Objective:To construct the plant expression vector of Der f1 allergen of Dermatophagoides pteronyssinu and expression in tobacco lamina.Methods:The Der f1 gene was amplified from the glycerin bacterium which contained pET28a(+)-Der f1 plasmid,cloned into the pMD 19-T plasmid,and then sequenced.The Der f1 gene was digested by ClaⅠand SalⅠ,and cloned into potato virus X (PVX) to construct plant expression vector PVX-Der f1,and then was transformed agrobacterium tumefaciens.The positive one was selected to infect tobacco lamina for expressing target protein.The protein was identified and analysed by SDS-PAGEand Western blot.Results:Digestion and sequence analysis confirmed that the plant expression vector was correct,and the SDS-PAGE and Western blot results showed that the molecular weight of the protein was about 34M_r and it could specific binding with positive serum.Conclusion:The plant expression vector of Der f1 is successfully constructed and the recombinant protein is also produced.

Objective To explore the changes of high-sensitivity C reactive protein(hs-CRP) and platelet parameter levels in newborns with hypoxic-ischemic encephalopathy(HIE)and its clinical significance.Methods Thirty-five infants with HIE and 16 healthy newborns were selected as study cases and controls respectively by automantc biochemistry analyzer.Serum hs-CRP content was measured by reaction rate method;Platelet parameter levels were detected by collecting blood samples from peripheral vascular of heel,and the activity of creatine kinase(CK),creatine phosphokinase isoenzyme(CK-MB),lactate dehydrogenase(LDH),alpha-hydroxybntyric dehydrogenase(?-HBDH),alanine transaminase(ALT),aspartate aminotransferase(AST) were assayed as well.Results 1.The hs-CRP levels in newborns with HIE increased obviously in acute stage,and significant difference were observed compared with controls(P0.05).2.PLT in newborns with HIE decreased significantly in acute stage compared with that of controls(P0.05).3.The values of CK,CK-MB,LDH,?-HBDH,AST,ALT in newborns with HIE were significantly higher than those in controls in acute stage(P

Objective To observe the expression of vimentin (Vim) after silence of interleukin-1β (IL-1β) in rats with spinal cord contusion (SCC). Methods The model of SCC was established in 30 Sprague-Dawley rats with Allen's method. The rats were randomized into vector group (n=15) and silence group (n=15), which were injected blank lentivirus vector and vector of IL-1β siRNA, respectively; and divided in three, seven and 28 days subgroups. The relationship between IL-1βand Vim was predicted with GeneMANIA bioinformatics. The expression of Vim protein and mRNA in spinal cord was detected with immunohistochemistry and real-time quantitative polymerase chain reaction. Results GeneMANIA bioinformatic analysis indicated that there was some direct and indirect relationship between IL-1β and Vim. The Vim protein and mRNA expressed in the spinal cord, and was less in the silence group than in the vector group (t>2.875, P<0.05). Conclusion Silence of IL-1β can inhibit the expression of Vim in SCC rats, which may promote the recovery of spinal cord function.

Objective To investigated the effect of TangNaiKang (TNK) on VEGF protein expression of GK rats Thoracic aorta. Methods 51 male GK rats were divided into five groups randomly: model group, pioglitazone group, and TNK treatment group (low, immediate and high dose). Another 10 male Wistar rats were served as normal control group. GK rats were fed with high-grease forage, while normal control group was fed with a standard diet. Fasting blood glucose, general HE staining and VEGF protein expression were detected by immunohistochemistry. Results The fasting glucose had a significant decline in TNK treatment groups. HE staining showing TNK can ameliorate intima thickness, reduce hyperplasia of shallow vascular smooth muscle cell, and improve wavy and plexiform arrangement of elastic lamina. Immunohistochemistry also showed that TNK decreased VEGF protein expression of great vessels. Conclusion TangNaiKang can prevent and cure diabetic vascular complication of GK rats.

OBJECTIVETo study the prognosis and the factors affecting the prognosis in children with acute respiratory distress syndrome (ARDS).

METHODSSeventy-eight children with ARDS were enrolled. The states of their survival within 30 days were followed-up.

