Analysis of the pedigree of a child with interstitial lung disease caused by a novel mutation of p. K114N in SFTPC gene / 中国小儿急救医学

Objective:To analyze the clinical and chest CT features in a family with interstitial lung disease(ILD), and assess the probable causative gene mutations for the family.Methods:In order to identify the etiology of the proband′s ILD, the pedigree was investigated.The clinical data of five proband′s pedigree members were collected, and the chest HRCT examination was performed on four proband′s pedigree members with respiratory symptoms.The human whole exon sequencing was performed on the proband′s blood samples, then its deleterious effects were assessed.Subsequently, the strong pathogenic mutation was validated by Sanger sequencing.Results:According to the family survey, there were five patients with ILD in the family, including three males and two females.One of them died.The surfactant protein C(SFTPC)gene(exon4, c.342G>T, p.K114N)was found in all four surviving patients.The mutation was considered to be originated from the father of the proband, and the pathogenic mutation was considered, which was not included in the databases and was a noval mutation.In addition, the clinical manifestations of different patients in the family were significantly different.Conclusion:The novel mutation of p. k114n in SFTPC gene can lead to ILD in children, and the mutation has incomplete exons in family members.Chest CT and whole exon sequencing play an important role in the diagnosis of ILD in children.

Protective effects of bone marrow mesenchymal stem cell-derived exosomes on smoke inhalation injury in rats / 医学研究生学报

Objective Acute lung injury induced by variety causes can be reduced by mesenchymal stem cells.Some studies have shown that mesenchymal stem cell-derived exosomes have similar features with mesenchymal stem cell,but its role in acute lung injury is less studied.The study was to investigate the protective role and underlying mechanisms of bone marrow mesenchymal stem cell-derived exosomes (BMSC-DEs) on smoke inhalation injury (SⅡ) in rats.Methods Thirty Wistar rats were randomly divided into 3 equal groups:normal control group,smoke inhalation injury (SⅡ) model group and bone marrow mesenchymal stem cell-derived exosomes (BMSC-DEs) treated group.12 h after establishing the SⅡ model,BMSC-DEs treated group was injected with 0.5 mL BMSC-DEs (derived from 4× 106 BMSCs),and normal control group and SⅡ model group were injected with equivalent volume of normal saline.7 days later,samples were collected.The histopathologic changes of lung were observed after HE staining;BCA was used to test the amounts of total protein in bronchoalveolar lavage fluid (BALF);Enzyme linked immunosorbent assay was used to test the levels of tumor necrosis factor-α (TNF-α) and keratinocyte growth factor (KGF) in the lung tissue;Immunohistochemical was used to test the levels of pulmonary surfactant protein C(SP-C).Results The BALF levels of total protein of SⅡ group was significantly higher than those of normal control group (P＜0.01) and BMSC-DEs groups(P＜0.05);Compared with normal group [(0.164±0.021) ng/L],the levels of tumor necrosis factor-α of SII and BMSC-DEs groups [(0.355±0.106)、(0.234±0.024) ng/L] (P＜ 0.05) were significantly higher,and SⅡ group was higher than that of BMSC-DEs group(P＜0.01);Compared with normal group,the KGF protein expression level in lung tissue of SⅡ group was significantly lower (P＜0.05),but BMSC-DEs group was higher (P＜0.05).BMSC-DEs group was higher than SⅡ group (P＜0.01);Immunohistochemistry showed that the SP-C expression level in lung tissue of SⅡ group was significantly lower than those of other groups (P＜0.05).There was no statistically difference between BMSC-DEs group and control group (P＞0.05).Conclusion BMSC-DEs has a protective effect of smoke inhalation injury rats,the underlying mechanism may be related to BMSC-DEs to reduce inflammation and promote restoration of the alveolar epithelial type Ⅱ.

