Objective To investigate the possible mechanism of the gap junctional influence on the change in permeability of the blood-brain barrier(BBB)after reperfusion subsequent to cerebral ischemia.Methods In the test laser scanning confocal microscope(LSCM)was used to investigate the change of Cx43 levels and distribution.The MCAO/R model was induced using intraluminal suture technique first described by Longa with a little modification.A total of 60 Wistar rats were divided into 4 groups:the sham-operation group,control group,octanol-treatment group and DMSO vehicle control group. Control group were further divided into seven subgroups at different time points of reperfusion after middle cerebral artery occlusion.To observe the change in permeability of BBB,Evans blue(EB)in the brain tissue was surveyed by the means of EB fluorescent quantitation.Octanol-treatment group and DMSO vehicle control group were done at the point of the peak of permeability of BBB.Octanol,the specific blocker for gap junctions(GJ)was used in an intervention study.To compare the amount of EB with the same point of groups,the influence of octanol on BBB permeability was investigated.Results At 3 h of reperfusion after cerebral ischemia for 2 h,the permeability of BBB began to increase,reached the peak at 24 h of reperfusion and was still elevated at 72 h.The Cx43 expression formed into bigger plague and remained linear disposition in the penumbra after reperfusion subsequent to cerebral ischemia.Octanol group was done at 24 h of reperfusion after cerebral ischemia.The amount of EB of octanol group((4.924?0.296)?g/g)was significantly lower than that of corresponding operation control group(5.543?0.506)?g/g.Conclusions (1)Cx43 expression is concentrated around vessels in brain.The Cx43 forms into bigger plague and the function maybe strengthens after reperfusion.Gap junction might aggravate the disruption of BBB.(2) Octanol,the specific blocker of gap junctions,could effectively prevent the permeability of BBB from increasing and has a protective effect on BBB.

ObjectiveTo investigate the efficacy of combined therapy of mild hypothermia and hibernation to treat severe brain injury. Methods24 patients with severe brain injury were randomly divided into combined therapy group and normothermia group. Glasgow Coma Scale scores of all the patients were in the range of 3 to 8. No later than 10 hours after their injury, hypothermia patients were given half dosage of No.1 hibernation cocktail and had been cooled by cooling blankets to 32℃-34℃ (rectal temperature) for 5 days, then to 35℃ for 24 hours, and slowly increased to their normal level. 3 days and 7 days after their admission, intracranial pressure,creatine phosphate kinase,partial pressure of arterial O2 and CO2, platelet and Na+,K+ were measured.7 days after their admission, Glasgow Outcome Scale scores of each patient and mortality of each group were measured. ResultsThe mortality of combined therapy group(25.0%) was significantly lower than that of normothermia group (66.6%,P<0.05). The decreased values of intracranial pressure, creatine phosphate kinase and platelet number of combined therapy group were all significantly higher than that of normothermia group respectively (P<0.05). There were no significant difference in mean artery pressure, blood electrolyte, and partial pressure of arterial O2 and CO2 between these two groups(P>0.05). ConclusionThe combined therapy of mild hypothermia and hibernation can effectively reduce the mortality of patients with severe brain injury as it is much easier, less invasive and with less complications.

In order to clarify material basis of effective parts of liangxue tongyu prescription, blood-heat and blood-stasis rat model induced by dry yeast was established. The changes of rectal temperature, blood viscosity and plasma viscosity were used to evaluate the cooling-blood and activating-blood effects of liangxue tongyu prescription and its parts. Compared with the model group, the extract from liangxue tongyu prescription, its volatile oil and n-butanol part could significantly reduce rectal temperature (P<0.01), and also reduce blood viscosity and plasma viscosity to various degrees (P<0.01 or P<0.05). So volatile oil and n-butanol part were primarily identified as effective parts of liangxue tongyu prescription. By using GC-MS with normalization method of area to analyze volatile oil of liangxue tongyu prescription, 70 compounds were identified, accounting for about 92.54%, mainly as β-asarone, paeonol, α-asarone and shyobunone. 42 compounds such as peony glycosides, tannins, and iridoid glycosides were identified by HPLC-MS techniques and standard comparison. The study determined the effective parts of liangxue tongyu prescription and clarified the chemical composition providing the foundation for further studies on material basis of liangxue tongyu prescription.
Acetophenones ; chemistry ; Animals ; Anisoles ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Gas Chromatography-Mass Spectrometry ; Oils, Volatile ; chemistry ; Rats ; Tannins ; chemistry

Lipoma is one of the unusual benign breast neoplasms and usually manifests at fatty breast of women at the age of 40 to 60. We experienced a case of large breast lipoma nearly replacing the whole left breast parenchymal tissue with mammographic finding of well-defined radiolucent mass, sonographic finding of hyperechoic mass with disorganized echopattern and computerized tomographic finding of very low attenuation mass, characteristic to adipose tissue, in a young woman of her dense breast.
Adipose Tissue ; Breast Neoplasms ; Breast* ; Female ; Humans ; Lipoma* ; Ultrasonography