RESULTSOf the 78 children with ARDS, 51 cases demised, 27 cases survived, with a 30-days survival rate of 35%. The average survival time was 14.4 days (median: 8 days). The peak of death appeared within 3 days after ARDS. There were significant differences in aspects of age, primary disease, percentage of neonatal hyaline membrane disease, pediatric critical illness score (PCIS), duration of mechanical ventilation, oxygenation index (PaO(2)/FiO(2)), white blood cell count and number of involved organs between the died and survived children (P<0.05 or 0.01). The Cox multiple factors analysis showed that the age (HR 3.924~3.938), primary disease (HR=1.817) and PCIS (HR=0.469) were the risk factors of death.

CONCLUSIONSThe peak of death usually appears within 3 days after ARDS. Age, primary disease and PCIS are the independent factors of prognosis in children with ARDS.

Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Multivariate Analysis ; Prognosis ; Respiratory Distress Syndrome, Adult ; mortality ; Survival Rate

BACKGROUND: Cell-free stem cell therapy has been an issue of concern, but there is no conclusion on how to extract high-quality exosomes. OBJECTIVE: To extract exosomes from human umbilical cord mesenchymal stem cells by using three different methods, and then to screen the optimal method. METHODS: Exosomes were extracted from human umbilical cord mesenchymal stem cells by using the Total Exosome Isolation test kit, Exo Quick test kit and differential ultracentrifugation method, respectively. Then, transmission electron microscopy was used for morphological observations, BCA was utilized to quantify the protein, and western blot assay was applied to detect surface markers CD9, CD81 and CD63. RESULTS AND CONCLUSION: Extraction of exosomes was completed by all the three methods, and round or oval membranous vesicles were observed under the transmission electron microscope. The protein content and purity of exosomes was highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group, and there were significant differences among the three groups (P < 0.05). Under the same protein concentration, surface specific markers, CD81, CD63 and CD9, were expressed highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group. The operating time was significantly lower in the Exobiology Quick kit group compared with the other two groups (P < 0.05). To conclude, despite a longer operating time, the differential ultracentrifugation method is a rational method to extract enough exosomes with relative high purity.

ObjectiveUsing dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to evaluate the hemodynamic perfusion characteristics of bone marrow infiltration in patients with acute leukemia (AL). MethodsForty-seven patients with AL received coronal pelvic T1WI DCE-MRI with fast low angle shot (FLASH) sequence. Among them, 25 were initial onset untreated (IOU) patients, 22 were treated AL patients, including 14 with complete remission (CR) and 8 with non-remission ( NR). The hemodynamic perfusion parameters including maximum percentage of enhancement ( Emax ) and slope were determined based on enhancement-time curves ( ETCs ) of iliac and lumbar vertebra. The proportion of marrow myeloblasts was recorded.For all patients, quantitative perfusion parameters of bone marrow infiltration in ilium were compared with those in lumbar. The values of Emax and ES were compared among IOU,CR and NR patients.Correlations between perfusion parameters and histopathological results were assessed. ResultsIn all the 47 patients, the Emax values of bilateral iliac bone marrow ( 15.70 ± 7.06)were slightly higher than that of lumbar bone marrow ( 11. 28 ± 5.52 ), and the difference was statistically significant (P ＜0. 01 ).There was no significant difference in the slop value between bilateral iliac bone marrow (0. 82 ± 0. 12 ) and lumbar bone marrow (0. 80 ± 0. 09 ) ( P ＞ 0. 05 ). In the 25 untreated patients,the Emax and slop values were 17. 15 ± 5.75 and 0. 98 ± 0. 13, respectively; in the 14 CR patients, they were 8. 76 ±3.93 and 0. 26 ± 0. 04, respectively, and in the 8 NR patients, they were 21.62 ± 6. 50 and 1. 38 ± 0. 02, respectively. There was significant difference in the Emax and slop values among the three groups (P＜0. 05).Compared with IOU and NR patients, both the Emax and slop values decreased significantly in iliac bone marrow of AL patients with CR (P ＜ 0. 05 ). There was no significant difference between IOU and NR patients ( P ＞ 0. 05 ). A significant positive correlation was found between Emax value of iliac bone marrow and the proportion of marrow myeloblasts ( r ＝0. 501 ,P ＜0. 05 ). There was a negative correlation between slop value of iliac bone marrow and the proportion of marrow myeloblasts ( r ＝0. 235 ,P ＞0.05).ConclusionsDCE-MRI can beused for evaluating the hemedynamic characteristics of microcirculation of bone marrow infiltration in patients with AL, which can provide useful information in evaluating prognosis and monitoring therapeutic effect.

