 100 ethnically matched healthy controls. Patients carried this mutation were characterized by the frequently acute exacerbation of IPF phenotype, with poor prognosis. The mean time to death was 2.8 years after diagnosis.</p><p><b>Conclusion</b>Using next-generation sequencing technology in familial IPF patients, we identified the heterozygous rare variant in TERT gene, and strengthened the importance of genetic variants in telomere-related pathogenesis in Chinese IPF patients.</p>
Subject(s)
Female , Humans , Male , Middle Aged , China , Idiopathic Pulmonary Fibrosis , Genetics , Mutation , Mutation, Missense , Pulmonary Fibrosis , Telomerase , Genetics , TelomereABSTRACT
Objective To explore the effect of heme oxygenase-1 (HO-1) on expressions of hypoxia inducing factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF)and regeneration of hepatic vascular plexus after orthotopic liver transplantation ischemia-reperfusion injury in rats.Methods Theexperimental study was conducted.According to the random number table,240 SD rats were divided into the 3 groups,80 rats in each group.Empty virus group:rats were transfected with the empty virus.Induced group:rats were transfected with HO-1 overexpression adenovirus.Inhibited group:rats were transfected with HO-1 RNAi adenovirus.Rats were made pairs (1 ∶ 1) and established rat liver transplantation model according to two cuffs method.Rats with less weight and with heavier weight were respectively chosen as donor rats and recipient rats,and then recieved tail intravenous injection of adenovirus at 24 hours before operation.(1) Detection of transfection efficiency of adenovirus before operation:HO-1 expression of liver tissue of rats in each group was detected by Western blot at 12 and 24 hours after injection of adenovirus.(2) Liver function test of recipient rats after liver transplantation:liver functions of recipient rats [alanine transaminase (ALT),aspartate transaminase (AST),alkaline phosphatase (ALP),gamma-glutamyl transferase (GGT)] were detected at l,3,7 and 14 days postoperatively.(3) Pathological histology of liver tissue and injury scores of recipient rats in the 3 groups after liver transplantation:paraffin sections of recipient rats in the 3 groups at postoperative 1 and 14 days were stained by HE staining and observed by light microscope,and were evaluated by Suzuki damage score standard.(4) Relative expressions of HIF-1α,VEGF and HO-1 in liver tissue of recipient rats were detected by Western blot.(5) Von Willebrand factor (vWF) in liver tissue of recipient rats at 14 days postoperatively was detected by immunofluorescence staining and small vessels were counted.Measurement data with normal distribution were represented as x ±s.Comparison between groups was analyzed by the independent-sample t test,comparison among groups was done using one-way ANOVA,and pairwise comparison was analyzed by the LSD test.Results (1) Detection of transfection efficiency of adenovirus before operation:the relative expression of HO-1 of liver tissue of rats at 12 and 24 hours preoperatively after injection of adenovirus was 1.08±0.16 and 1.08±0.26 in the empty virus group,1.18±0.21 and 1.39±0.19 in the induced group,0.87±0.26 and 0.57±0.12 in the inhibited group,respectively,with statistically significant differences in different time points (F =4.232,36.513,P＜ 0.05).(2) Liver function test of recipient rats after liver transplantation:level of ALT at 3 days postoperatively in the empty virus group,induced group and inhibited group was (504±67)U/L,(438±47)U/L and (490±39)U/L,with a statistically significant difference (F=3.517,P＜0.05).Levels of ALT,AST and ALP at 7 days posto-peratively were (443±49) U/L,(430± 34) U/L,(455± 38) U/L in the empty virus group and (382± 49) U/L,(372±50) U/L,(394±25) U/L in the induced group and (493±44) U/L,(455±62) U/L,(470±72) U/L in the inhibited group,respectively,with statistically significant differences (F =10.950,5.667,5.398,P＜0.05).Levels of ALT,AST,ALP and GGT at 14 days postoperatively were (394±46)U/L,(361 ±68)U/L,(417 ±17)U/L,(4.5±1.1)U/L in the empty virus group and (283±47) U/L,(288±60) U/L,(332±46) U/L,(2.5±0.5) U/L in the induced group and (446± 43) U/L,(422± 51) U/L,(423± 63) U/L,(4.3 ± 1.3) U/L in the inhibited group,respectively,with statistically significant differences (F=26.906,9.924,8.013,9.279,P＜ 0.05).(3) Pathological histology of liver tissue and injury scores of recipient rats in the 3 groups after liver transplantation:liver cell swelling,loose cytoplasm and a varying quantity of inflammatory cell infiltration in the portal regions in the liver tissue of 3 groups were observed at 1 day postoperatively.A few inflammatory cell infiltrations in the portal regions,basically normal liver cell arrangement and a slightly swelling of liver cell were found in the empty virus group at 14 days postoperatively.Reduced liver cell swelling and basically normal structure of liver lobule were observed in the induced group.There were small patchy or focal necrosis of liver cell,masses of inflammatory cell infiltration in the portal regions and damage of bile duct in the inhibited group.Suzuki score at 1 day postoperatively in the empty virus group,induced group and inhibited group were respectively 6.7± 1.7,6.1 ± 1.2 and 7.6± 1.3,with no statistically significant difference (F=2.257,P＞0.05).Suzuki score at 14day postoperatively in the empty virus group,induced group and inhibited group were respectively 4.0±0.8,2.9± 0.8 and 5.1± 1.4,with a statistically significant difference (F=9.776,P＜0.05).