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BACKGROUND: It has been proved that the rare earth compounds can obviously restrain the growth of tumor in vitro and in vivo.OBJECTIVE: To check the effect of chlorinated lanthanum (LaCl3) on the expressions of cyclin D1 and cyclin dependent kinase 4 (CDK4) of hepatocellular carcinoma cells.DESIGN: Controlled observation.SETTING: Department of Nephropathy, First Clinical Hospital, Jilin University. MATERIALS: The experiment was carried out in the cell culture laboratory (province-level) of Department of Histology and Embryology, Jilin University from July 2002 to June 2003. Wells of the microwell plate seeded with CBRH-7919 cells were randomly divided into 4 groups according to the dose of LaCl3: control, 0.01, 0.1, 1.0 mmol/L LaCl3. And each group was further divided into three subgroups according to the culture time: 1, 3, 5 days, with 6 wells for each.METHODS: The growth of CBRH-7919 cells was monitored following treated with 0.01, 0.1, 1.0 mmol/L LaCl3 for 1, 3, 5 days in vitro, respectively. ① Absorbance(A) of each well was measured by MTT assay.② The changes of distribution of cells into different phases of cell cycle were detected by flow cytometry. ③ Cyclin D1 and CDK4 expression was detected by immunocytochemical analysis.MAIN OUTCOME MEASURES: The expression of cyclin D1 and CDK4 in CBRH-7919 cells of different doses of LaCl3 at different time.RESULTS: Effect of LaCl3 on the growth of CBRH-7919: On days 3 and 5, the A value of each well in the 0.1 and 1.0 mmol/L LaCl3 groups was respectively significantly lower than that in the control group (day 3: 1.140±0:070, 0.706±0.092,1.461±0.087; day 5: 1.888±0.020,0.625±0.037,2.544±0.032, respectively) (P ＜ 0.05,0.001 ). ②Effect of LaCl3 on the percentage of CBRH-7919 cells at G0/G1 stage: On days 1, 3, and 5, the percentage of CBRH-7919 cells at G0/G1 stage in the 1.0 mol/L LaCl3 group was respectively significantly higher than that in the blank control group [(60.70±0.20)%, (39.49±0.67)%; (61.66±0.97)%, (45.56±1.00)%; (69.92±0.18)%, (49.24±0.27)%, (P ＜ 0.01,0.001 )]; On day 5, the percentage of CBRH-7919 cells at G0/G1 stage in the 0.1 mol/L LaCl3 group was respectively significantly higher than that in the blank control group[(58.88±0.73)%, (49.24±0.27)%, P ＜ 0.05]. ③ Effect of LaCl3 on the expressions of cyclin D1 and CDK4 of CBRH-7919 cells: On day 1, the expressions of cyclin D1 and CDK4 of CBRH-7919 cells in the 0.1 and 1.0 mmol/L LaCl3 groups were respectively lower than those in the blank control group (expression of cyclin D1: 562±35,453±22,860±82;expression of CDK4: 705±84,680±28,762±16, P ＜ 0.05, 0.01 and 0.001).CONCLUSION: LaCl3 inhibited the growth of CBRH-7919 cells correlating down-regulating cyclin D1 and CDK4 as reflected on the cell cycle arrest of CBRH-7919 cells from G1 stage to S stage.
