Bioinformatic analysis on differentially expressed genes in A549 cells exposed to silica dust / 中国职业医学

OBJECTIVE: To analyze the differentially expressed genes(DEGs), and screen the key genes and signaling pathways in human lung epithelial A549 cells exposed to silica dust using bioinformatics and gene chip. METHODS: The GSE30215 gene expression profiles of A549 cells exposed to silica dust were downloaded from Public Gene Expression Omnibus database developed by the National Center for Biotechnology Information. The DEGs were screened by using GEO2 R analysis tools. Then, the DEGs were imported into the biological information annotation database for Gene Ontology(GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. The protein-protein interaction(PPI) network was constructed with the Search Tool for the Retrieval of Interacting Genes database and visualized using the software Cytoscape. Real-time quantitative polymerase chain reaction(PCR) was used to verify the expression of key DEGs in A549 cells. RESULTS: Of the 52 DEGs screened, 45 were up-regulated and 7 were down-regulated. The results of GO analysis showed that the DEGs were mainly distributed in extracellular region, associated with regulating biological functions such as chemotaxis, transcription factor activity and so on. KEGG pathway enrichment analysis showed that these DEGs were mainly involved in the tumor necrosis factor(TNF) signaling pathway and nucleotide-binding oligomerization domain like receptor signaling pathway. The top 10 key DEGs screened by PPI network were C-C motif chemokine ligand(CCL)2, prostaglandin-endoperoxide synthase 2, interleukin 6, C-X-C motif chemokine ligand(CXCL) 8, CXCL2, jun proto-oncogene, colony stimulating factor 2(CSF2), CCL20, TNF-α induced protein 3(TNFAIP3), and CXCL5. Real-time quantitative PCR results revealed that the changes of key genes were in consistent with the screening results, except the CCL2. CONCLUSION: We found 10 key DEGs that are related to the toxicity caused by exposure to silica dust in A549 cells by bioinformatics. Among them, CSF2, CCL20 and TNFAIP3 may provide new research direction for the mechanisms of the development of multiple pulmonary fibrotic diseases including pneumoconiosis.

Analysis on differentially expressed microRNAs for TGF-β1-induced trans-differentiation in MRC-5 cells / 中国职业医学

OBJECTIVE: To investigate the differentially expressed microRNAs(miRNAs) in human embryonic lung fibroblast MRC-5 cells stimulated by transforming growth factor-β1(TGF-β1) using microarray chip, and screen for key genes and signaling pathways of fibroblast trans-differentiation. METHODS: The miRNA expression gene chip dataset GSE43992 on TGF-β1 stimulated MRC-5 cells were downloaded from high-throughput Gene Expression Omnibus(GEO) database of National Center for Biotechnology Information of the United States. The R language Limma package was used to screen the differentially expressed miRNAs. Corresponding target genes were predicted by miRWalk database performed by Gene Ontology(GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway enrichment analysis. The protein-protein interaction(PPI) network was constructed by the search tool for the Retrieval of Interacting Genes database. RESULTS: A total of five differentially expressed miRNAs were identified, including four up-regulated miRNAs and one down-regulated miRNA; and 42 corresponding differentially expressed target genes were predicted. GO analysis indicated that the target genes were significantly enriched in collagen catabolic process, extracellular matrix organization, membrane organization, collagen fibril organization, and cellular response to amino acid stimulus. The results of KEGG pathway analysis showed that the signaling pathways corresponding to miRNAs and target genes were mainly concentrated in 18 signaling pathways, that were mainly related to the age-ethnic signaling pathways and protein digestion and absorption miRNAs in tumors and diabetic complications. The core genes transfected into the myofibroblasts by the three fibroblasts screened by the PPI network were threonine kinase 1, estrogen receptor 1 and β-catenin. CONCLUSION: Five differentially expressed miRNAs, 42 target genes, 18 signaling pathways, and 3 core genes related to TGF-β1-induced MRC-5 cell trans-differentiation were screened. It can provide new reference for the treatment and research of many diseases including pneumoconiosis and pulmonary fibrosis.