Decreased expression of surfactant protein C in lung tissue of chronic obstructive pulmonary disease rats / 基础医学与临床

Objective To study the role of surfactant protein C ( SP-C) in rat lung of chronic obstructive pulmonary disease (COPD).Methods Forty healthy conventional Sprague-Dawley (SD) rats were randomly divided into four groups , normal control group ( control group ) , smoke exposure group ( smoking group ) , lipopolysaccharide group (LPS group), smoke exposure +Lipopolysaccharide group (COPD group).The arterial partial pressure oxygen (PaO2) and arterial partial pressure of carbon dioxide pathological (PaCO2) were detected.The ultrastructure of lung tissue was observed by transmission electron microscope .Enzyme-linked immuno sorbent assay (ELISA) were performed to determine protein expression of SP-C in lung and bronchoalveolar lavage fluid ( BALF).RT-qPCR were performed to determine mRNA expression of SP-C in lung.Results Compared with control group , smoking group and LPS group, the PaO2 of COPD group was obviously lower , the PaCO2 of COPD group was obviously higher;the ultrastructure and histological analysis of lung tissues showed chronic inflammatory injury ; Compared with control group , the expression of SP-C protein in was reduced , as well as SP-C mRNA expression .Conclusions The expression of SP-C in lung of rats COPD model is down-regulated.SP-C may be involved in COPD .

Pediatric Case Report on an Interstitial Lung Disease with a Novel Mutation of SFTPC Successfully Treated with Lung Transplantation

Mutations of the surfactant protein (SP)-C gene (SFTPC) have been associated with neonatal respiratory distress syndrome (RDS) and childhood interstitial lung disease (ILD). If accurate diagnosis and proper management are delayed, irreversible respiratory failure demanding lung transplantation may ensue. A girl was born at term but was intubated and given exogenous surfactant due to RDS. Cough and tachypnea persisted, and symptoms rapidly progressed at 16 months of age despite treatment with antibiotics, oral prednisolone, methylprednisolone pulse therapy, and intravenous immunoglobulin. At 20 months, she visited our hospital for a second opinion. A computed tomography scan showed a diffuse mosaic pattern with ground-glass opacity and subpleural cysts compatible with ILD. A video-assisted thoracoscopic lung biopsy revealed ILD with eosinophilic proteinaceous material and macrophages in the alveolar space. Bilateral lung transplant from a 30-month-old child was done, and she was discharged in room air without acute complications. Genetic analysis revealed a novel c.203T>A, p.Val68Asp mutation of SP-C, based on the same exon as a known pathogenic mutation, p.Glu66Lys.

Development of a Synthetic Surfactant Using a Surfactant Protein-C Peptide Analog: In Vitro Studies of Surface Physical Properties

PURPOSE: Pulmonary surfactant (PS) replacement has been the gold standard therapy for neonatal respiratory distress syndrome; however, almost all commercial PSs contain animal proteins. We prepared a synthetic PS by using a human surfactant protein (SP) analog and evaluated its in vitro properties. MATERIALS AND METHODS: A peptide sequence (CPVHLKRLLLLLLLLLLLLLLLL) of human SP-C was chosen to develop the peptide analog (SPa-C). The new synthetic SP-C PS (sSP-C PS) was synthesized from SPa-C, dipalmitoyl phosphatidylcholine, phosphatidyl glycerol, and palmitic acid. Physical properties of the sSP-C PS were evaluated by measuring the maximum and minimum surface tensions (STs), surfactant spreading, and adsorption rate. In addition, we recorded an ST-area diagram. The data obtained on sSP-C PS were subsequently compared with those of purified natural bovine surfactant (PNBS), and the commercial product, Surfacten(R). RESULTS: The sSP-C PS and Surfacten(R) were found to have maximum ST values of 32-33 mN/m, whereas that of PNBS was much lower at 19 mN/m. The minimum ST values of all three products were less than 10 mN/m. The values that were measured for the equilibrium ST of rapidly spreading sSP-C PS, Surfacten(R), and PNBS were 27, 27, and 24 mN/m, respectively. The surface adsorptions were found to be the same for all three PSs (20 mN/m). ST-area diagrams of sSP-C PS and Surfacten(R) revealed similar properties. CONCLUSION: In an in vitro experiment, the physical properties exhibited by sSP-C PS were similar to those of Surfacten(R). Further study is required to evaluate the in vivo efficacy.