Male Breast cancer is an uncommon disease with an incidence of I per cent of all breast cancers. Male breast cancer usually appears as a small mass with well-defined contour which is eccentrically located in relation to the nipple on mammogram. We report a case of breast cancer in a 51-year-old man with mammographic appearance of large hyperdense mass with nipple inversion and axillary lymphadenopathy, gray-scale sonographic finding of homogeneous solid mass and mu Itiple tumor vessels with in the mass on color Doppler ultrasound.
Breast ; Breast Neoplasms ; Breast Neoplasms, Male* ; Humans ; Incidence ; Lymphatic Diseases ; Male ; Male* ; Middle Aged ; Nipples ; Ultrasonography

Thanatophoric dysplasia is the most common lethal congenital chondrodysplasia with characteristic features of narrow thorax, short rib, severe platyspondyly, short bowed limbs and skull deformity, etc. It is not a hereditary disorder and there is usually no family history of dysplasia. We experienced a case of thanatophoric dysplasia at 38 weeks of gestation with antenatal sonographic and abdominal radiographic findings of small thorax, short bowed extremities with surrounding thickened soft tissues and marked platyspondyly. Soon atter delivery, the baby died and post-mortem radiographs showed the characteristic findings of thanatophoric dysplasia.
Congenital Abnormalities ; Extremities ; Humans ; Pregnancy ; Ribs ; Skull ; Thanatophoric Dysplasia* ; Thorax ; Ultrasonography

Background Arresting the overexpression of vascular endothelial growth factor (VEGF) will be a new approach to the inhibition of neovascularization.RNA interference (RNAi) can inhibit the expression of specific gene,and its application in eye has little interference to other gene expression.Objective This study was to investigate the effect of small interference RNA (siRNA) targeting VEGF on the expression of VEGF and retinal neovascularization in oxygen-induced retinopathy (OIR) model.Methods psi-HITM/enhanced green fluorescent protein (EGFP)/VEGF siRNA was designed and prepared in vitro.Mouse endothelioma (EOMA) were cultured in DMEM without antibiotic and divided into 5 groups.The cells were incubated in DMEM only in the blank control group;while 1 μl of LipofectamineTM 2000 + psi-HITM/EGFP,1 μl LipofectamineTM 2000 + 40,50 or 60 nmol/L of psi-HITM/EGFP/VEGF siRNA was added into DMEM in the negative control group and siRNA groups,respectively.The expression of VEGF mRNA and protein was detected by real time PCR (RT-PCR) and Western blot.The optimal effective concentration of VEGF siRNA was assessed.OIR models were established in 48 7-day-old C57BL/6J mice by raising them at an oxygen concentration of (75±2) ％ for 5 days and then to normal air.The mice were randomized into the model group,null vector group and VEGF siRNA group.1 μl of a mixture of psi-HITM/EGFP or VEGF siRNA (60 nmol/L) and LipofectamineTM 2000 was intravitreally injected,respectively,in the null vector group and VEGF siRNA group.The normal mice were used as the normal control group.Expression of VEGF mRNA and protein in the mouse retinas was detected by RT-PCR and Western blot,respectively,and FITC-dextran stretched retinal preparation was examined to evaluate the neovascularization,and retinal sections were examined to quantify the number of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM).Results The in vitro transfection test showed that the expression of VEGF mRNA and protein in the EOMA cells was significantly different among the blank control group,negative control group and 40,50,60 nmol/L VEGF siRNA groups (F =148.890,P ＜ 0.001; F =306.960,P ＜ 0.001),and the expression of VEGF mRNA was lower in different concentrations of VEGF siRNA groups than that in the blank control group (t=73.950,119.890,156.480,all at P＜0.001).Also,the expression of VEGF protein was less in different concentrations of VEGF siRNA groups than that in the blank control group (t =15.452,23.344,42.119,all at P＜0.001).The optimal inhibitory concentration of VEGF siRNA was 60 nmol/L.In vivo study determined that compared to the model group and null vector group,the non-perfusion zones and neovascular net in the retina were decreased,and the number of vascular endothelial cell nuclei extending beyond the ILM was less in the VEGF siRNA group.The relative expression level of VEGF mRNA in the retinas was 1.23±0.18,4.02±0.16,3.98±0.19 and 1.98±0.12 in the normal control group,model group,null vector group and VEGF siRNA group,respectively,with a significant difference among them (F=369.780,P＜0.001),and the relative expression levels of VEGF mRNA in the model group and null vector group were higher than that in the normal control group (t=37.880,37.336,both P＜0.001),and the expression of VEGF mRNA in the VEGF siRNA group declined by 50.8％ (t=10.183,P＜0.001).The difference in the expression levels of VEGF protein also was assayed among the various groups (F=408.980,P＜0.001),and VEGF level in the retina was lowered by 68.0％ in the VEGF siRNA group compared to the model group (t =11.473,P＜0.001).However,VEGF level in the VEGF siRNA group remained at a high level in comparison with the normal control group (t =2.422,P＜0.001).Conclusions Intravitreal injection of VEGF siRNA can attenuate retinal neovascularization by effectively downregulate the expression VEGF mRNA and protein in the retina.