Construction and application of PGRN and Rev-erbβ double genes knockout HEK293 cell lines / 生物工程学报

In order to study the molecular mechanism and physiological significance of the interaction between PGRN and Rev-erbβ, the PGRN gene in HEK293 (Rev-erbβ-/-) marked as C3-6 cell lines was knocked out by CRISPR/Cas9 system to generate the Rev-erbβ and PGRN double genes knockout HEK293 cell lines. First, four sgRNAs were designed for PGRN gene, and PGRN sgRNA2 and sgRNA3 with the higher activity were used to construct the Lentiviral vector, pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro. Then, the lentivirus vector carrying Cas9 and double PGRN sgRNA were used to infect HEK293 C3-6 cells. Through drug screening, cloning and sequencing, we obtained the monoclonal HEK293 (Rev-erbβ-/-; PGRN-/-) marked as C3-6/23 cell lines. Using qRT-PCR and Western blotting, we detected PGRN mRNA and protein expression in C3-6/23 cell lines. Finally, genetic complementation was used to study the effect of PGRN-mediated Rev-erbβ on the regulation of the target gene promoter transcriptional activity in the C3-6/23 cell lines. In HEK293 C3-6/23 cell lines, the two DNA chains of PGRN gene were both deletion mutagenesis, and the expression mRNA and protein of PGRN did not reach the detection level. At the same time, the interaction between PGRN and Rev-erbβ enhanced the regulation of Rev-erbβ on the transcription of target gene promoter in the cell lines. Using CRISPR/Cas9 system, we successfully constructed the double knockout HEK293 (Rev-erbβ-/-; PGRN-/-) monoclonal cell lines. The study found that PGRN could affect Rev-erbβ on the regulation of target gene promoter transcription in the C3-6/23 cell lines; however, the mechanism of PGRN involvement in mediating Rev-erbβ in transcriptional regulation remains to be further studied.

The medium-term efficacy of total hip replacement with CFP handle prosthesis / 中国医师进修杂志

Objective To investigate the medium-term efficacy of total hip replacement with CFP handle prosthesis.Methods Thirty-two patients (34 hips) undergoing total hip replacement with CFP handle prosthesis were selected.The preoperative diagnosis was:femoral neck fracture in 6 cases,femoral head necrosis in 12 cases,osteoarthritis in 10 cases,ankylosing spondylitis in 3 cases,and pigmentation villi nodular synovitis in 1 case.Ten days,1 month,6 months,and 1 year (a year later) after surgery,the hip joint function and X ray film performance were followed up.Results There was no complication such as infect and injury of blood vessel and nerve.Postoperative X ray film showed the femoral handle prosthesis position was good.The average Harris hip score was 32.6 (20-53) scores before surgery,and back to 95.3(90-100) scores after surgery,no patients needed revision surgery.Postoperative Engh score was above 10 scores after half a year.Conclusion The medium-term efficacy of total hip replacement with CFP handle prosthesis is good,and can be used as one of the choice of primary total hip replacement.

Comparison of proximal femoral nail anti-rotation and anatomical locking plate in fixation of unstable intertrochanteric fracture / 中华创伤杂志

Objective To compare the outcome of proximal femoral nail anti-rotation (PFNA)versus anatomical locking plate (ALP) in treatment of unstable intertrochanteric fracture.Methods The study included 89 patients who had received PFNA fixation (PFNA group,n =45) or ALP fixation (ALP group,n =44) for unstable intertrochanteric fracture between February 2008 and September 2009.Operation time,amount of bleeding,drainage volume,postoperative complications,fracture healing time and hip joint score after fracture healing were compared between two groups.Results Mean operation time in PFNA group was obviously shorter than that in ALP group (61.4 minutes vs 114.8 minutes,P ＜0.01).Mean amount of bleeding and drainage volume were significantly lower in PFNA group than in ALPgroup (119.3 mlvs 136.8ml,P＜0.01; 74.9mlvs80.3 ml,P＜0.01).While,PFNA group was not significantly different from ALP group with regard to average fracture healing time and Harris hip joint score after fracture healing (4 months vs 4.1 months,P ＞ 0.05; 91.2 points vs 89.8 points,P ＞0.05).Two patients had poor fracture reduction in PFNA group,but all patients had good fracture reduction in ALP group.No obvious adverse postoperative complications occurred in PFNA group,but one patient had fracture nonunion in ALP group.Conclusions PFNA and ALP fixation of unstable intertrochanteric fracture present insignificant differences in fracture healing time,postoperative complications and hip joint function after fracture healing.Whereas,PFNA is more suitable for the elder patients on account of shorter operation time and less intraoperative bleeding.

