AIM: To study the endothelial progenitor cell markers and biological characteristics of human umbilical cord blood CD133+ cells isolated by immunomagnetic bead (IMB) method. METHODS: The experiment was carried out in the Laboratory of Burn Surgery, Xijing Hospital of the Fourth Military Medical University of Chinese PLA from November 2002 to August 2003.①CD133+ cells were enriched from human umbilical cord blood of placenta through spontaneous delivery or cesarean delivery (offered by Department of Gynecology and Obstetrics, Xijing Hospital of the Fourth Military Medical University of Chinese PLA), with the informed consents of parturients.②Human umbilical cord blood was anticoagulated by ACD-B and then diluted, 7 mL of cord blood combined with 3 mL lymphocyte isolation liquid were centrifuged for 30 minutes at 2 100 r/min. Low-density mononuclear cells coloring buffy were selected to wash off platelet and inoculated for 30 minutes by the IMB and FITC-CD133 antigen at room temperature to wash excessive antigens. The FITC-CD 133-coated IMB was added into mononuclear cells which were cultured for 30 minutes at room temperature and placed in magnetic field for 1 minute to remove the cell suspension. IMB-cell isolation liquid was supplemented to release CD133+ endothelial progenitor cells, which were cultured with EGM-2MV media in dishes coated with collagen I. And 24 hours later those unadherent cells were detached, culture liquid was removed half after 3 hours, and total culture duration was 3-4 weeks.③The morphologic change of CD133+ cells isolated with IMB were observed; The percentage of CD133+ cells in cord blood monocytes and sorting efficiency of IMB were analyzed by fluorescence activated cell sorting (FACS). The cells cultured in vitro were identified by fluorescence immunoassay and enzymo-histochemistry while transmission electron microscope (TEM) was used to observe the W-P body of mature vascular endothelial cells. RESULTS: ①Morphologic observation of CD133+ cells culture in vitro: The adherent cells expanded pseudopods on days 4 and 5, and grew rapidly in clonal manner 7 days later. The cells showed a typical spindle-shaped morphology during 2-3 weeks and cluster areas of cobblestone-like cells after 2-week culture and confluence.②Isolation rate of CD133+ cells: The percentage of CD133+ cells in cord blood monocytes was 0.91%. And after IMB sorting, the percentage of CD133+ cells was 85.52%.③On the fifth day, few cells were positive for CD133 while most cells were positive for CD 34 and vWF; On the tenth day, all cells were negative for CD133 and most cells were still positive for CD 34 and vWF.④The cells after 10-day culture had typical W-P body in TEM. CONCLUSION: Immunomagnetic sorting can efficiently select enriched CD133+ endothelial progenitor cells from human umbilical cord blood. Our data of CD133, CD34, vWF antibodies and TEM support the hypothesis that endothelial progenitor cells may contribute to mature vascular endothelial cells.

Objective To investigate the clinical value of serum homocysteine (Hcy),Cystatin C (CysC) detection in coronary heart disease prevention,diagnosis and treatment.Methods A total of 200 cases of coronary heart disease patients including 50 cases of acute myocardiac infarction (AMI) (AMI group),85 cases of unstable angina pectoris (UAP)(UAP group.) and 65 cases of stable angina pectoris (SAP) (SAP group) were selected,and 120 cases of healthy controls were selected as control group.The serum levels of Hcy and CysC were detected and compared.Results The serum levels of Hcy and CysC in AMI group,UAP group and SAP group were significandy higher than those in control group[Hcy:(22.35 ± 8.18),(16.54±7.56),(14.52±6.38) μmol/Lvs.(9.35±3.23) μmol/L; CysC:(1.32±0.27),(1.88± 0.66),(1.19 ± 0.46) mg/L vs.(0.80 ± 0.33) mg/L],and there were significant differences between two groups (P＜ 0.01).The serum level of Hcy in AMI group was higher than that in UAP group and SAP group [(22.35 ± 8.18) μ mol/L vs.(16.54 ± 7.56),(14.52 ± 6.38) μ mol/L],and there was significant difference (P ＜ 0.01).Conclusions The relationship between Hcy,CysC level and coronary heart disease is close,and Hcy levels increase with increasing degree of coronary heart disease.Detection of Hcy and CysC has some clinical value on prevention,diagnosis,treatment of coronary heart disease.