Differentiation of bone marrow mesenchymal stem cells induced by alveolar epithelial cells of rats exposed to silica dust / 中国职业医学

OBJECTIVE: To observe whether bone marrow mesenchymal stem cell( BMMSC) could be induced by alveolar epithelial cell( AEC) of rats exposed to silica dust or not. METHODS: BMMSCs were isolated and cultivated from 6specific pathogen free healthy male SD rats through bone marrow adherent method. The AECs from other 6 rats randomly selected from the same batch were cultivated by immune adherent purification method. Three rats were treated with 1. 0 m L( 40 g/L mass concentration) of silicosis dust suspension by one time intratracheal injection as silicosis dust exposure model,and the other 3 rats were given 0. 9% sodium chloride solution as normal. Experimental group was the co-culture of BMMSCs and AECs from silicosis dust exposure rats. Control group A was the co-culture of BMMSCs and AECs from normal rats. Control group B was the culture of BMMSCs alone. The morphology changes were observed by the inverted phase contrast microscope at the time points of the 4th and the 8th day. Double immunofluorescence staining using aquaporin 5( AQP5) and surfactant protein C( SP-C) was performed on the treated BMMSCs. The fluorescence staining was observed using the inverted fluorescence microscope( IFM) and laser scanning confocal microscope( LSCM). Integral optical density( IOD) analysis was conducted on fluorescence of 2 kinds of proteins by Image-pro plus 6. 0 graphic analysis software. RESULTS: After the co-culture,the BMMSCs in experimental group and control group A changed from long spindle shape to cubic and polygonal shape,the variation of morphology on day 8 was more obvious than that on day 4,and the change in control group A was less obvious than that of experimental group. There was no obvious morphology change in BMMSCs of control group B. By IFM and LSCM,on day 4 and day 8,the expression of green fluorescence AQP5 and red fluorescence SP-C were all observed in BMMSCs of experimental group and control group A. The BMMSCs of control group B only showed a little green fluorescence expression of AQP5,no expression of red SP-C fluorescence was seen. Both by IFM and LSCM,on day 4 and day 8,the 2 kinds of IOD of BMMSCs in experiment group were higher than those of control group A and B at the same time points( P < 0. 01); the IOD of control group A was higher than that of control group B at the same time point( P < 0. 01). The IOD of experiment group and control group A on day 8 were higher than those on day4 in the same group( P < 0. 01). CONCLUSION: AEC of rats exposed to silica dust can effectively induce BMMSC to be differentiated into AEC.

Association between gene mutations of surfactant protein C gene exon Ⅱ and neonatal respiratory distress syndrome / 中华实用儿科临床杂志

Objective To investigate association between gene mutations of surfactant protein C (SP-C) exon Ⅱ and neonatal respiratory distress syndrome(NRDS).Methods From January 2013 to December 2014 in Neonatal Intensive Care Unit of Bayi Children's Hospital Affiliated to Beijing Military General Hospital,a total of 90 cases with NRDS were selected as NRDS group,and they were grouped into 3 subgroups,30 cases in each subgroup.Clinical data were collected by observation figures.A total of 90 cases without NRDS as control group were matched with NRDS group as for the gestational age (GA),gender and birth weight(BW).The study samples were from the clinically diagnosed venous sampling 1 mL,and genomic DNA was extracted with routine technique.After the polymerase chain reaction (PCR) amplification of sequencing was performed.Chromas software and Vector NTI Advanc software were used for analysis,the results were compared with the normal SP-C sequence of GenBank.Results Heterozygous missense mutations included c.115G ＞ T and c.139G ＞ C existed in exon Ⅱ of SP-C gene,which changed protein sequence.GT genotype frequency(0.067) of c.115G ＞T mutation in NRDS group was higher than that(0) in control group (x2 =7.283,P ＜ 0.05).With c.115 G ＞ T gene mutations in patients with NRDS,Ⅲ-Ⅳ grade chest changed and mortality was higher than that of control group.GC genotype frequency (0.067) of c.139G ＞ C mutation in NRDS group was higher than that(0) in control group (x2 =6.207,P ＜ 0.05).Among NRDS group,64 cases of cesarean section patients,GC genotype frequencies were lower than GG genotype frequency (x2 =9.276,P ＜ 0.01),55 cases of patients needed 3-4 PS pulmonary surfactant treatment,GC genotype frequencies were lower than GG genotype frequency (x2 =5.343,P ＜ 0.05).Conclusions Two heterozygous mutations were located in SP-C gene of exon Ⅱ in Chinese Han population of this study.These mutations have never been reported before.c.115G ＞ T of SP-C gene mutation is associated with pathogenesis and severity of NRDS and increases the risk of death in NRDS;while c.139G ＞ C of SP-C gene mutation is associated with pathogenesis of NRDS,but it does not increase the risk of severity and death in NRDS.

