Objective Combine olfactory ensheathing glia (OEG) implantation with ex vivo non-viral vector-based neurotrophin- 3 (NT-3) gene therapy in attempting to enhance regeneration after thoracic spinal cord injury (SCI...Objective Combine olfactory ensheathing glia (OEG) implantation with ex vivo non-viral vector-based neurotrophin- 3 (NT-3) gene therapy in attempting to enhance regeneration after thoracic spinal cord injury (SCI). Methods Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1 (+)-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord (T9) contusion by the New York University impactor. The animals in 3 different groups received 4x 1050EG transfected with pcDNA3.1 (+)-NT3 or pcDNA3.1 (+) plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. Results NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1 (+)-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. Conclusion Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT- 3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.展开更多
Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor (GDNF), and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by...Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor (GDNF), and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells (BMSCs). Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration (800 pg/mL) than BMSCs did (less than 100 pg/mL). The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin (10 μmol/L). Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.展开更多
Objective To observe the activating effect of ciliary neurotrophic factor (CNTF) on astrocyte in vitro. Methods Astrocytes cultured purely from newborn rats. Cerebral cortex was raised in normal and serum deprivatio...Objective To observe the activating effect of ciliary neurotrophic factor (CNTF) on astrocyte in vitro. Methods Astrocytes cultured purely from newborn rats. Cerebral cortex was raised in normal and serum deprivation condition with different concentrations (in ng/ml: 0, 2, 20, or 200) of CNTF. After cultured for 24 h, the shape and the cell cycle of astrocytes were examined by immunocytochemistry and flow cytometer, respectively. Results The immunoactivity of glial fibrillary acidic protein (GFAP) and the nuclear size of astrocytes were increased when CNTF was applied, whether cells were cultured in medium with or without serum. CNTF promoted astrocytes to enter the cell cycle in medium with serum, but had no this effect in medium without serum. Conclusion In medium without serum, astrocytes could differentiate into activated state ceils with CNTF application, but could not proliferate; in medium with serum, astrocytes could proliferate with aid of CNTF.展开更多
Objective To investigate the expression of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) in monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood ...Objective To investigate the expression of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) in monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. Methods The monkeys were immediately removed brain after death in operation of group A (identical temperature perfusion group) and group B (ultraprofound hypothermia perfusion group). Immunohistochemical technique was used to determine frontal cellular expression of NGF and GDNF. Statistics were analyzed by ANOVA analyses with significance level at P 〈 0.05. Results The expressions of NGF and GDNF in the group B were significantly higher than those in the group A (P 〈 0.05). Conclusion NGF and GDNF increased significantly in the monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. It may be a protective mechanism for neuron survival and neural function recovery.展开更多
Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein (GFAP) production and glial cell line-derived neurotrophic factor (GDNF) secretion of astrocytes under isch...Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein (GFAP) production and glial cell line-derived neurotrophic factor (GDNF) secretion of astrocytes under ischemic insult and the related signaling pathways. Methods Using transient right middle cerebral artery occlusion (tMCAO) and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, enzyme linked immunosorbent assay (ELISA) were used to investigate location of P2Y1 receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules. Results Blockage of P2Y1 receptor with the selective antagonist N^6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate diammonium (MRS2179) reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y1 receptor caused elevation of phosphorylated Akt and cAMP response element binding protein (CREB), and reduction of phosphorylated Janus kinase2 (JAK2) and signal transducer and activator of transcription3 (STAT3, Ser727). After blockage of P2Y1 receptor and deprivation of oxygen-glucose-serum, AG490 (inhibitor of JAK2) reduced phosphorylation of STAT3 (Ser727) as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase 1/2 (MEK 1/2) U0126, an important molecule of Ras/extracellular signal- regulated kinase (ERK) signaling pathway, decreased the phosphorylation of JAK2, STAT3 (Ser727), Akt and CREB. Conclusion These results suggest that P2Y1 receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.展开更多
Objective: To detect the expressions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in purified rat choroid plexus epithelial cells in vitro. Methods: Primary and passage choroid plexu...Objective: To detect the expressions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in purified rat choroid plexus epithelial cells in vitro. Methods: Primary and passage choroid plexus epithelial cells were obtained from newborn, one-day Spragne-Dawley rats. The expressions of BDNF and NGF were measured by qRT-PCR and Western blottingting. The secretions of BDNF and NGF were detected by ELISA. Cell supematants of primary cells, purified cells and passage 1 cells were harvested. Results: The expression of BDNF in the purified cells was significantly lower than that in the primary cells (P〈0.05), and it in the primary cells and the purified cells was significantly higher than that in the passage 1 cells (P〈0.05). The expression of NGF was significantly higher in the purified cells than in the primary cells and the passage 1 cells (P〈0.05). It in the passage 1 cells was significantly higher than that in the primary cells (P〈0.05). Conclusion: The time of CPECs transplantation for central nervous system diseases should be selected based on their secretory function and features,which could lead to better and more effective treatment.展开更多
