AIM: To establish a HSP90 highly expressing cell line and study the effect of high level of HSP90 on cell stress response. METHODS: The recombined plasimid pSmycHSP, which contains the full length DNA coding for human HSP90?, was introduced into mouse fibroblast cell line NIH-3T3 by electroporation after being subcloned, purified and identified by limited enzyme digestion. Screened by G418, the positive clones were selected and identified by immunofluorescence, immunocytochemistry and Western-blotting. NIH-3T3 cells transfected with empty plasmid served as control, hyperthermia(44 ℃, 20 min, 40 min)was used to simulate oxidative stress. The activity of lactate dehydrogenase (LDH) in supernatant and damage of DNA were detected by automatic biochemistry analyzer and flow cytometer separately to analyze the effect of high-level HSP90 on cell membrane and DNA injuries under stress condition. RESULTS: The rising level of HSP90 was shown by immunofluorescence, immunocytochemistry and Western-blotting in HSP90 overexpressing cell line. There was no difference in the leakage of LDH between HSP90 overexpressing cell line and control, but the damage of DNA was more severe at 44 ℃for 20 min in HSP90 overexpressing cell line than control. Compared with control, the above indices were relieved at 44 ℃ for 40 min in HSP90 overexpressing cell line. CONCLUSION: The NIH-3T3 derived cell line, which stably expressed high level of HSP90, was established. The effect of high-level HSP90 on cells is complex at different intensity of stress, and the protection may be shown at more severe stress circumstance.

Objective To approach the influence of erythropoietin on proliferation and differentiation of neural stem cells originated from the hippocampi of adult rats in vitro. Methods Neural stem cells were isolated from the hippocampi of adult rats and cultured in vitro with serum-free incubation and single-cell cloning technique; immunochemistry technique was employed to identify the neural stem cells and neural stem cells-derived progeny. Erythropoietin (EPO) were added to cultures in different concentrations during proliferation and differentiation of neural stem cells, respectively. Results Compared with the control group, EPO supplementation group displayed an average twofold to fourfold increase in Nestin positive cells over a wide range of plating densities during proliferation, whereas showed a striking decrease in Nestin positive cells during differentiation,following an early appearance of Tuj1 or GFAP positive cells. The percentage of Tuj1 positive cells increased significantly to 57% in EPO supplementation group versus 47% in control group (n=12, P

Objective To prove the existence of dry eye in children and to learn the clinical characteristics of them. Design Prospective observational case series. Participants 38 cases(76 eyes) of suspected children of dry eye diagnosed by adult standard and 38 normal children subjects. Methods 38 suspected child patients were studied who were diagnosed clinically with dry eye by adult standard, and were followed up 6 months. The control group consisted of 38 normal children with no significant difference in age. Dry eye examinations including Schirmer test, break-up time(BUT) and fluorescein staining were performed on these two groups. Following items were recorded in 38 suspected children of dry eye, including symptoms and causations. Main Outcome Measures Symptoms, Schirmer test, BUT and fluorescein staining. Results In the 38 suspected patients, frequent blinking was the most common symptom in 21 cases(55.26%), followed by dryness (15 cases, 39.47%), redness (14 cases, 36.84%) and photosensitivity (14 cases, 36.84%). The Schirmer test and BUT were decreased remarkably in the suspected patients compared to those of normal subjects (P=0.0000). In both groups, right eyes were correlated with left eyes in both Schirmer test and BUT. Between these two groups, Schirmer test was nol correlated with BUT. In the suspected patients, the results of BUT and fluorescein staining were improved (P

AIM: To investigate the effect of Ba 2+ concentration on L-type of Ca 2+ channel in hypothalamic neurons.METHODS: The cell acute isolation technique and cell-attached patch-clamp technique were used. RESULTS : The slope conductance of L-type Ca 2+ channel were 28.6 pS (110 mmol/L) and 19.1 pS (10 mmol/L), and the open probability (NP 0) obviously different with different Ba 2+ concentration as carrier. CONCLUSION: Ba 2+ concentration had the obvious effect on the L-type Ca 2+ channel.