OBJECTIVETo characterize the role of Smads proteins in alpha 2 (I) collagen (COL1A2) gene expression induced by bone morphogenetic protein-2 (BMP-2) in odontoblast cell line MDPC-23.

METHODSEndogenous Smad protein expression was determined by immunocytochemistry. Smads function and their role in COL1A2 gene expression were investigated in cotransfection experiments using promoter-luciferase reporter gene construct.

RESULTSMDPC-23 cells expressed Smad1, Smad5 and Smad6. BMP-2 promoted the activation of COL1A2 promoter reporter construct. Transient overexpression of Smad1 or Smad5 was enhanced, while overexpression of Smad6 inhibited BMP-2-induced COL1A2 promoter activity. BMP-2 inducibility could be blocked by overexpression of Smad1 or Smad5 dominant negative mutant.

CONCLUSIONSSmad signaling is functioning and appears to be involved in BMP-2-induced COL1A2 collagen transcription in MDPC-23. Smad signaling may play an important role in odontoblast differentiation and dentin extracellular matrix formation mediated by BMP-2.

Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; genetics ; Cell Line ; Collagen ; genetics ; Collagen Type I ; Mice ; Odontoblasts ; cytology ; metabolism ; Smad Proteins ; physiology ; Transforming Growth Factor beta ; genetics

This study was purposed to investigate the effect of phycocyanin at different concentration on proliferation of K562 cells, to detect the changes of integrin beta1 expression and intracellular focal adhesion kinase (FAK) gene expression on the surface K562 cells treated with phycocyanin, and to explore the possible mechanism of integrin beta1 effect on phycocyanin inhibiting proliferation of K562 cells. The expression level of integrin beta1 on the surface of K562 cells was evaluated by flow cytometry (FCM); the growth of K562 cells treated with phycocyanin was measured by MTT assay; the expression level of FAK mRNA was analyzed by relatively quantitative RT-PCR after four-day culture of K562 cells with phycocyanin of 40 microg/ml, 80 microg/ml and 160 microg/ml, respectively. The results showed that integrin beta1 expression on the surface of K562 cells was significantly higher than that in bone marrow mononuclear cells (BMMNC) from normal subjects. Phycocyanin could not change the level of integrin beta1 expression. Phycocyanin could increase the expression of FAK gene on K562 cells and inhibit the proliferation of K562 cells. It is concluded that phycocyanin can inhibit the proliferation of K562 cells through enhancing the conjunction of cell stroma with integrin beta1 on K562 cell surface, up-regulating the expression level of FAK gene in K562 cells, restoring the signaling pathway of proliferation inhibition mediated by integrin beta1. The possible mechanism of phycocyanin in the proliferation inhibition of K562 cells is to increase the expression of FAK gene. The phycocyanin may be considered as a potential agent for inhibition of cancer cell proliferation.
Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Proliferation ; drug effects ; Focal Adhesion Kinase 1 ; biosynthesis ; genetics ; Humans ; Integrin beta1 ; biosynthesis ; genetics ; K562 Cells ; Phycocyanin ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the role of c-Jun and c-Fos as transcriptional factors in regulation of dentin sialophosphoprotein (DSPP) gene by a promoter-luciferase reporter gene construct in odontoblast cell line MDPC-23.

METHODSEndogenous c-Jun or c-Fos protein was determined by immunocytochemistry. The role of c-Jun or c-Fos in transcription of DSPP was investigated in co-transfection experiments using promoter-luciferase reporter gene construct containing the sequence between -791 bp and +54 bp of mouse DSPP gene.

RESULTSc-Jun and c-Fos was expressed by MDPC-23 cells, and located in the nucleus of MDPC-23 cells. Overexpression of c-Jun or c-Fos significantly inhibited luciferase activity of DSPP promoter.

CONCLUSIONThese findings suggest c-Jun and c-Fos down-regulated the transcription of DSPP gene as a transcriptional factor in odontoblast.

Animals ; Cell Line ; Extracellular Matrix Proteins ; Gene Expression Regulation ; Mice ; Odontoblasts ; Phosphoproteins ; Promoter Regions, Genetic ; Sialoglycoproteins ; Transfection