BACKGROUND: The study aimed to explore the effects of hypothermia state induced by 4 oC normal saline (NS) on liver biochemistry, enzymology and morphology after restoration of spontaneous circulation (ROSC) by cardiopulmonary resuscitation (CPR) in swine. METHODS: After 4 minutes of ventricular fibrillation (VF), standard CPR was carried out. Then the survivors were divided into two groups: low temperature group and normal temperature group. The low temperature (LT) group (n=5) received continuously 4 oC NS at the speed of 1.33 mL/kg per minute for 22 minutes, then at the speed lowering to 10 mL/kg per hour. The normal temperature (NT) group (n=5) received NS with normal room temperature at the same speed of the LT group. Hemodynamic status and oxygen metabolism were monitored and the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured in blood samples obtained at baseline and at 10 minutes, 2 hours and 4 hours after ROSC. At 24 hours after ROSC, the animals were killed and the liver was removed to determine the Na+-K+-ATPase and Ca2+-ATPase enzyme activities and histological changes under a light or electron microscope. RESULTS: Core temperature was decreased in the LT group (P<0.05), while HR, MAP and CPP were not significantly decreased (P>0.05) compared with the NT group (P>0.05). The oxygen extraction ratio was lower in the LT group than in the NT group (P<0.05). The serum levels of ALT, AST and LDH increased in both groups but not significantly in the LT group. The enzyme activity of liver ATP was much higher in the LT group (Na+-K+-ATP enzyme: 8.64±3.32 U vs. 3.28±0.71 U; Ca2+-ATP enzyme: 10.92±2.12 U vs. 2.75±0.78 U, P<0.05). The LT group showed less cellular edema, inflammation and few damaged mitochondria as compared with the NT group. CONCLUSION: These data suggested that infusing 4 oC NS continuously after ROSC could quickly lower the core body temperature, while maintaining a stable hemodynamic state and balancing oxygen metabolism, which protect the liver in terms of biochemistry, enzymology and histology after CPR.

AIMTo establish methods and requirements for quality control of recombinant human tumor necrosis factor receptor Fc fusion protein (rhTNFR-Fc).

METHODSBiological potency of rhTNFR-Fc was determined by neutralizing the bioactivity of TNF-alpha. rhTNFR-Fc samples were reduced by beta-mercaptoethanol and the peptide map was performed by tryptic digestion. Residual protein A and the host cell protein content were detected by ELISA. Anti-TNFR and anti-Fc antibodies were used in ELISA for detection of the rhTNFR-Fc content.

RESULTSThe quality control methods, such as bioassay, peptide map, residual protein A detection, were established and used for quality control of rhTNFR-Fc. The unit of rhTNFR-Fc (AU) was defined according to the international unit of TNF-alpha. The specific activity was up to 8 x 10(4) AU.mg-1. The requirements for quality control of rhTNFR-Fc were established.

CONCLUSIONThe methods and requirement were used for quality control of rhTNFR-Fc products.

Animals ; Biotechnology ; methods ; Cell Division ; drug effects ; Etanercept ; Immunoglobulin G ; biosynthesis ; chemistry ; pharmacology ; Mice ; Peptide Mapping ; Quality Control ; Receptors, Tumor Necrosis Factor ; biosynthesis ; chemistry ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; pharmacology ; Technology, Pharmaceutical ; standards ; Tumor Cells, Cultured

AIMTo establish the quality control methods for recombinant human endostatin.

METHODSBiological activity was determined by endothelial cell migration assays. Peptide mapping was tested by trypsin digestion and RP-HPLC. Purity was determined by non-reduced SDS-PAGE and RP-HPLC. Other tests including molecular weight, isoelectrical point, etc. were done according to the National Requirements for Biological Products (2000).

RESULTSThe method of bioassay was established and used for determining activity of endostatin. Specific activity of the three batchs of drug substance was 1.45 x 10(6), 1.57 x 10(6) and 2.73 x 10(6) u.mg-1 proteins. Peptide mappings of the three batches of drug substance were completely identified. Both purity results of the products tested by SDS-PAGE and RP-HPLC were more than 99%.

CONCLUSIONThe established methods can effectively control the quality of recombinant human endostatin.

Cell Movement ; drug effects ; Cells, Cultured ; Endostatins ; pharmacology ; Endothelium, Vascular ; cytology ; Humans ; Quality Control ; Recombinant Proteins ; pharmacology ; Technology, Pharmaceutical ; methods