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BACKGROUND: It has been suggested that amniotic epithelial cells (AECs) express almost all the markers of neural cell and secrete biologically active neurotrophins such as brain derived neurotrophin factor (BDNF) and neurotrophin-3 (NT3).If AECs can substitute neural cells, its neurotrophic effect will bring expansive prospect in treating spinal cord injuries and degenerative neural disease.OBJECTIVE: To observe the survival, migration and secretory function of AECs after transplanted into the injured spinal cord.DESIGN: An observational experiment.SETTING: Department of Histology and Embryology, School of Basic Medical Science, Jilin University.MATERIALS: Embryonic rat of 12-14 days (n =1) and adult Wistar rats (n =18, 300-350 g) were provided by the Experimental Animal Center of Jilin University. Immunohistochemical reagents: Mouse anti-rat BrdU monoclonal antibody was bought from Sigma Company. Rabbit anti-rat NT3 polyclonal antibody and rabbit anti-rat BDNF polyclonal antibody were bought from Boster Company. SP immunohistochemistry reagents were purchased from Maixin Company.METHODS: The experiment was made in the Department of Histology and Embryology, Basic Medical Science of Jilin University from July to October 2005. ① Wistar rats were anesthetized by intraperitoneal injection of chloral hydrate, subcutaneous tissue and muscle were separated, spinous process and lamina of vertebra were removed by bone ribbing rongeur. to expose the spinal cord. The spinal cords were clamped at the twelfth thoracic vertebra (T12) for 3 minutes.After surgery, the wounds were smeared with penicillin G, then muscle and skin were sutured. The rats were anesthetized by inhaling ether if necessary. ② Obtaining and culture of AECs: Amniotic membrane was peeled from the placenta of a pregnant Wistar rat of 12-14 days. The amnictic membrane was dissected into small pieces of 1 mm×1 mm×1 mm, then digested and cultured, and mechanically made into single cell suspension, finally plated in bottles. ③ Transplantation of AECs into injured spinal cord: The initial wound was slit and injected with 5 μL Brdu labeled AECs (1×1012 L-1) to the exposed injured spinal cord at 3.0 mm anterior to the injured site. The injections were made at a rate of 5 μL per 3 minutes with a microsyringe. The syringe was slowly pulled out after 5 minutes, then muscle and skin were sutured. ④ Sampling and immunohistochemical analysis: Three animals were sacrificed at 1 week and the other three at 2 weeks postoperatively. The sections were fixed with 40 g/L paraformaldehyde in phosphate buffer solution (PBS) for 20 minutes at room temperature, followed by incubation with primary antibodies at 4 ℃ overnight. The samples were treated with secondary antibodies, biotinylated anti-mouse or rabbit immunoglobulin (IgG) at 37 ℃ for 20 minutes; Followed by incubation of horseradish peroxidase (HRP) labeled third antibodies at 37 ℃ for 20 minutes, then stained with 0.2 g/L diaminobenzidine (DAB) or AEC.MAIN OUTCOME MEASURES: Survival, migration and expression of AECs after transplanted into the injured spinal cord. RESULTS: After transplantation, most of the AECs gather beneath the pia mater of injured spinal cord at 1 week. But they migrated more extensively and many positive nuclear cells (brown) were observed in the center cannel and surrounding gray mater. Meantime, it was also detected that the transplanted AECs could express NT3 (positive cells stained as red) and BDNF in the injured spinal cord.CONCLUSION: AECs could survive for at least 3W after transplanted into the injured spinal cord of adult rats and could migrate widely; Furthermore, they could secrete neurotrophic factors such as NT-3 and BDNF.
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Objective To isolate the rat insulin gene enhancer binding protein 1(Islet-1)gene and construct plEGFP-C1-Islet-1 recombinant retroviral expression vector.Methods The cDNA encoding the rat Islet-1 gene was isolated by RT-PCR method,the cDNA was first cloned into PGET-1 TA vector to facilitate the sequence and then subcloned into the retroviral vector plEGFP-C1.plEGFP-C1-Islet-1 was transfected into PA317 packaging cells with lipofectamine 2000.Transformants were selected in medium containing G418.Results A 1 050 bp DNA fragment was obtained by RT-PCR;plEGFP-C1-Islet-1 recombinant retroviral expression vector was identified by restrictive enzymes digestion,PA317 cells transfected with recombinant vector expressed enhancer green fluorescent protein(EGFP).Conclusion The gene encoding the rat Islet-1 is obtained and plEGFP-C1-Islet-1 expression vector is constructed successfully.
