AIM: To investigate the kinetics of interleukin-6(IL-6) protein and mRNA expressions in the co-culture of rat brain vascular endothelial cells(VECs) with fibrin.METHODS: The IL-6 concentration were measured by rat IL-6 ELISA kit and the IL-6 mRNA was measured by real-time PCR after development an in vitro model of in situ fibrin polymerization on rat brain vascular endothelial cells.RESULTS: Fibrin induced IL-6 expression.The IL-6 antigen concentration increased significantly in 1.0 g/L media group compared to the low dose fibrin group or control media group(P

AIM: Integrins act as receptors for molecules of the extracellular matrix (ECM) and play a major role in mediating cell morphology change,metabolism,function,migration,proliferation and differentiation. It is still unclear that whether ECM is associated with regulation of microglial activation and integrin expression. The study investigated the effects of three different ECM (fibronectin, vitronectin and laminin) on the expression of rat microglia integrin. METHODS:Experiments were performed in Research Center for Clinical Neurology, Tongji Medical College of Huazhong University of Science and Technology from May 2006 to August 2007. ①Neonatal Wistar rats were bought from Experimental Animal Center of Tongji Medical College. Animal intervention met the animal ethical standard. ②Rat microglia was cultured and purified. Culture plate was coated with fibronectin, vitronectin and laminin, and non-ECM served as control. ③The influence of individual ECM on microglia integrin expression and microglia surface expression of MHC Ⅰ by flow cytometry. RESULTS:①MHC classⅠexpression on rat microglia was significantly increased by fibronectin and vitronectin(P

To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.
Carbon Monoxide/metabolism ; Cell Hypoxia ; Cyclic GMP/metabolism ; Heme Oxygenase (Decyclizing)/biosynthesis ; Heme Oxygenase (Decyclizing)/*genetics ; Heme Oxygenase-1 ; PC12 Cells ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Up-Regulation

Objective To study the enzymatic progress of amyloid precursor protein (APP), and construct the recombinant eukaryotic expression plasmid encoding the double mutations of APP and 2 different types of fluorescent protein. Methods The last 300 bases of APP named as C99 containing APP717 mutation was amplified by polymerase chain reaction (PCR) with the template pcDNA3.0-APP.The cyan and yellow fluorescence sequences named as CFP and YFP respectively were obtained by PCR with the template of digestion of the plasmid pcDNA3.0-CFP-CaM-YFP.The 54 bases in the middle of APP named as 54 bp containingSwedish mutation were synthesized. The 4 fragments, CFP, YFP, C99 and 54 bp were inserted into the vector pcDNA3.0.By genetic engineering the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP were constructed and finally identified by enzyme digestion, PCR and sequencing. Then the 2 constructs were transfected into SH-SY5Y cells respectively. The expression of the fusion gene was examined by multi-photon con-focal microscopy and the fluorescence resonance energy transfer (FRET) signal was collected. The amyloid beta (Aβ)deposition was detected by immunocytochemistry. Results (1) DNA sequencing showed that the sequence of the construct was correct.(2)The cyan and yellow fluorescence could be seen by the multi-photon con-focal fluorescence microscopy. The fusion gene could be correctly expressed.(3)FRET was present in the cells expressing CFP-54bp-YFP,but absent in the cells expressing CFP-54bp-YFP-C99.(4)A13 labeled by YFP was detected by multi-photon con-focal microscope and deposited in the cytoplasm, membrane and in the intercellular space.(5)It was shown that by immunocytochemistry, the CFP-54bp-YFP-C99 expressionproduct could generate Aβ. The Aβ deposition was detected in the cell membrane, cytoplasma and intercellular space. Conclusions (1) The fusion protein could generate Aβ peptide by β and γ proteolytic processing.(2)Aβ may be generated in the conrse of APP transportation from cell plasma to cell membrane.(3)C99 is very important for the correct cleavage of APP. Our test data strongly suggest that C99 may function as the signal-like peptide. It may guide and direct the APP to the right location for the cleavage.(4)Before the Aβ deposition is formed outside of the cell, Aβ intracellular accumulation results in the change of cell shape.

In order to better understand the clinical manifestation of systemic lupus erythematosus (SLE) with intracranial hypertension syndrome (IHS), we analyzed the clinical features and treatment of a typical SLE patient with IHS. SLE is one of the most unpredictable autoimmune diseases involving multiple organ systems that is defined clinically and associated with antibodies directed against cell nuclei. IHS is an uncommon manifestation of neuropsychiatric SLE (NPSLE) and is characterized by an elevated intracranial pressure, papilledema, and headache with occasional abducens nerve paresis, absence of a space-occupying lesion or ventricular enlargement, and normal cerebrospinal fluid chemical and hematological constituents. IHS has been reported in a few sporadic cases in patients with SLE worldwide, but rarely has been reported in China. In this study, a 34-year-old female SLE patient with IHS was reported and pertinent literature reviewed. The clinical presentation, image logical features, and investigatory findings were discussed.
Diagnosis, Differential ; Intracranial Hypertension/diagnosis ; Intracranial Hypertension/*etiology ; Lupus Erythematosus, Systemic/*complications ; Lupus Erythematosus, Systemic/diagnosis

Objective To establish the model of distraction osteogenesis for rabbit femur bone defect, and to observe the effect of icariin on regenerate ossification after distraction osteogenesis, thus to find a method for promoting regenerate ossification after distraction osteogenesis. Methods After the rabbit model of bone defect had been established successfully, the rabbits were equipped with distraction device. And then the 24 modeled rab bits were randomly divided into 2 groups. The experimental group was injected with icariin extract of Herba Epimedii into the interspace of bone distraction, and the control group was given local injection of recombinant human bone morphogenetic protein-2 (rhBMP-2, 100μg/kg) . On week 0, 1, 4 and 6 after the resting period, X-ray photography was carried out in both groups. On week 6 after distraction osteogenesis, the bone specimens of distraction osteogenesis region in both groups were observed by micro-computerized tomography ( CT) for the comparison of bone mass, and bone mineral content and mineral density of the newly-formed bone. Results The results of the features of imageology, and the statistical data of the bone mineral content and density showed that osteogenesis speed and osteogenic quality of the experimental group were superior to those of the control group. Conclusion The rabbit model of distraction osteogenesis for femur bone defect has been established preliminarily, and icariin can promote the speed and quality of regenerate ossification after distraction osteogenesis.