Objective: The aim of this study was to analyze the capacity of Artemin promoting the motility and invasiveness of MIA PaCa-2 pancreatic cancer (PAC) cells. Methods: The PAC cell line MIA PaCa-2 was cultured in vi...Objective: The aim of this study was to analyze the capacity of Artemin promoting the motility and invasiveness of MIA PaCa-2 pancreatic cancer (PAC) cells. Methods: The PAC cell line MIA PaCa-2 was cultured in vitro and studied using Transwell chamber analysis. The motility and invasiveness ability affected by different concentrations of Artemin and its receptor GFRa3 were determined. Expression level of matrix metalloproteinase-2 (MMP-2), epithelial cadherin (E-cadherin) were quantitative analysis using RT-PCR and Western blot in MIA PaCa-2 cells stimulated with Artemin and receptor GFRa3. Results: MIA PaCa-2 PAC cell motility and invasiveness was significantly increased with Artemin and its receptor GFRa3 increasing concentrations than control (P 〈 0.01). 150 ng/mL was the best of both the role of concentration. MMP-2 was increased significantly (t = 6.35, t = 7.32), while E-cadherin was significantly lower (t = 4.27, t = 5.61), after affected by the 150 ng/mL Artemin and GFRα3,respectively. The difference was statistically significant compared with the control group (P 〈 0.01). Conclusion: Artemin and its receptor GFRa3 can promote PAC cell motility and invasiveness ability and contribute to the aggressive behavior. The mechanism may be related to increased expression of MMP-2 molecule and E-cadherin downregulation expression.展开更多
Objective: To study the sequence and function of the glial cell line-derived neurotrophic factor (GDNF) transcript in subjects of Han nationality. Methods: The Han nationality GDNF transcript was amplified by RT-PCR a...Objective: To study the sequence and function of the glial cell line-derived neurotrophic factor (GDNF) transcript in subjects of Han nationality. Methods: The Han nationality GDNF transcript was amplified by RT-PCR and expressed by baculovirus expression system. Biological activity of the expressed product was measured by the primary culture of midbrain dopaminergic neurons. Results: There only existed the shorter GDNF transcript of 555 bp in the Han nationality. The secretory expression product of the shorter transcript in insect cells promoted the survival and differentiation of dopaminergic neurons. Conclusion: It is found that there is a 78 bp deletion in the Han nationality GDNF transcript compared with the reported 633 bp GDNF transcript. The 78 bp deletion does not affect the secretory expression and biological activity of GDNF mature protein.展开更多
文摘Objective Combine olfactory ensheathing glia (OEG) implantation with ex vivo non-viral vector-based neurotrophin- 3 (NT-3) gene therapy in attempting to enhance regeneration after thoracic spinal cord injury (SCI). Methods Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1 (+)-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord (T9) contusion by the New York University impactor. The animals in 3 different groups received 4x 1050EG transfected with pcDNA3.1 (+)-NT3 or pcDNA3.1 (+) plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. Results NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1 (+)-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. Conclusion Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT- 3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.
基金This work was supported by the Natural Science Foundation of Shanghai Municipality(No.03ZR14016).
文摘Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor (GDNF), and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells (BMSCs). Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration (800 pg/mL) than BMSCs did (less than 100 pg/mL). The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin (10 μmol/L). Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.
文摘Objective To observe the activating effect of ciliary neurotrophic factor (CNTF) on astrocyte in vitro. Methods Astrocytes cultured purely from newborn rats. Cerebral cortex was raised in normal and serum deprivation condition with different concentrations (in ng/ml: 0, 2, 20, or 200) of CNTF. After cultured for 24 h, the shape and the cell cycle of astrocytes were examined by immunocytochemistry and flow cytometer, respectively. Results The immunoactivity of glial fibrillary acidic protein (GFAP) and the nuclear size of astrocytes were increased when CNTF was applied, whether cells were cultured in medium with or without serum. CNTF promoted astrocytes to enter the cell cycle in medium with serum, but had no this effect in medium without serum. Conclusion In medium without serum, astrocytes could differentiate into activated state ceils with CNTF application, but could not proliferate; in medium with serum, astrocytes could proliferate with aid of CNTF.
基金This work was supported by the Key Program of Natural Science Foundation of Yunnan Province, China (No. 2003C0010Z).
文摘Objective To investigate the expression of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) in monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. Methods The monkeys were immediately removed brain after death in operation of group A (identical temperature perfusion group) and group B (ultraprofound hypothermia perfusion group). Immunohistochemical technique was used to determine frontal cellular expression of NGF and GDNF. Statistics were analyzed by ANOVA analyses with significance level at P 〈 0.05. Results The expressions of NGF and GDNF in the group B were significantly higher than those in the group A (P 〈 0.05). Conclusion NGF and GDNF increased significantly in the monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. It may be a protective mechanism for neuron survival and neural function recovery.
