Objective To screen and analyze genes up regulated in human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide (LPS). Methods Suppression subtractive hybridization (SSH) was performed between the unstimulated HUVEC(driver) and HUVEC stimulated with LPS(tester) to generate subtractive cDNA library. The library was screened with colony dot hybridization to further verify the differentially expressed cDNA clones. Positive clones were sequenced and BLAST analyzed. The 3 novel cDNA sequences were verified by RT PCR. Results Twenty five up regulated genes related to inflammation, cellular cytoskeletal rearrangement, cellular proliferation and apoptosis, intercellular message transduction, and 3 new expression sequence tags (EST) were acquired. RT PCR indicated the expression of the new ESTs only in HUVEC stimulated by LPS. Conclusion SSH is a powerful technique of high sensitivity for the detection and clone of up regulated gene expressed in HUVEC stimulated by LPS, which may be helpful to clarify the mechanism of endothelial cells activation stimulated by LPS.

To clone the full length cDNA sequence of a novel expression sequence tag ST55 (GenBank Accession No. BI121646) from human umbilical vein endothelial cells stimulated by lipopolysaccharide, rapid amplification of cDNA ends (RACE) was used to extend the 3′and 5′ends of ST55 to obtain the full length cDNA sequence according to the known ST55 sequence. The cDNA was identified by Northern blot and analyzed with bioinformatics. A novel human full length cDNA was cloned and named endothelial overexpressed lipopolysaccharide associated factor 1( EOLA1 ) (GenBank Accession No. AY074889). This gene was located at chromosomal Xq27.3 and encodes a protein EOLA1 composed of 158 amino acids. Our data show that EOLA1 is a novel human gene associated with activated endothelial cells, and may play a role in intercellular signal transduction.

Objective To study the subcellular localization and the tissue expression of EOLA1(endothelial-overexpressed lipopolysaccharide-associated factor 1). Methods The fusion protein EOLA1-EGFP expressed vector was constructed and transfected into endothelial cells. After 24 hours posttransfection, the subcellular localization of EOLA1 was detected by laser-scanning microscopy. The tissue-specific distribution of EOLA1 was assessed with Multiple Tissue Northern Blots. Results The expression of EOLA1 was tissue-specific in various human tissues. With human multiple tissue Northern blot analysis, it was shown that EOLA1 could express in the heart, skeletal muscle, kidney, liver, placenta, colon, spleen, small intestine, but did not in the brain, lung, thymus, and peripheral blood leukocyte. EOLA1 was mainly localized in cytoplasm and could move into the nucleus. Conclusion EOLA1 is one of intercellular proteins and may play a role in intercellular signal transduction.

Objective To construct small interfering RNA(siRNA) expression vectors targeting EOLA1 (Endothelial-overexpressed lipopolysaccharide-associated factor 1) gene, and to assay their effects on the expression of EOLA1. Methods GFP-EOLA1 fusion protein expressive vector pEGFP-N2/EOLA1 was constructed and transfected into ECV304 cells. The transfected cells were cultured in M199 containing G418 (400?g/ml) for 5 weeks to screen the cell line stably expressing GFP-EOLA1 fusion protein. For interfering EOLA1 target gene, complementary oligonucleotides were desiqned and synthesized at different sites. The oligonucleotides were inserted into pSinencer3.1/H1 plasmid. Then, the vector was transfected into the ECV304 cells stably expressing GFP-EOLA1 fusion protein and the inhibiting affection to target gene EOLA1 was identitied by the observation of the green fluorescence in transfected cells with inverted fluorescent microscope and Western immunoblot assay. Results The ECV304 cell line stably expressing GFP-EOLA1 fusion protein was constructed, and the siRNA vector of EOLA1 can knockdown EOLA1 gene expression specifically. Conclusion The siRNA vectors specifically interfering EOLA1 expression were constructed successfully, which would be useful in the study of function of EOLA1 gene.

To screen genes in endothelial cells resulted from responding to lipopolysaccharide (LPS), mRNA was extracted from both untreated human umbilical endothelial cells (HUVEC) following and HUVEC which were treated with LPS for 6 hours. cDNAs of both populations were synthesized to generate cDNA libraries by suppression subtractive hybridization (SSH). The libraries were then screened with colony dot blots. Positive clones were sequenced and BLAST analysed. The results showed differential genes included 3 novel genes and 22 known genes. The 3 novel genes were confirmed by Northern blotting analysis. These 22 known genes were involved in the regulation of proinflammatory response, cell apoptosis, cytoskeleton, signal transduction and energy metabolism. These results suggest that SSH is an effective technique to detect differential gene expression in HUVEC, which may be helpful to evaluate molecular mechanisms of endothelial injury induced by LPS and provide potential therapeutic targets for LPS related disturbances.