AIM: To observe the effect of high temperature on the delayed rectifier K+ channel(I K) in hypothalamus neurons METHODS: The kinetic properties of I K were measured by cell-attached mode of patche-clamp technique RESULTS: The conductance, long-term open constant(? o2 ) and open probability(Po) of I K increased with increase of temperature ( P

Adaptation, Physiological ; Animals ; Female ; Gene Expression Regulation ; Hot Temperature ; Hypothalamus ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Transcriptome

Because of the aggressive threaten of heat stroke and a lack of understanding of the mechanism of action, mammal animal models for experimental heat stroke were well developed. During the past 5 decades, anesthetized mouse, rat, rabbit, dog, baboon and monkey were used as animal model for experimental heat stroke. However, anesthetized mammals models have some limitations, such as neuroprotective effect of anesthetic agents, possible disturbance on injury and recovery of stroke animals by anesthetic agents, difficulty of discussing animal behavior before and after heat stroke, it was also difficult for the models to evaluate cognitive function of animal under hot environment. Considering humanitarian, only awaked and unrestrained mouse heat stroke model was accepted so far. Therefore, we also developed an awaked and unrestrained rat heat stroke model, and found it was helpful to evaluate drug effectiveness for animal behavior and cognitive function under hot environment.
Animals ; Cognition ; Disease Models, Animal ; Heat Stroke ; physiopathology ; Mice ; Rats

AIM: To study the effect and mechanism of all-trans retinoic acid (atRA) on neural stem cell (NSCs) proliferation and differentiation from new born Sprague- Dawley rat striatum. METHODS: NSCs were isolated from the brains of new born Sprague-Dawley rat striatum, and the features of cells were characterized by immunofluorescence staining. The effects of different culture medium on cell cycle distribution and proliferation of NSCs were determined by flow cytometry (FCM) . The effects of atRA on differentiation of NSCs were determined by immunofluorescence staining and classified count of differentiated cells. RESULTS: FCM assay indicated that atRA inhibited the proliferation of NSCs. The percentage of cells in G0/G1 phase in atRA treatment group was significantly higher than that in control, and the proliferation index (PI) was significantly low. The percentage of neurons differentiated from NSCs in atRA group was 2.5 times of the control group after induced by adding 10% FCS in culture medium. CONCLUSION: atRA counteracts the effects of bFGF on the promotion of mitosis and inhibition of differentiation of NSCs. atRA also promotes NSCs to differentiate into neurons in vitro.

AIM: To induce preconditioning and oxidative stress by H_2O_2 in HepG2 cells. METHODS: The different doses of H_2O_2 were used to induce apoptosis in HepG2 cells, which was estimated by AO/EB staining, MTT assay and flow cytometry. RESULTS: The different group of HepG2 cells stained with AO/EB showed different staining state. The high dose of H_2O_2 resulted in the increase in apoptosis rate of HepG2 cells and made MTT activity decreased. However, after pretreated with low dose of H_2O_2, the apoptosis rate was decreased and MTT activity was increased. CONCLUSION: The high dose H_2O_2 induced apoptosis in HepG2 cells and the low dose H_2O_2 protected HepG2 cells against the oxidative stress.

AIM: To find the role of heat shock protein 90(HSP90) and tubulin in oxidative stress preconditioning in HepG2 cells.METHODS: The different doses of H2O2 were used to induce cell injury in HepG2 cells.MTT assay,Western blotting and confocal laser microscopy were also used.RESULTS: MTT colorimetry showed that preconditioning(50 mmo1/L H2O2) provided a temporary resistance against subsequent oxidative stress(500 mmol/L H2O2).Western blotting demonstrated that preconditioning increased the levels of HSP90 and tubulin in HepG2 cells,and lessen the declining of HSP90 and tubulin after stress.Tubulin and HSP90's colocalizations in cells with different doses of H2O2 were also observed under laser scanning confocal microscope.CONCLUSION: Tubulin might play important role in oxidative stress preconditioning in HepG2 cells by combining with HSP90.