The polyclonal antibodies against VLDL receptor were prepared and identified. Rabbits were immunized with polypeptide fragment of VLDL receptor as antigen. The collected blood serum of the immunized rabbits was analyzed and identified by using ELISA and Western Blot. The results showed that the rabbit against mouse and human VLDL receptor antibodies were obtained with high titer and could recognize the natural VLDL receptors through Western blot. The prepared polyclonal antibodies against VLDL receptor provide a new tool to study the protein of VLDL receptor.
Antibodies/chemistry ; Antibodies/*immunology ; Enzyme-Linked Immunosorbent Assay ; Peptides/*immunology ; Receptors, LDL/*immunology

OBJECTIVETo explore the effect of Sedum sarmentosum Bunge Extract (SSBE) on severe acute pancreatitis (SAP) induced acute lung injury (ALI) model rats and their excessive inflammatory reactions.

METHODSForty-two healthy adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups, the sham-operated control group (C), the SAP group (SAP), and the SSBE treated group (SSBE), 14 in each group. SAP induced ALl rat model was induced by retrograde injection of 5% sodium taurocholate (1 mL/kg) into the pancreatic duct. SSBE (100 m/kg) was administrated subcutaneously after the establishment of the SAP model. Equal dose of SSBE was injected again 12 h later. Equal volume of normal saline was administrated in the same way for rats in the C group and the SAP group. Rats were sacrificed after successful modeling and samples taken at 12 and 24 h. Pathological changes in the pancreas and the lung tissue were observed under light microscope. The ascites, serum amylase (AMS), wet/dry proportion (W/D) of the lung tissue, activities of myeloperoxidase (MPO), interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha) were also measured.

RESULTSAscites and serum AMS activities significantly increased; MPO, IL-1, IL-6, TNF-alpha contents, and W/D ratio also significantly increased in the SAP group, when compared with the C group (P<0.05). Compared with the SAP group, those parameters were all attenuated in the SSBE group at 12 and 24 h (P<0.05, P<0.01). Pathological changes in the pancreas and the lung tissue were alleviated in the SSBE group under light microscope. The injury degree ranged between that of the C group and the SAP group.

CONCLUSIONSSBE could relieve the ALl in SAP model rats, which could be achieved through alleviating inflammation responses of SAP rats.

Acute Lung Injury ; drug therapy ; etiology ; Animals ; Drugs, Chinese Herbal ; therapeutic use ; Interleukin-1 ; Interleukin-6 ; Lung ; Male ; Pancreas ; Pancreatitis ; complications ; drug therapy ; Peroxidase ; Rats ; Rats, Sprague-Dawley ; Sedum ; Taurocholic Acid ; Tumor Necrosis Factor-alpha

Objective To investigate the role of protein kinase C (PKC) in induction of vascular endothelial growth factor (VEGF) secretion by isoflurane in primary cultured rat cardiomyocytes. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into 6 groups ( n = 6 each): control group (group C), 3 different concentration isoflurane groups (group Ⅰ1-3 ), PKC inhibitor calphostin C group (group P), and PKC inhibitor + isoflurane group (group PI). The cells were exposed to 0.7%, 1.4% and 2.1% isoflurane for6 h in group Ⅰ1-3 respectivly. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L in group P. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L, then the cells were exposed to 1.4% isoflurane for 6 h in group PI. VEGF concentrations and expression of PKC isoforms were determined by ELISA and Western blot respectively. Results Compared with group C, the VEGF concentration was significantly increased in group Ⅰ2 and Ⅰ3, and PKCε expression was down-regulated in the cytoplasm while upregulated in the cytomembrane in group Ⅰ2 ( P ＜ 0.01 ), but no significant change was found in the parameters mentioned above in group Ⅰ2 ( P ＞ 0.05). PKCα, PKCδ and PKCζ expression was significantly higher in the cytoplasm than in the cytomembrane in group C and Ⅰ2. VEGF concentrations were gradually increased with the increase in isoflurane concentrations ( P ＜ 0.05). VEGF concentrations were significantly lower in group PI than in Ⅰ2 ( P ＜0.05) .Conclusion Isoflurane induces VEGF secretion in primary cultured rat cardiomyocytes through translocation of PKCε from the cytoplasm to the cytomembrane, suggesting that it is a mechanism of the cardioprotective effects of isoflurane.