Objective To investigate the clinical characteristics of diffuse alveolar hemorrhage. Methods 12 patients with diffuse alveolar hemorrhage hospitalized in Guangzhou NO.1 Hospital were included in the research, whose clinical characteristics were analyzed. Results 7 cases of the 12 diffuse alveolar hemorrhage cases were male and 5 cases were female. 8 cases were caused by ANCA associated vasculitis , 1 cases by connective tissue disease, 1 cases by poisoning, and 2 cases of unknown etiology (medication could be considered). The clinical manifestations were fever (91.67%), hemoptysis (100%), anemia (100%), and dyspnea (3.33%). Conclusion Diffuse alveolar hemorrhage is a life-threatening clinical syndrome. It can be caused by many causes. It should be considered if there is the presence of hemoptysis, dyspnea, anemia, etc. Timely examination and early intervention can effectively improve the prognosis of the disease.

Object To explore the beneficial effect of phytoecdysone (EDS) on myocardial infarction and its mechanism of action Methods Rat myocardial infarction model was prepared by ligating the left anterior descending coronary artery, and EDS was injected ip for seven consecutive days Serum creatine phosphokinase (CPK) glutamic oxalacetic transaminase (GOT), lactic dehydrogenase (LDH) activities, infarct size(IS), coronary blood flow, capillary vessel density and vascular endothelial growth factor (VEGF) expression were determined Results 0 5, 5, and 50 mg/kg of phytoecdysone were able to effect the activities of serum CPK, GOT, LDH in a dose depending manner with an optimal effect for improving cardiac zymogram at the dose of 5 mg/kg ip At this dosage EDS can markedly reduce IS, increase coronary blood flow, capillary vessel density and the expression of VEGF Conclusion ESD can alleviate myocardial infarction symptoms The mechanism of such beneficial effect may due to its ability to promote VEGF expression regeneration of capillary vessels and increase coronary blood flow

Objective To construct the ?-gal reporter genes containing the 5′-end flanking of endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) gene in different sequence lengths and identify the sequence, which regulates the gene expression of EOLA1 by the ?-gal analysis system. Methods The target sequences were amplified by the method of genome walker, and were inserted into the upstream of ?-gal gene located in the ?-gal enhancer vector by the directional clone technique respectively; the regulative sequence was identified by analyzing the ?-gal activities of reconstructed plasmid in ECV304 cells. Results The regions, containing 2 659 bp and 1 951 bp upstreaming from exon 1, significantly stimulated the reporter gene activity as compared with that of the ?-gal control vector in transfected cells. But the region, containing 361 bp upstreaming from exon 1, did not stimulate the reporter gene activity. Conclusion There is an up-regulative element of gene transcription in the region of -361 to -1 951 bp in EOLA1 gene upstream.

Objective To identify the promoter sequence of endothelial-overexpressed lipopolysaccharideassociated factor 1 ( EOLA1) gene and to elucidate the molecular mechanisms controlling EOLA1 expression. Methods A DNA fragment containing 1 723 bp 5' upstream of the EOLA1 gene and the transcription start site was generated by polymerase chain reaction and then cloned into a luciferase reporter gene vector,pGL3-basic. The relative luciferase activities driven by this 5'-upstream fragment and a series of deletion mutants were measured in transiently transfected human ECV304 cells,respectively. At last,the 1 723 bp upstream of the EOLA1 gene was analyzed online with Cluster Buster. Results A fragment 785 bp upstream of the EOLA1 coding region was sufficient to promote transcription. Further deletion analysis of the 785 bp fragment indicated that a 68 bp element from-738 to -676 was important for EOLA1 transcription in ECV304 cells. The 1 723 bp sequence contains binding sites for Sp1 and Myf. Conclusion We map the EOLA1 promoter by deletion analysis and reveal that the proximal region ( -738 to -676 bp) ,which contains binding sites for Sp1 and Myf,is essential for human EOLA1 promoter activity in ECV304 cells.

Objective To investigate the therapeutic effect of different methods of dressing on the wound after local debridement of dia-betic foot. Methods A total of 53 patients which underwent local debridement of diabetic foot were divided into control group and treatment group. Patients in control group were dressed on traditional measurement,while patients in the treatment group were dressed on the external medicinal wine for the diabetic foot basic on the traditional treatment. The local transcutaneous oxygen partial pressure,ulcer healing rate,av-erage healing time and the amputation rate were observed. Results The healing rate and percutaneous oxygen partial pressure of the treat-ment group were significantly increased than those in the control group (P<0. 05). The average healing time and amputation rate of the treat-ment group were significantly less than those in the control group (P<0. 05). Conclusion Dressing on external medicinal wine after local debridement of diabetic foot can improve the wound healing in the diabetic foot.