(4) Western blot results:the relative expressions of HIF-1α and VEGF (43 KD) in liver tissue of recipient rats at 1 day postoperatively were 0.21±0.10,0.30±0.12 in the empty virus group and 0.23±0.09,0.34±0.14 in the induced group and 0.17± 0.06,0.29±0.11 in the inhibited group,respectively,with no statistically significant difference (F =0.902,0.410,P＞0.05).The relative expressions of VEGF (24 KD) and HO-1 in liver tissue of recipient rats at 1 day postoperatively were 1.21 ±0.25,0.55±0.12 in the empty virus group and 2.13±0.40,0.72±0.12 in the induced group and 0.91±0.22,0.26±0.07 in the inhibited group,respectively,with statistically significant differences (F=35.158,39.082,P ＜ 0.05).The relative expressions of HIF-1α,VEGF (43 KD),VEGF (24 KD) and HO-1 in liver tissue of recipient rats at 7 days postoperatively were 0.49±0.22,0.46±0.13,0.98± 0.37,0.98±0.37 in the empty virus group and 0.83±0.26,0.63±0.19,1.60±0.33,1.49±0.46 in the induced group and 0.24±0.09,0.30±0.12,0.64±0.18,0.75±0.26 in the inhibited group,respectively,with statistically significant differences (F=16.853,10.021,20.756,8.156,P＜0.05).(5) Immunofluorescence staining results:number of small vessels at 14 days postoperatively in the empty virus group,induced group and inhibited group was respectively 7.9±2.0,10.6± 1.9 and 7.6 ± 1.9,with a statistically significant difference (F=5.921,P＜0.05).Conclusion HO-1 could promote expressions of HIF-1α and VEGF in liver tissue after liver transplantation ischemia-reperfusion injury and regeneration of intrahepatic vascular plexus,and it also alleviate bile duct ischemia-reperfusion injury after liver transplantation.
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Ten phenanthrenes, two organic acids, one organic acid ester and one flavonoid were isolated from the aerial part of Juncus setchuensis by various chromatographic techniques usingsilica gel, polyamide, Sephadex LH-20 as solid phases, and preparative HPLC. Their structures were identified by MS and NMR spectroscopic data as effusol(1), juncusol(2), juncuenin D(3), dehydroeffusol(4), dehydrojuncusol(5), juncuenin B(6),dehydrojuncuenin B(7), 2-methoxyl-7-hydroxyl-1-methyl-5-vinyl phenanthrene(8), 2-hydroxyl-7-carboxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene(9), 2-hydroxyl-7-carboxyl-1-methyl-5-vinylphenanthrene(10), luteolin(11), vanillic acid(12), daphnetin(13), p-coumaric acid(14), respectively. Compound 13 was isolated from the genus Juncus for the first time and compounds 5, 8-12 were isolated from J. setchuensis for the first time. The elevated plus-maze(EPM) was used to evaluate the anxiolytic activity of compounds 6 and 7. Compound 6 at 5 mg•kg⁻¹ and 10 mg•kg⁻¹ showed anxiolytic activity as well as compound 7 at 10 mg•kg⁻¹ and 20 mg•kg⁻¹.
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A novel and sensitive HPLC-UV method has been developed for the simultaneous determination of twelve major compounds in Longdan Xiegan Pill.The chemical profile of the twelve compounds,including geniposidic acid (1),geniposide(2),gentiopicroside(3),liquiritin(4),crocin(5),baicalin(6),wogonoside(7),baicalein(8),glycyrrhizic acid (9),wogonin (10),oroxylin A ( 11 ) and aristolochic acid A (12),was acquired using high-performance liquid chromatography-diode array detector coupled with an electrospray tandem mass spectrometer (HPLC-DAD-ESI-MS).The analysis was performed on a Dikma Platisil ODS C18 column (250 mm × 4.6 mm,5 μm ) with a gradient solvent system of acetonitrile-0.1％ aqueous formic acid.The validation was carried out and the linearities ( r ＞ 0.9996),repeatability (RSD＜1.8％),intra- and inter-day precision (RSD＜1.3％),and recoveries (ranging from 96.6％ to 103.4％ ) were acceptable.The limits of detection (LOD) of these compounds ranged from 0.29 to 4.17 ng.Aristolochic acid A,which is the toxic ingredient,was not detected in all the batches of Longdan Xiegan Pill.Furthermore,hierarchical cluster analysis was used to evaluate the variation of the herbal prescription.The proposed method is simple,effective and suitable for the quality control of this traditional Chinese medicine (TCM).
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Objective To study cortactin function in cancer cell endocytosis.Methods We applied cortactin siRNA interference to MDA-Mb-231,a human breast adenocarcinoma cell line in which cortactin was over-expressed,and introduced anti-cortactin immunoreagents into the cells with BioPorter system to interfere with cortactin function in vivo.Capture-ELISA assay was used to measure transferrin uptake.We used immunoblot assay to assess the effect of cortactin knock-down and immunoflurescence microscopy to examine the effect of cortactin down-regulation on transferrin uptake.Results Interference of cortactin function in cells resulted in impairment of transferrin endocytosis.Transferrin fluorescent intensity in cytoplasm in cortactin siRNA treated-cells was significantly reduced in comparison to that of mock-treated cells.Less than 50% of cells subjected to cortactin siRNA treatment had normal transferrin uptake.Endocytosis in MDA-Mb-231 cells introduced with cortactin antibodies was impaired as well,showing a 30%～ 60% reduction in transferrin uptake.Conclusion Crtactin,an actin-binding protein,plays an essential role in cell endocytosis.