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Objective To study the effects of LaCl3 on P16 and P21 expressions of hepatocellular carcinoma cells.Methods The experiment was divided into 4 groups.In experiment groups,CBRH-7919 cells were cultivated with 0.01,0.10 and 1.00 mmol?L-1 LaCl3 in DMEM containing 10% calf serum.In control group,CBRH-7919 was cultivated in DMEM containing 10% calf serum without LaCl3.The growth of CBRH-7919 cells was observed following treated with 0.01,0.10,1.00 mmol?L-1 LaCl3 for 1,3,5 d in vitro, respectively,the changes of cell cycle were detected by flow cytometry.At the same time,P16 and P21 were detected by immunocytochemical analysis.Results Compared with control group,the inhibitory rate of growth of CBRH-7919 cells was increased evidently when treated with 0.1 and 1.00 mmol?L-1 LaCl3 for 3 and 5 d(P
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BACKGROUND: It has been suggested that amniotic epithelial cells (AECs) express almost all of the markers of neural cell and secret a lot of neurotrophic factors and neurotransmitters. If AECs could substitute neural cells, its neurotrophic effect will bring promising prospect in treating neuron injuries and degenerative neural disease.OBJECTIVE: To detect specific proteins of neural cells in rat's cultured AECs.DESIGN: Repeated measurement design.SETTING: Second Clinical Medical College , Jilin University; Department of Histology & Embryology, School of Basic Medical Science, Jilin University.MATERIALS: This experiment was conducted at the Department of Histology & Embryology, School of Basic Medical Science, Jilin University from October 2004 to October 2005. The rat amniotic epithelial tissue was mechanically peeled from an embryonic 12 to 14days Wistar rats. Mouse anti Nestin was purchased from Chemicon Co.,and anti-ChAT rabbit anti-NSE and anti-NT-3 antibodies from Wuhan Boshide Company. Mouse anti-Musashi antibody was donated by Pro.Okano.METHODS: AECs were dissociated and purified from the amnion of pregnancy 12-14 day rats. AECs were treated with trypsin for 5 minutes,then cultured in DMEM/F12 medium at a humidified atmosphere of 0.05 volume fraction of CO2 in air at 37 ℃. Cells were inoculated at a concentration of 5×109 cells/L in culture flask. After 3 days, cells were inoculated onto poly-lysine-treated 35 mm culture Petri dish at a density of 1 × 108 cells/L for immunocytochemically staining. The cells were fixed with 40 g/L paraformaldehyde for 20 minutes. Immunocytochemical staining method was used to detect the expression of microtubule-associated protein-2 (MAP-2),neuron specific enolase(NSE), glial fibrillary acidic protein (GFAP) and choline acetyl transferase(ChAT).MAIN OUTCOME MEASURES: ① Morphological observation of rat'AECs at different culture time. ② Expression of specific protein of neural cells in rat' cultured AECs.RESULTS: ① After cultured for 24 hours, the AECs were flat and presented fibroblast-like morphology. 3 to 5 days later, cell bodies were well stacked; AECs had a big and round nucleus and were connected with each other by flourishing dendrites. ② Immunocytochemical staining results after culture for 4 days showed that AECs expressed Nestin, ChAT,NSE, Musashi, MAP-2, GFAP.CONCLUSION: AECs are homologous to neural cells in morphology, and it may be a new cell source to treat nervous system disease.
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Objective:To discuss the effects of Rhodosin,the extractant of medical plants na med Rhodiola Sachinonsis A.Bor,on aging rats and the model rats of Alzheier′s d isease (AD).Methods:Step down and water maze tests were used to determine the content of ace tylcholine (Ach) and the activity of acetylcholine transferase (ChAT) of hippoca mpus,lipid peroxide (LPO) and activity of superoxide dismute (SOD) of the cerebr al cortex,and cerebellar cortex and spinal cord were observed respectively.At th e same time,the general light microscope and eletron microscope were used to obs erve the changes in morphology.Results:The Rhodosin as an antioxidant could enhance the content of Ach and the activity of ChAT,reduce the formation of LPO,and increase activity of SOD.The Rh odosin could facilitate metabolism,enhance activity,and inhibit degeneration of aging rat cells.Conclusion:Rhodosin had obvious anti-aging effects on aing rats and also had p reventive,protective,anti-dementia effects on experimental dementia.
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Objective To investigate the probabilities of brain-derived stem cells from fetal rats differentiating into neurons and astrocytes by velvet antler polypeptide(VAP) in vitro. Methods Neural stem cells from E12-14d rats were cultured for 7 days until neural stem cells (NSCs) aggregations were formed into neurospheres. The neurospheres were cultured at different concentrations of VAP, and immunocytochemistry was used to detect the differentiation of neural stem cells. Results The differentiated cells in 50?g/L VAP group are more than that in control group; the number of NSE positive cells in 50?g/L,100?g/L and 200?g/L groups is more than that in control group.Conclusion Neural stem cells can be successfully induced into neurons by VAP in vitro, which could provide a basis for regeneration of nerve system.;