To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.
Animals ; Carbon Monoxide ; metabolism ; Cell Hypoxia ; Cyclic GMP ; metabolism ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; PC12 Cells ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Up-Regulation

In order to investigate the pathogenesis of Alzheimer disease (AD) and study the enzy-matic progress of amyloid precursor protein (APP), the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP(which was named C99 containing APP717 mutation), together with the fragment encoding yellow fluorescence protein (which was named YFP) were amplified by PCR. The two fragments (YFP andC99) were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β (Aβ) was de- tected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, mem-brane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the depo-sition in the cell space and the cell shape was rough. It was suggested that Aβ could be generatedwithin the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells.Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to fur-ther study the cleavage mechanism of APP and to explore the pathogenesis of AD.

In order to better understand the clinical manifestation of systemic lupus erythematosus (SLE) with intracranial hypertension syndrome (IHS),we analyzed the clinical features and treatment of a typical SLE patient with IHS.SLE is one of the most unpredictable autoimmune diseases in-volving multiple organ systems that is defined clinically and associated with antibodies directed against cell nuclei.IHS is an uncommon manifestation of neuropsychiatric SLE (NPSLE) and is characterized by an elevated intracranial pressure,papilledema,and headache with occasional ab-ducens nerve paresis,absence of a space-occupying lesion or ventricular enlargement,and normal cerebrospinal fluid chemical and hematological constituents.IHS has been reported in a few sporadic cases in patients with SLE worldwide,but rarely has been reported in China.In this study,a 34-year-old female SLE patient with IHS was.reported and pertinent literature reviewed.The clinical presentation,image logical features,and investigatory findings were discussed.

Objective: To understand the prevalence, diagnosis and treatment of chronic critical illness (CCI) in China. Methods: The clinical data of 472 adult patients admitted to ICU in 53 hospitals, including basic information, disease-related data, nutrition program, etc., were collected on May 10, 2019, by means of multi-center cross-sectional study. If surgical intervention was needed or the occurrence of the disease was directly related to the surgery, ICU patients were regarded as surgical ICU cases (n=211). In this study, the diagnostic criteria for CCI were: (1) admission to ICU >14 days;(2) combined with persistent organ dysfunction. The prevalence,distribution and treatment of CCI and surgery-related CCI were recorded and analyzed. The Mann-Whitney U test, chi-square test or Fisher exact test were used for comparative analysis. Results: Among the 472 ICU patients from 53 hospitals, 326 were male (69.1%) and 146 were female (30.9%). The prevalence of CCI was 30.7% (145/472). Among 211 surgery-related ICU patients, 57 developed CCI with a prevalence of 27.0%. As compared to non-CCI patients, higher APACHE II score [median (IQR) 13.5 (10.0, 18.3) vs. 11.0 (7.0, 16.0), U=2970.000, P=0.007], higher Charlson comorbidity index [median (IQR) 4.0 (2.0, 7.0) vs. 3.0 (1.0, 5.0), U= 3570.000, P=0.036] and higher ratio of breath dysfunction [68.4% (39/57) vs. 48.1% (74/154), χ²=6.939, P=0.008] and renal dysfunction [42.1% (24/57) vs. 18.2% (28/154), χ²=12.821, P<0.001] were found in surgery-related CCI patients. While SOFA score, Glasgow coma score and other visceral function were not significantly different between surgery-related CCI and non-CCI patients (all P>0.05). NUTRIC score showed that surgery-related CCI patients had higher nutritional risk [43.9% (25/57) vs. 26.6%(41/154), U=5.750, P=0.016] and higher ratio of mechanical ventilation [66.7% (38/57) vs. 52.3% (79/154), χ²=3.977, P=0.046] than non-CCI patients. On the survey day, the daily caloric requirements of 50.2% (106/211) of surgery-related ICU patients were calculated according to the standard adult caloric intake index (104.6 to 125.5 kJ·kg^-1·d^-1, 1 kJ=0.239 kcal), and the daily caloric requirements of 46.4% (98/211) of patients were calculated by physicians according to the severity of the patient′s condition. 60.2% (127/211) of nutritional support therapy was enteral nutrition (including a combination of enteral and parenteral nutrition), while the remaining patients received parenteral nutrition (24.6%, 52/211), simple glucose infusion (9.0%, 19/211), or oral diet (6.2%, 13/211). The target calorie of CCI group was 104.6 (87.9, 125.5) kJ·kg^-1·d^-1, and the actual calorie intake accounted for 0.98 (0.80, 1.00) of the target calory. In the non-CCI group, the target calorie was 104.6 (87.9, 125.5) kJ·kg^-1·d^-1, and the actual calorie consumed accounted for 0.91 (0.66, 1.00) of the target calorie. There was no statistically significant difference between two groups (P=0.248, P=0.150). Conclusion The prevalence of CCI and surgery-related CCI in ICU is high, along with severe complications, respiratory and renal dysfunction and mechanical ventilation. Surgical patients admitted to ICU are at high nutritional risk, and active and correct nutritional support is essential for such patients.