OBJECTIVETo investigate the protective effect of mouse astrocyte-conditioned medium (ACM) on hypoxic and mechanically injured neurons by a cell model in vitro, and to explore the possible mechanism.

METHODSThe model of hypoxic neuronal injury was caused by 3% O2 in three-gas incubator. Neurons were cultured with ordinary medium or 20% ACM respectively and randomly divided into hypoxic group (hypoxia for 4, 8, 24 h and marked as H4R0, H8R0, H24R0) and hypoxia reoxygenation group (H4R24, H8R24, H24R24). Mechanical injury model was developed by scratching neurons cultured in 20% ACM or ordinary medium to different degrees. Neurons in both medium were divided into normal control group, mild, moderate and severe injury groups. The 20% ACM was added 24 h before hypoxia/reoxygenation or mechanical injury. The morphology and survival of neurons were observed and counted by trypan blue staining. The concentration of NO, lactic dehydrogenase (LDH) and membrane ATPase activity were detected by corresponding kits.

RESULTSIt was showed that 20% ACM can obviously promote the survival rate of hypoxia/reoxygenated neurons and scratched neurons as well. The morphology and number of neurons exposed to hypoxia or scratch injury showed great difference between groups with or without ACM treatment. Compared with control group, the concentration of NO and LDH was much lower in hypoxic/reoxygenated neurons treated with 20% ACM, and the ATPase activity was higher. For the mechanical injury model, neurons with moderate injury also revealed a lower NO and LDH concentration than the control group. All the differences were statistically significant (P less than 0.05).

CONCLUSIONACM can promote the survival and functional recovery of neurons following hypoxia or scratching to a certain degree. The mechanism may be associated with reducing the synthesis and release of NO and LDH as well as increasing the activity of membrane ATPase.

Animals ; Astrocytes ; physiology ; Cell Hypoxia ; Cell Survival ; Cells, Cultured ; Culture Media, Conditioned ; Mice ; Nerve Growth Factors ; physiology ; Neurons ; physiology

OBJECTIVETo investigate the regulation of different hypoxia on cell survival and autophagy.

METHODSPC12 cells were treated with different hypoxia. The cell survival was measured by MTT assay, expressions of LC3 and p62 were marked for autophagy detected by Western Blot, and the level of reactive oxygen species (ROS) was analyzed by flow cytometry.

RESULTSThe cell viability was different under different hypoxia: moderate hypoxia promoted cell viability, and severe hypoxia caused a decrease in cell viability; autophagy marker molecules, p62 and LC3-II expressions were different: moderate hypoxia increased p62 and LC3-II expressions, in contrast, severe hypoxia led to the decrease of p62 and LC3-II expressions; compared to normoxia, moderate hypoxia did not change the levels of ROS, while severe hypoxia increased the levels; 3-MA, the inhibitor of autophagy, elevated the levels of ROS in the three oxygen concentrations, additionally, the increased amplitudes in the moderate and severe hypoxia groups were higher than that in the normoxia group.

CONCLUSIONModerate hypoxia promotes cell survival, severe hypoxia causes the cell death, and the autophagy activity may mediate the effects of different hypoxia.

Animals ; Autophagy ; physiology ; Cell Death ; Cell Hypoxia ; Cell Survival ; PC12 Cells ; Rats ; Reactive Oxygen Species ; metabolism

This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type II collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at Gl phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G1 phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.
Animals ; Apoptosis ; physiology ; Cell Cycle ; physiology ; Cell Proliferation ; Cell Size ; Cell Survival ; physiology ; Cells, Cultured ; Intervertebral Disc ; cytology ; physiopathology ; Intervertebral Disc Degeneration ; pathology ; physiopathology ; Rabbits ; Reference Values

BACKGROUNDCalreticulin (CRT) is major Ca 2+ -binding chaperone mainly resident in the endoplasmic reticulum (ER) lumen. Recently, it has been shown that non-ER CRT regulates a wide array of cellular responses. We previously found that CRT was up-regulated during hypoxia/reoxygenation (H/R) and this study was aimed to investigate whether CRT nuclear translocation aggravates ER stress (ERS)-associated apoptosis during H/R injury in neonatal rat cardiomyocytes.

METHODSApoptosis rate and lactate dehydrogenase (LDH) leakage in culture medium were measured as indices of cell injury. Immunofluorescence staining showed the morphological changes of ER and intracellular translocation of CRT. Western blotting or reverse transcription polymerase chain reaction was used to detect the expression of target molecules.