The measurement of the anatomic parameter of the proximal femur and the comparative study of the two methods of defining the morphology of marrow cavity / 中国医师进修杂志

Objective To identify the relationship of the femoral morphology with the sex and age, analyze and compare the two methods of defining the morphology of marrow cavity.Methods AP and lateral radiography of the hip joint was made in 45 patiens who accepted total hip arthroplasty.The anatomic parameters of the proximal femur were measured on those radiography.The femoral morphology was analyzed using Dorr criteria and Noble class respectively.Results (1)The anatomic parameters of the proximal femur were not correlated with the age in male.(2)The inner diameter of canal 10 em distal to the lesser trochanter, isthmus width and canal to calar isthmus rate were positively correlated with age in female.(3)The medial cortical and lateral cortical thickness 10 cm distal to the lesser trochanter,cortical index and canal-flare index were negatively correlated with age in female.(4)According to Dorr criteria,13 femora were classified as type A,20 as type B,and 12 as type C.According to Noble class,4 femora were classified as champagne type,34 as normal type,and 7 as stove pipe type.Conclusions The femoral morphology don't change with age in male.The width of femoral canal increase with age in female.Type C of Dorr criteria contains all of stove pipe type and a portion of normal type.

The early results of total hip replacement with a proximally hydroxyapatite-coated femoral component / 中华创伤骨科杂志

Objective To summarize the early results of total hip replacement with a proximally hydroxyapatite-coated femoral component. Methods From February 2000 to October 2003, 65 cases (70 hips) underwent uncemented total hip replacement with a proximally hydroxyapatite-coated femoral component (U2, United Orthopedic Corporation, Taiwan, China). The diagnosis included femoral neck fractures in 30 cases, osteoarthritis in 15 cases, avascular necrosis of the femoral head in 9 cases, loosening of artificial femoral head in 8 cases, rheumatoid arthritis in 2 cases, and arthrodesis in 1 case. Clinical and radiographic evaluations were performed at 1 week, 3 and 6 months postoperatively, and yearly afterwards. Bone mineral density (BMD) of the proximal femur was measured with dual-energy X-ray Absorptiometry (DEXA) for 44 cases. Results Postoperative X-ray films showed that the initial fixation was excellent or good in all the stems. The follow-ups averaged 3 years (2 to 5.5 years). The mean preoperative Harris hip score was 32 (25 to 52) points, which were improved to 96 points at the latest follow-ups. There was no reported incidence of thigh pain at any time throughout our study. And no case needed revision. By the measurements on the X-ray films taken within 1 year postoperatively, 7 of the 70 stems had subsidence of less than 1.5 mm. No hip experienced extensive proximal end osteolysis, intramedullary osteolysis, or loosening. DEXA showed that the BMD values decreased after operation, and then returned rapidly to the normal or near normal ones 3 to 6 months after operation, especially in zones Ⅱand Ⅵwhere the increase was even more significant than at the control side. All the stems were radiographically stable with evidence of osseous ingrowth typically seen in HA-coated zones, according to the criteria proposed by Engh et al. Conclusions Since HA-coating on femoral implants can enhance the initial fixation of implants, accelerate bone ingrowth and osseous fixation of the femoral component, and seal the interface between the implant and bone to prevent ingress of polyethylene particles, it is helpful in improving clinical results of the cementless total hip replacement.