Objective:To remove murine embryonic stem cells(mESC)from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting(MACS).Methods:Neural differentiation of mESC was induced by a 5-stage method.The specific cell surface marker,SSEA-1,was used to identify ES cells in the differentiating cells.The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test.The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate.After the optimization,stage 4 cells were dissociated into single cell suspension,incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM,and then sorted through the magnetic field.The rate of SSEA-1 positive cells in pre-and post-separation groups was assessed by flow cytometry,and the viability of cells was evaluated by trypan blue staining counting under light microscopy.Results:The proportion of SSEA-1 positive cells in the separated cells can be reduced from(7.19?1.36)% to(1.34?0.80)%.The survival rate of sorted cells was more than 92%,similar to that of pre-separation cells.Conclusions:The MACS system we used can effectively remove mESC from the differentiated cells.The sorted cells will be well provided for the subsequent studies about transplantation therapy.

Obstructive sleep apnea-hypopnea syndrome(OSAHS) is a common sleep-related breathing disorder,which has a series of impact on the cardiovascular system.The dctection of some biochemical indicators plays an important role in predicting this kind of cardiovascular damage.The role of inflammatory predicting factors such as TNF-?,IL-6,CRP,IL-10,MMPs and ICAM-1 is reviewed in this paper.

0.05). Conclusion The exon 8+488C/T polymorphism of Aiolos gene exists in this population of Han ethnics,however,it is not associated with bronchial asthma.

Objective To investigate the benefits of long-term home noninvasive positive pressure ventilation(NIPPV) for patients with stable chronic obstructive pulmonary disease(COPD). Methods From 2006 to 2007,46 patients with chronic respiratory failure due to stable COPD receiving NIPPV in Croix Rousse Hospital were retrospectively analysed.The arterial blood gas analysis of before treatment,1,3,6,12,24 and 36 months after treatment were compared,and the lung function of before treatment,6,12,24 and 36 months after treatment were also compared. Results PaCO2 of 1,3,6,12,24 and 36 months after receiving NIPPV significantly decreased(P

OBJCETIVETo investigate the method of separation of culture of bone microvascular endothelial cells (BMECs) of human femoral head in vitro.

METHODSFrom October 2013 to January 2014,15 femoral heads without pathologic change from patients resected during hip replacement were selected involving 2 males and 13 females with a mean age of 71.2 years old ranging from 38 to 92. Cancellous bone in femoral head was bited into broken bone grain and transfered into medium in aseptic contidion. Cells were isolated by the methods of enzymic digestion and density gradient centrifugation,purified by differiential attachment. The characteristics of cells was observed by inverted microscope. vWF and CD31 immunofluorescence analysis was applied for identification of cells.

RESULTSThe number of cells was positively correlated with patients' age after 24 hours in primary culture. The older patients had the less cells numbered. After 4 to 5 days' culture, primary cells appeared short spindle,polygon shaped and cobblestone-like morphology. After 7 to 10 days' culture, primary cells proliferated densely, became fusion, arranged in swirl, and contact inhibition appeared significantly. Immunofluorescence staining revealed the cells were 100% positive for vWF and CD31, and it showed that the cultured cells were BMECs.

CONCLUSIONIt was a simple, steady, effective method with good reproducibility, by which highly purified human BMECs can be obtained.

Adult ; Aged ; Aged, 80 and over ; Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; methods ; Cells, Cultured ; Endothelial Cells ; cytology ; Female ; Femur Head ; blood supply ; Humans ; Male ; Microvessels ; cytology ; Middle Aged

OBJECTIVETo evaluate the inhibitory effect of tumor suppressor PTEN on cell growth of endometrial carcinoma.

METHODSThe exogenous wild PTEN cDNA via an adenoviral vector (Ad-PTEN) was introduced into Ishikawa cells. The expression of PTEN protein was detected by Western blot. The growth of Ishikawa cells was evaluated by trypan blue exclusion method and MTT.

RESULTSThe expression of PTEN protein was induced on day 1, and greatly increasing on day 3 - 5 after Ad-PTEN infection. The expression of PTEN significantly inhibited the growth of Ishikawa cells, and also significantly inhibited the growth of Ishikawa cells induced by IGF-II.

CONCLUSIONAdenovirus-mediated introduction of exogenous PTEN into human endometrial carcinoma cells can induce growth suppression. PTEN gene may be a novel therapeutic agent for endometrial carcinoma.

Adenoviridae ; genetics ; Cell Proliferation ; Endometrial Neoplasms ; metabolism ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Insulin-Like Growth Factor II ; pharmacology ; PTEN Phosphohydrolase ; Phosphoric Monoester Hydrolases ; biosynthesis ; genetics ; physiology ; Recombination, Genetic ; Transfection ; Tumor Suppressor Proteins ; biosynthesis ; genetics ; physiology