基金the National Natural Science Foundation of China (No. 30500189)
文摘Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein (GFAP) production and glial cell line-derived neurotrophic factor (GDNF) secretion of astrocytes under ischemic insult and the related signaling pathways. Methods Using transient right middle cerebral artery occlusion (tMCAO) and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, enzyme linked immunosorbent assay (ELISA) were used to investigate location of P2Y1 receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules. Results Blockage of P2Y1 receptor with the selective antagonist N^6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate diammonium (MRS2179) reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y1 receptor caused elevation of phosphorylated Akt and cAMP response element binding protein (CREB), and reduction of phosphorylated Janus kinase2 (JAK2) and signal transducer and activator of transcription3 (STAT3, Ser727). After blockage of P2Y1 receptor and deprivation of oxygen-glucose-serum, AG490 (inhibitor of JAK2) reduced phosphorylation of STAT3 (Ser727) as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase 1/2 (MEK 1/2) U0126, an important molecule of Ras/extracellular signal- regulated kinase (ERK) signaling pathway, decreased the phosphorylation of JAK2, STAT3 (Ser727), Akt and CREB. Conclusion These results suggest that P2Y1 receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.
基金Supported by grants from the National Natural Science Foundation of China(No.30973099 and No.81271341)
文摘Objective: To detect the expressions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in purified rat choroid plexus epithelial cells in vitro. Methods: Primary and passage choroid plexus epithelial cells were obtained from newborn, one-day Spragne-Dawley rats. The expressions of BDNF and NGF were measured by qRT-PCR and Western blottingting. The secretions of BDNF and NGF were detected by ELISA. Cell supematants of primary cells, purified cells and passage 1 cells were harvested. Results: The expression of BDNF in the purified cells was significantly lower than that in the primary cells (P〈0.05), and it in the primary cells and the purified cells was significantly higher than that in the passage 1 cells (P〈0.05). The expression of NGF was significantly higher in the purified cells than in the primary cells and the passage 1 cells (P〈0.05). It in the passage 1 cells was significantly higher than that in the primary cells (P〈0.05). Conclusion: The time of CPECs transplantation for central nervous system diseases should be selected based on their secretory function and features,which could lead to better and more effective treatment.
基金Supported by grants from the National Science Foundation of Shandong Province (No. ZR2010HL053)the Surface Project of Jining Medical University
文摘Objective: The aim of this study was to analyze the capacity of Artemin promoting the motility and invasiveness of MIA PaCa-2 pancreatic cancer (PAC) cells. Methods: The PAC cell line MIA PaCa-2 was cultured in vitro and studied using Transwell chamber analysis. The motility and invasiveness ability affected by different concentrations of Artemin and its receptor GFRa3 were determined. Expression level of matrix metalloproteinase-2 (MMP-2), epithelial cadherin (E-cadherin) were quantitative analysis using RT-PCR and Western blot in MIA PaCa-2 cells stimulated with Artemin and receptor GFRa3. Results: MIA PaCa-2 PAC cell motility and invasiveness was significantly increased with Artemin and its receptor GFRa3 increasing concentrations than control (P 〈 0.01). 150 ng/mL was the best of both the role of concentration. MMP-2 was increased significantly (t = 6.35, t = 7.32), while E-cadherin was significantly lower (t = 4.27, t = 5.61), after affected by the 150 ng/mL Artemin and GFRα3,respectively. The difference was statistically significant compared with the control group (P 〈 0.01). Conclusion: Artemin and its receptor GFRa3 can promote PAC cell motility and invasiveness ability and contribute to the aggressive behavior. The mechanism may be related to increased expression of MMP-2 molecule and E-cadherin downregulation expression.
基金National Natural Science Foundation of China (No.30000048) National Basic Research Program of China (G1999054000)
文摘Objective: To study the sequence and function of the glial cell line-derived neurotrophic factor (GDNF) transcript in subjects of Han nationality. Methods: The Han nationality GDNF transcript was amplified by RT-PCR and expressed by baculovirus expression system. Biological activity of the expressed product was measured by the primary culture of midbrain dopaminergic neurons. Results: There only existed the shorter GDNF transcript of 555 bp in the Han nationality. The secretory expression product of the shorter transcript in insect cells promoted the survival and differentiation of dopaminergic neurons. Conclusion: It is found that there is a 78 bp deletion in the Han nationality GDNF transcript compared with the reported 633 bp GDNF transcript. The 78 bp deletion does not affect the secretory expression and biological activity of GDNF mature protein.