Surfactant protein C gene mutations associated with children interstitial lung disease / 国际儿科学杂志

Surfactant protein C(SP-C)is found to be expressed only in the alveolar type Ⅱ cells(AECⅡ),surfactant protein C gene mutations are found to be associated with Children interstitial lung disease.This review discusses the mechanism,diagnosis and therapy of the interstitial lung disease induced by the surfactant protein C gene mutations.

Investigation of surfactant protein-C gene on respiratory distress syndrome in the Han nationality new-borns of the Inner Mongolia / 中国小儿急救医学

Objective To investigate the relationship between single nucleotide polymorphisms of surfactant protein C(SP-C)gene and respiratory distress syndrome(RDS)in the Han nationality newborns in Inner Mongolia and whether there is a mutation occurs on SP-C gene exon 4 and 5.Methods One hundred newborns with RDS(case group)and 100 newborns without RDS(control group)were selected.PCR gene analysis was used to establish the genotype and allele frequencies of exon 4 (T138N)and 5 (S186N)on SP-C.Results In the Han nationality newborns of Inner Mongolia region,there was no mutation on SP-C gene exon 4 and 5.Exon 4(T138N)on SP-C could be checked out three genotypes:namely AA,AC and CC.The genetic polymorphisms of exon 4 on SP-C were not statistically different between the case group and the control group(χ2 ﹦0.744,P ﹦0.689).Besides,exon 5(S186N)on SP-C could also be checked out three genotypes:namely AA,AG and GG.The genetic polymorphisms of exon 5 on SP-C were also not statistically different between the case group and the control group(χ2 ﹦0.770,P ﹦0.681 ).Conclusion There is no mutation on SP-C gene exon 4 and 5.The genetic polymorphism of exon 4 and 5 on SP-C displays no signifi-cant correlation with RDS of the Han nationality newborns in Inner Mongolia.

Correlation analysis of surfactant protein-C genetic polymorphisms and neonatal respiratory distress syndrome of the Mongol nationality in Inner Mongolia / 中国小儿急救医学

Objective To investigate the distribution of surfactant protein-C( SP-C) gene single nu-cleotide polymorphisms and to study the association between the SP-C gene polymorphisms and neonatal respiratory distress syndrome( NRDS) in infants. Methods Fifty-one infants with NRDS( NRDS group) and 51 infants without RDS( control group) were selected. PCR gene analysis and polymerase chain reaction were used to establish the genotype and allele frequencies of SP-C exon 4(T138N) and exon 5(S186N),SP-C exon 4 and 5 for the mutation,and then the association between the polymorphisms and NRDS was analyzed. Results SP-C gene mutations were not found in exon 4 and 5. In the Mongol nationality of the Inner Mon-golia region,SP-C exon 4(T138N) genotypes could check out three genotypes:namely AA,AC and CC. The frequencies of allele A and allele C of SP-C exon 4(T138N) were not statistically different between NRDS group and control group(χ2 =0. 454,P=0. 797). In the Mongol nationality,SP-C exon 5(S186N) genotypes could check out three genotypes:namely AA,AG and GG. The frequencies of allele A and allele G of SP-C exon 5(S186N) were not statistically different between NRDS group and control group(χ2 =0. 493,P =0. 782). Conclusion SP-C exon 4(T138N) and exon 5(S186N) gene polymorphism in Inner Mongolia newborns displays no significant correlation with sex,birth weight or gestational age. SP-C gene mutations are not found in exon 4 and 5. SP-C gene exon 4(T138N) and exon 5(S186N) polymorphisms are not found to be associated with NRDS in Mongol nationality of the Inner Mongolia.