Objective To improve the diagnosis and treatment of asymptomatic adrenal pheochromocytoma. Method The clinical data of 33 patients with asymptomatic adrenal pheochromocytoma were reviewed. Results All the patients were not preut with typical signs and symptoms of pheochromocytoma, 16 patients were found adrenal mass by routine physical examination, 15 patients presented with superior abdominal or flank pain, 1 patient was fever and 1 patient was diarrhea. Most of patients were prent with round mass with low density in the center of the tumor,5 patients had elevated level of plasma epinephrine and norepinephrine (16% ,5/31),24 hours urine CA and VMA were elevated in 6 patients (24%, 6/25) and 5 patients (20%, 5/25) respectively, 26 patients who were suspected pheochromocytoma preoperatively were treated with α-adrenergic blockade (prazosin) to control the blood pressure or with intravenous colloid fluids and blood transfusion to expand intravascular volume before operation. The tumors were removed successfully in 32 patients, and biopsy was done in 1 patient because of the serious invasion to the vena cava by the tumor. All the patients were diagnosed pheochromocytoma histopathologically,and followed up for 1 month to 7 years, no tumor recurrence. Conclusions The patients with asymptomatic adrenal pheochromocytoma have lower catecholamine excretion in their plasma and urine. Combination of modem imaging examinations are useful methods to diagnose asymptomatic adrenal pheochromocytoma. To lower the risk of operation, the control of blood pressure and the expanding of intravascular volume are important for the patients with huge adrenal mass or typical suspected appearance of pheochromocytoma.

Objective To explore the relationship between K-ras gene and cholangiocarcinoma by detecting the K-ras gene mutation of bile duct tissues.Method We studied all the patients who presented to our hospital from June 2010 to June 2012 with stenosis of the bile duct.There were 17 cases of cholangiocarcinoma and 17 cases of benign stenosis.From the DNA extracted from the paraffin tissues,we used the HRM assay for K-ras gene mutation.Result We found that the HRM method and the DNA typing had exactly the same result for the DNA content which confirmed the effectiveness of the HRM assay.Of note,the K-ras mutation rate was found to be significantly higher in the cholangiocarcinoma cases (11/17) when compared with the benign cases (3/17).Conclusion The mutation of the K-ras gene was closely related to cholangiocarcinoma.Our results suggest a new way to diagnosis cholangiocarcinoma.

Objective T o prom ote the further research on body stature estim ation and the innovative ap-plications based on the distances betw een the anatom ical landm arks on body torso surface. Methods A specification for the collection of distances betw een the anatom ical landm arks on body torso surface w as established. T he data of 933 cases of adult population in Y angtze R iver D elta region w ere collected. M ultiple linear regression m ethod w as used to statistical analyse and establish the regression equation of stature estim ation. Results A regression equation about 5 variables including gender (x1), cervical verte-brae-coccyx line (x2), sterna-pubis line (x3), distance betw een acrom ion and iliospinale anterius (x4) and shoulder breadth (x5), and stature (y) w as established, y=105.406+5.414 x1+0.436 x2+0.286 x3+0.225 x4+0.193 x5. Conclusion T he m ethod is suitable for the rapid, sim ple and accurate estim ation of stature for the forensic experts.

Autophagy dysregulation, mitochondrial dynamic abnormality and cell cycle re-entry are implicated in the vulnerable neurons of patients with Alzheimer's disease. This study was designed to testify the association among autophagy, mitochondrial dynamics and cell cycle in dividing neuroblastoma (N2a) cells. The N2a cells were cultured in vitro and treated with different concentrations of 3-methyladenine (3-MA). The cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. They were randomly divided into control group (cells cultured in normal culture medium) and 3-MA group (cells treated with 10 mmol/L 3-MA). The cell cycle was analyzed in the two groups 3, 6, 12, and 24 h after treatment by flow cytometry. Western blotting was used to evaluate the expression levels of mitofission 1 (Fis1), mitofusin 2 (Mfn2), microtubule-associated protein 1 light chain 3 (LC3), cell cycle-dependent kinase 4 (CDK4) and cdc2. The flow cytometry revealed that the proportion of cells in G(2)/M was significantly increased, and that in G0/G1 was significantly reduced in the 3-MA group as compared with the control group. Western blotting showed that the expression levels of Fis1, LC3, and CDK4 were significantly up-regulated in the 3-MA group at the four indicated time points as compared with the control group. Mfn2 was initially decreased in the 3-MA group, and then significantly increased at 6 h or 12 h. Cdc2 was significantly increased in the 3-MA group at 3 h and 6 h, and then dropped significantly at 12 h and 24 h. Our data indicated that 3-MA-induced suppressed autophagy may interfere with the cell cycle progression and mitochondrial dynamics, and cause cell death. There are interactions among cell cycle, mitochondrial dynamics and autophagy in neurons.

Crigler-Najjar Syndrome ; complications ; diagnosis ; Female ; Humans ; Infant ; Jaundice ; etiology