RESULTSCompared with control, H/R increased apoptosis rate and LDH activity. The ER became condensed and bubbled, and CRT translocated to the nucleus. Western blotting showed up-regulation of CRT, Nrf2, activating transcription factor 4 (ATF4), CHOP and caspase-12 expression after H/R. Exogenous CRT overexpression induced by plasmid transfection before H/R increased cell apoptosis, LDH leakage, ER disorder, CRT nuclear translocation and the expression of ERS-associated molecules. However, administration of the ERS inhibitor, taurine, or CRT siRNA alleviated cell injury, ER disorder, and inhibited ERS-associated apoptosis.

CONCLUSIONSOur results indicated that during H/R stress, CRT translocation increases cell apoptosis and LDH leakage, aggravates ER disorder, up-regulates expression of nuclear transcription factors, Nrf2 and ATF4, and activates ERS-associated apoptosis.

Animals ; Apoptosis ; genetics ; physiology ; Calreticulin ; genetics ; metabolism ; Cell Hypoxia ; genetics ; physiology ; Cell Survival ; genetics ; physiology ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Myocytes, Cardiac ; cytology ; metabolism ; RNA Interference ; Rats

In order to study the physicochemical characteristics of cytosolic PrP (CytoPrP) and evaluate its possible influence on cell viability, a recombinant plasmid expressing human CytoPrP eukaryoticly was constructed and transfected into human neuroblastoma cell line SH-SY5Y transiently. Proteinase-resistant activities of CytoPrP were evaluated by a proteinase K (PK) digestion and cytotoxic effects of CytoPrP were tested by MTT assay and Trypan Blue cell-counting. The presence of CytoPrP in cytoplasm after transfection was controlled by the presence of protease inhibitor. Compared with wild-type PrP, CytoPrP possessed relatively stronger PK-resistant activities. Obvious cytotoxic effects were observed in the cells after inducement of CytoPrP in cytoplasm by protease inhibitor, showing a dose-dependent manner. The results provide useful scientific evidences for further studies of potential role of CytoPrP in pathological mechanism of prion disease.
Cell Line, Tumor ; Cell Survival ; Cytosol ; chemistry ; Endopeptidase K ; pharmacology ; Humans ; Prions ; genetics ; physiology ; Transfection

BACKGROUNDEndothelial cell damage is an important pathophysiological step of restenosis after angioplasty and stenting. Cell transplantation has great therapeutic potential for endothelial recovery. We investigated the effect of transplanting endothelial progenitor cells (EPCs) derived from human early fetal aortas in rat injured arteries.

METHODSThe carotid arterial endothelium of Sprague-Dawley rats was damaged by dilatation with a 1.5 F balloon catheter, and then EPCs derived from human early fetal aortas (<14 weeks) were injected into the lumen of the injured artery in transplanted rats, with an equal volume of normal saline injected into control rats. Rats were sacrificed at 2 and 4 weeks after treatment and transplanted cells were identified by immunohistochemical staining with anti-human CD31 and anti-human mitochondria antibodies. Arterial cross-sections were analyzed by pathology, immunohistochemistry, and morphometry.

RESULTSGreen fluorescence-labeled EPCs could be seen in the endovascular surface of balloon-injured vessels after transplantation. The intimal area and intimal/medial area ratio were significantly smaller in the transplanted group than in the control (P < 0.05) and the residual lumen area was larger (P < 0.05). After EPC transplantation, a complete vascular endothelial layer was formed, which was positive for human von Willebrand factor after immunohistochemical staining, and immunohistochemical staining revealed many CD31- and mitochondria-positive cells in the re-endothelialized endothelium with EPC transplantation but not control treatment.

CONCLUSIONEPCs derived from human early fetal aorta were successfully transplanted into injured vessels and might inhibit neointimal hyperplasia after vascular injury.