Correlation of pulmonary surfactant protein B, C and neonatal respiratory distress syndrome / 国际儿科学杂志

Neonatal respiratory distress syndrome is one of the common critically ill newborn's disease, the pathogenesis of which is closely related to gene mutation of the pulmonary suffactant protein(SP).Surfactant protein is an important component of the pulmonary surfactant, which plays an important role in the structure, metabolism, and function of pulmonary surfactant.SP-B and SP-C in pulmonary surfactant is extremely important and closely related to each other.Study on relationship between the SP gene allelic variation and neonatal respiratory distress syndrome could contribute to early genetic intervention, promote the clinical diagnosis and treatment, and bring far-reaching significance to reduce the neonatal mortality rate.

Expression of microRNA-646 in A549 cells treated by lipopolysaccharide and its function / 中华实用儿科临床杂志

Objective To measure the expression of microRNA-646 (miR-646) in A549 cells under different concentrations of lipopolysaccharide(LPS) treatment,and to explore the possible mechanism of miR-646 in A549 cells under LPS treatment.Methods A549 cells were divided into control group and experimental groups.A549 cells from the control group were treated with RPMI-1640 and A549 cells from the experimental groups were treated with LPS (5 mg/L,10 mg/L,15 mg/L) in a duration of 24 hours.Immunocytochemical method and Western blot were used to detect the changes in surfactant protein A (SP-A) and surfactant protein C (SP-C),and quantitative real-time polymerase chain reaction was used to detect the changes in miR-646 in all groups.Results Compared with control group,the expression of SP-A in cytoplasm of A549 cells were decreased in experimental groups (all P ＜ 0.05),and LPS in different concentrations induced the expression of SP-A in A549 cells in a dose-dependent manner.Compared with control group,the expression of SP-C in cytoplasm of A549 cells was decreased in experimental groups (all P ＜ 0.05).The expression of SP-C was the lowest in 10 mg/L LPS treatment group.The relative expression level of the control group of miR-646 was 0.9597 ±0.0200,in 5 mg/L LPS treatment group it was 1.6319 + 0.1325,in 10 mg/L LPS treatment group it was 2.4762 ±0.1380,and in 15 mg/L LPS treatment group it was 1.6642 ±0.0938.There were statistically significant differences between the experimental groups and control group (all P ＜ 0.01).Conclusion miR-646 may have a biological function in acute respiratory distress syndrome through inhibiting the transcription of SP-C.

Alveolar damage and the alveolar epithelial cell change in neonatal rats with chronic lung disease induced by hyperoxia / 中国小儿急救医学

Objective To explore the changes of alveolar morphology and alveolar epithelial cells in rats with hyperoxia-induced chronic lung diseases (CLD). Methods CLD model in neonatal rats was established by inhalation of high concentration oxygen(85%～90% ). Eighty neonatal rats were randomly exposed to hyperoxia (model group) and to room air (control group) (n =40 each). Radical alveolar counts and the alveolar septum thickness were used to evaluate alveolar development. The site and intensity of expression of SPC,AQP5 protein were detected by immunohistochemical staining,the dynamic expression of SPC mRNA,AQP5mRNA was detected by RT-PCR on day 1,3,7,14 and 21 after exposure. Results There were no significant differences about alveolar wall thickness and RAC between experimental groups and control group on day 1～3 ( P > 0. 05 ). But there was significant difference between the model group and the control groups on day 7 and 14 (P <0. 01 ). For model group,alveolar septum thickness peaked on day 21, the difference was significant compared with control group ( 10. 62±5.01 vs 3.62±0. 88, P < 0. 001 ), but RAC decreased to the lowest level, the difference was significant compared with control group ( 3.57±1.24 vs 10. 47±0. 88,P <0. 001 ). The expression of SPC decreased on day 3 manifestedly but increased on day 7 and the levels of SPC were higher than that in the control group. Experimental group showed gradual decrease in AQP5 expression as the lung impairment devastated. Conclusion Alveolar development was delayed and alveolar epithelial cell (AEC) was damaged in the neonatal CLD rats. The changes of SPC,AQP5 expression suggested AECI was severely damaged and failed in full recovery, meanwhile the quantity of AEC Ⅱ was increased but the ability of its differentiation and transformation was decreased.