Background Increasingly,evidence from population,clinic-based and laboratory studies supports an independent association between obstructive sleep apnea syndrome (OSAS) and an increased risk of type 2 diabetes; however,this observation has yet to be replicated in China and the potential mechanisms that link these two conditions are not clear.Methods A total of 179 Han Chinese subjects were enrolled in this study.All subjects underwent polysomnography,the oral glucose tolerance-insulin releasing test (OGTT-IRT) and serum HbA1c measurement.Indexes including homeostasis model assessment-IR (HOMA-IR),Matsuda index,HOMA-β,early phase insulinogenic index (△I30 / △G30),AUC-I180 and oral disposition index (DIo) were calculated for the assessment of insulin resistance and pancreatic β-cell function.Results Based on OGTT,25.4％,44.6％ and 54.5％ subjects were diagnosed having glucose metabolic disorders respectively in control,mild to moderate and severe OSAS groups (P ＜0.05).Serum HbA1c levels were highest in subjects with severe OSAS (P ＜0.05).In contrast,compared with normal subjects,HOMA-β,△I30/△G30 and DIo were lower in severe OSAS group (P ＜0.05).In stepwise multiple linear regressions,0-min glucose and HbA1c were positively correlated with the percentage of total sleep time below an oxyhemoglobin saturation of 90％ (T90) (Beta =0.215 and 0.368,P ＜0.05); 30-min and 60-min glucose was negatively correlated with the lowest SpO2 (LSpO2) (Beta =-0.214 and -0.241,P ＜0.05).HOMA-β and Dlowere negatively correlated with T90 (Beta =-0.153 and-0.169,P ＜0.05) while body mass index (BMI) was the only determinant of HOMA-IR and Matsuda index.Conclusions OSAS is associated with impairment in glucose tolerance and pancreatic β-cell function in Han Chinese subjects while insulin sensitivity is mainly determined by obesity.

OBJECTIVETo culture and amplify the young rabbit's bone marrow stromal cells (BMSCs) in vitro, and to observe the effect of hypothermia on the cells' growing behavior and biological function.

METHODSBMSCs were acquired from the rabbit' tibia bone marrow and induced to mature osteoblasts in vitro. The cultured cells growing well in vitro were preserved in liquid nitrogen. The anabiotic cells having cryopreserved for 1 week were chosen as the experimental group, and the routine 7th generation as the control group. Their biological function in comparion by the examination of morphological changes, cells' proliferation ability, colone forming ratio, synthesis ability of ALP and protein, mineralized nodes forming ability were observed.

RESULTSAs contrast to the control groups, the anabiotic cells also grew and proliferated well in vitro except a little more slowly than before. They had the similar general shape in all the time segments, but a little differences in cells' ultrastructure. The experimental groups also had the typical characters of mature osteoblasts, and high abilities of the synthesis of ALP and proteins. The statistic data showed that these two groups had no significant difference (P > 0.05).

CONCLUSIONThe cryopreserved osteoblasts had the same biological functions and the similar growing behaviors as before. These results suggest that it is practical to use the cryopreserved osteoblasts for further study on bone tissue engineering.

Animals ; Bone Marrow Cells ; Bone and Bones ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; In Vitro Techniques ; Osteoblasts ; Rabbits ; Tissue Engineering

BACKGROUNDAlthough flunarizine has been widely used for migraine prophylaxis with clear success, the mechanisms of its actions in migraine prophylaxis are not completely understood. The aim of this study was to investigate the effects of flunarizine on tetrodotoxin-resistant Na(+) channels and high-voltage activated Ca(2+) channels of acutely isolated mouse trigeminal ganglion neurons.

METHODSSodium currents and calcium currents in trigeminal ganglion neurons were monitored using whole-cell patch-clamp recordings. Paired Student's t test was used as appropriate to evaluate the statistical significance of differences between two group means.

RESULTSBoth tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were blocked by flunarizine in a concentration-dependent manner with the concentration producing half-maximal current block values of 2.89 µmol/L and 2.73 µmol/L, respectively. The steady-state inactivation curves of tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were shifted towards more hyperpolarizing potentials after exposure to flunarizine. Furthermore, the actions of flunarizine in blocking tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were use-dependent, with effects enhanced at higher rates of channel activation.

CONCLUSIONBlockades of these currents might help explain the peripheral mechanism underlying the preventive effect of flunarizine on migraine attacks.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Female ; Flunarizine ; pharmacology ; Male ; Mice ; Patch-Clamp Techniques ; Sensory Receptor Cells ; drug effects ; metabolism ; Sodium ; metabolism ; Trigeminal Ganglion ; cytology ; drug effects ; metabolism

OBJECTIVETo investigate the effect of hepatocyte growth factor (HGF) on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) after allogeneic bone marrow transplantation (allo-BMT) and related mechanism in acute lymphoblastic leukemia (ALL) mice.

METHODSTwenty nude mice were randomly divided into control (group A) and test (group B) groups for monitoring relapse, and 20 BALB/c mice into control (group C) and test (group D) groups for GVHD. HGF as injected from day 0 to day 7 after BMT for groups B and D, while PBS for A and C. CD4(+) and CD8(+) T cell were evaluated by flow cytometry. The survival of mice after BMT was recorded. The level of tumor necrosis factor-alpha (TNF-alpha) was evaluated by ELISA.

RESULTSThe median past-BMT survival were 7.00 +/- 1.58, 9.00 +/- 1.58, 11.00 +/- 3.95 and 24.00 +/- 13.44 days for groups A, B, C, D, respectively, being prolonged in group D. HGF could decrease the quantity of CD4(+) T cells [group D (10.39 +/- 1.15)% vs group C (13.50 +/- 1.80)%, P < 0.01] and increase CD8(+) T cell [group D (12.25 +/- 2.85)% vs group C (6.12 +/- 1.99)%, P < 0.01], decrease the level of TNF-alpha in transplanted ALL mice [group D (112.10 +/- 18.99) pg/ml vs group C (143.90 +/- 25.35) pg/ml, P < 0.01] and reduce the degree of GVHD.

CONCLUSIONHGF could alleviate post-allo-BMT GVHD but retain GVL effect.

Animals ; Bone Marrow Transplantation ; Disease Models, Animal ; Female ; Graft vs Host Disease ; prevention & control ; Graft vs Leukemia Effect ; drug effects ; Hepatocyte Growth Factor ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; surgery ; Random Allocation ; Transplantation, Homologous