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BACKGROUND:Neural stem cell(NSC) survival is closely associated with cell apoptosis in ischemic-hypoxic regions following transplantation.Numerous studies have revealed that X-box binding protein 1(XBP1) is a transcription factor during endoplasmic reticulum unfolded protein response and is essential for cell survival,differentiation,and anti-apoptotic effects.OBJECTIVE:To determine the effects of the XBP1 gene on NSC proliferation and apoptosis under hypoxic conditions following XBP1 gene transfection into rat embryonic hippocampal NSCs using recombinant adenovirus vector.DESIGN,TIME AND SETTING:In vitro experiments were performed at the Laboratory of Cell Biology of Jilin University and Laboratory of Proteomics,Department of Neurology,Jilin University,China from September 2008 to November 2009.MATERIALS:Recombinant adenovirus package XBP1 gene and Ad-XBP1-enhanced green fluorescent protein plasmid(Guangzhou Easywin BioMed Technology,China),rabbit anti-XBP1 and its target gene estrogen receptor degradation-enhancing α-mannosidase-like protein(EDEM),glucose-regulated protein 78(GRP78),anti-apoptotic molecule Bcl-2 and proapoptotic molecule Bax polyclonal antibody(Santa Cruz Biotechnology,Inc.,Santa Cruz,CA,USA),and CoCl2(Sigma,St.Louis,MO,USA) were used in the present study.METHODS:Hippocampi from embryonic,Sprague Dawley rats on gestational day 16 were harvested for NSC isolation and cloning,followed by immunofluorescence for Nestin and sub-culturing.The recombinant adenovirus Ad-XBP1-enhanced green fluorescent protein plasmid was transfected into rat embryonic hippocampal NSCs,and then CoCl2 was applied to induce hypoxia.MAIN OUTCOME MEASURES:Cell quantification and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay were utilized to detect proliferation in XBP1-transfected NSCs for 7 consecutive days.Western blot assay was utilized to quantify XBP1,GRP78,EDEM,Bcl-2,and Bax expression.Flow cytometry was used to measure apoptosis.RESULTS:NSC proliferation was significantly enhanced following XBP1 gene transfection(P < 0.05).Under hypoxic conditions,GRP78,EDEM,and Bcl-2 levels increased,but Bax levels decreased.In addition,NSC apoptosis decreased following transfection(P < 0.05).CONCLUSION:The XBP1 gene was successfully transfected into rat embryonic hippocampal NSCs using a recombinant adenovirus vector.NSC proliferation following transfection,as well as anti-apoptotic effects under hypoxia,was significantly increased.
BACKGROUND: Neural stem cell (NSC) survival is closely associated with cell apoptosis in ischemic-hypoxic regions following transplantation. Numerous studies have revealed that X-box binding protein 1 (XBP1) is a transcription factor during endoplasmic reticulum unfolded protein response and is essential for cell survival, differentiation, and anti-apoptotic effects. OBJECTIVE: To determine the effects of the XBP1 gene on NSC proliferation and apoptosis under hypoxic conditions following XBP1 gene transfection into rat embryonic hippocampal NSCs using recombinant adenovirus vector. DIGNIGN, TIME AND SETTING: In vitro experiments were performed at the Laboratory of Cell Biology of Jilin University and Laboratory of Proteomics, Department of Neurology, Jilin University, China from September 2008 to November 2009. MATERIALS: Recombinant adenovirus package XBP1 gene and Ad-XBP1-enhanced green fluorescent protein plasmid (Guangzhou Easywin BioMed Technology, China), rabbit anti-XBP1 and its target gene estroge (GRP78), anti-apoptotic molecule Bcl-2 and proapoptotic molecule Bax polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) USA), and CoCl2 (Sigma, St. Louis, MO, USA) were used in the present study. METHODS: Hippocampi from embryonic, Sprague Dawley rats on gestational day 16 were harvested for NSC isolation and cloning, followed by immunofluorescence for Nestin and sub-culturing. recombinant adenovirus Ad-XBP1-enhanced green fluorescent protein plasmid was transfected into rat embryonic hippocampal NSCs, and then CoCl2 was applied to induce hypoxia. MAIN OUTCOME MEASURES: Cell quantification and 3- (4,5-dimethylthiazol-2 -yl) -2,5-diphenyltetrazolium bromide colorimetric assay were utilized to detect proliferation in XBP1-transfected NSCs for 7 consecutive days. Western blot assay utilized to quantify XBP1, GRP78, EDEM, Bcl-2, and Bax expression. Flow cytometry was used to measure apoptosis.RESULT S: NSC apoptosis was significantly enhanced following the XBP1 gene transfection (P <0.05) .Nuder hypoxic conditions, GRP78, EDEM, and Bcl-2 levels increased, but Bax levels decreased. CONCLUSION: The XBP1 gene was successfully transfected into rat embryonic hippocampal NSCs using a recombinant adenovirus vector. NSC proliferation following transfection, as well as anti-apoptotic effects under hypoxia, was significantly increased.