STAT3 participates in the injury of alveolar type Ⅱ epithelial cells during severe acute pancreatitis:an in vitro study / 第三军医大学学报

Objective To study the role of signal transducer and activator of transcription3 (Stat3) in alveolar type Ⅱ epithelial cells (AT-Ⅱ) treated with the serum from rat model of severe acute pancreatitis. Methods AT-Ⅱ cells of primary culture were treated with serum from rat model of severe acute pancreatitis (SAP group),or SAP serum+AG490 (JAK inhibitor),while the normal cell control was set. AT-Ⅱ cells after treatment were collected to determine activation of Stat3 by EMSA,Stat3 mRNA expression by RT-PCR,and surfactant protein C (SP-C) level in AT-Ⅱ cells by flow cytometry. Results Stat3 protein and mRNA levels were enhanced in SAP group (P

Cultivation,identification and sorting of bronchoalveolar stem cells derived from mouse lung / 第三军医大学学报

Objective To cultivate,identify and sort bronchoalveolar stem cells(BASC)derived from normal adult mouse lung.Methods After enzymatic digestion of lung tissue with dispase and collagenase in combination,the Sca-1+ cells were isolated from the obtained pulmonary cells by magnetic cell sorting.These Sca-1+ cells were cultured in dishes coated with collagen and mouse fibroblast cell line Swiss-3T3 under a serum-free culture system for BASC,which were identified by the dual-color immunofluorescent staining clara cell specific antigen(CCA)and surfactant protein C(SP-C).Finally,these pure BASC were isolated by the flow cytometry.Results One lung of normal adult mouse could yield(1.6-1.8)?107 nucleated cells in this enzyme digestion procedure.The percentage of Sca-1+ cells we sorted from lung tissue was much higher than the unsorted [(87.3?5.9)% and(9.6?1.8)%,P

Comparative Quantitative Study of Surfactant Protein C mRNA by Filter Hybridization and Solution Hybridization in Rats / 결핵

BACKGROUND: Surfactant protein C(SP-C) is a hydrophobic 5,000 dalton molecule. SP-C has the primary roles in accelerating surface spreading of a surfactant phospholipid. The filter hypbridization and solution hybridization assays are both rapid and sensitive and can be used to measure the RNAs complementary to any cloned DNA sequence. METHODS: The authors measured the SP-C mRNA levels quantitatively using solution hybridization and filter hybridization assays to obtain a standard curve equation to quantify the mRNA of unknown samples comparatively. RESULTS: 1. The minimum level of the specimens by solution hybridization was 3 pg for SP-C mRNA. 2. The standard curve equation of the solution hybridization assay between the counts per minute(Y) and the SP-C mRNA transcript input(X) was Y=6.46 X+244. The correlation coefficient was 0.9. 3. The minimum detection level of specimens by filter hybridization was 0.1 ng for SP-C mRNA. 4. The standard curve equation of the filter hybridization assay between the counts per minute(Y) and SP-C mRNA transcript input(X) is Y=2541.6 X+252.7. The correlation coefficient was 0.99. CONCLUSION: A comparison of CPM/filter in the linear range allowed an accurate and reproducible estimation of the SP-C mRNA copy number. Filter hybridization and solution hybridization assays are both rapid and sensitive and can be used to measure the RNAs complementary to any cloned DNA sequence. It is ideally suited to situations where accurate quantitation of multiple samples is required.