Animals ; Carotid Arteries ; pathology ; Cell Adhesion ; physiology ; Cell Survival ; physiology ; Cell Transplantation ; Endothelial Progenitor Cells ; cytology ; physiology ; Humans ; Immunohistochemistry ; Microscopy, Fluorescence ; Neointima ; therapy ; Rats ; Rats, Sprague-Dawley

Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be selectively isolated and induced to express bone phenotype in vitro. The osteoblastic progenitor can be estimated by counting the number of cells attach using the haemacytometer. This study was undertaken to test the hypothesis that human aging is associated with a significant change on the number of osteoblastic progenitors in the bone marrow. Bone marrow aspirates were harvested from 38 patients, 14 men (age 11-70) and 24 women (age 10-70) and cultured in F12: DMEM (1:1). In total 15 bone marrow samples have been isolated from patients above 40 years old (men/women) of age. Fourteen (93.3%) of this samples failed to proliferate. Only one (6.7%) bone marrow sample from a male patient, aged 59 years old was successfully cultured. Seventy percent (16/23) of the samples from patient below than 40 years old were successfully cultured. However, our observation on the survival rate for cells of different gender from patient below 40 years old does not indicate any significant difference. From this study, we conclude that the growth of bone marrow stromal cells possibly for bone engineering is better from bone marrow aspirates of younger patient.
Age Factors ; Bone Marrow Cells/*cytology ; Cell Aging/*physiology ; Cell Division/*physiology ; Cell Survival/physiology ; Mesenchymal Stem Cells/*cytology ; Osteoblasts/*cytology ; Sex Factors ; Stromal Cells/cytology ; *Tissue Engineering

Totipotent and regionally non-specified embryonic stem (ES) cells provide a powerful tool to understand mechanisms controlling stem cell differentiation in different regions of the adult brain. As the development capacity of ES cells in the adult brain is still largely unknown, we grafted small amounts of mouse ES (mES) cells into adult rat brains to explore the survival and differentiation of implanted mES cells in different rat brain regions. We transplanted the green fluorescent protein (GFP)-positive mES cells into the hippocampus, septal area, cortex and caudate nucleus in rat brains. Then the rats were sacrificed 5, 14 and 28 d later. Of all the brain regions, the survival rate of the transplanted cells and their progeny were the highest in the hippocampus and the lowest in the septal area (P<0.01). The grafted ES cells could differentiate into nestin-positive neural stem cells. Nestin-positive/GFP-positive cells were observed in all brain regions with the highest frequency of nestin-positive cells in the hippocampus and the lowest in the medial septal area (P<0.01). mES cells differentiated into end cells such as neurons and glial cells in all transplantation sites in recipient brains. In the hippocampus, the ES cells differentiated into neurons in large amounts. These results demonstrate that only some brain regions permit survival of mES cells and their progeny, and form instructive environments for neuronal differentiation of mES cells. Thus, because of region specific presence of microenvironmental cues and their environmental fields, the characteristics of the recipient tissue were considerably important in formulating cell replacement strategies for neural disorders.
Animals ; Brain ; cytology ; Cell Differentiation ; physiology ; Cell Survival ; Embryonic Stem Cells ; cytology ; transplantation ; Female ; Graft Survival ; Mice ; Rats ; Rats, Sprague-Dawley ; Transplantation, Heterologous ; physiology

OBJECTIVEThe present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells.

METHODS AND RESULTSMutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type.

CONCLUSIONThese data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Proliferation ; Cell Survival ; genetics ; physiology ; Colorimetry ; HeLa Cells ; Humans ; Mutation ; Oligopeptides ; genetics ; Plasmids ; genetics ; Prion Proteins ; Prions ; genetics ; metabolism ; physiology ; Transfection

OBJECTIVEIn this study, human bronchial epithelial cells were inoculated with positive sputum specimens of HBoV. After four days' infection, cytopathic effects (CPE) were observed by inverted microscopy. These viruses all cause typical cell damages such as rounded and shrivelled, fusion and fallout. These damages got quick following increased future degenerations. The other assay result of CPE within the infected cells were observed by inverted microscopy, have typical "owl's eye" plaque and above 90 percent hemadsorption within the infected cells by erythrocytes for hemadsorption technique. The typical fluorescence lump of nucleus within the infected cells was found by indirect immunofluorescence technique.

CONCLUSIONIsolation and identification of HBoV could be done in the human bronchial epithelial cell, and we found some characterizing CPE in the human bronchial epithelial cell after HBoV infection. The above studies pave a way for studying pathogenicity of human bocavirus.

Bocavirus ; physiology ; Bronchi ; cytology ; Cell Death ; physiology ; Cell Survival ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; virology ; Fluorescent Antibody Technique, Indirect ; Host-Pathogen Interactions ; Humans ; Microscopy, Fluorescence