ABSTRACT
ObjectiveTo explore the changes of astrocytes in vitro on the expression of Cyclin D1 under normal and hypoxia/reoxygenation conditions.MethodsThe primary astrocytes were isolated from the cortex of SD fetal rats(less than 24 hours) and identified by immunosytochemical method with anti-GFAP antibody after 3 passages.Astrocytes of passage 3 were divided into normal group ( control group),hypoxia/reoxygenation group ( H/R 37℃ ),and mild hypothermiaintervention group( H/R 32℃ )separately.Astrocytes from the later tow groups were reoxygenated with 4,12,and 20 hours separately after exposed to hypoxia conditions for 8 hours.Trypan blue staining was employed to detect the survival rates and immunofluorescence,western-blot were used to analyse the expressin of Cyclin D1 of of astrocytes of different groups and time points.Results 1.The GFAP positive astrocytes from passage 3 exceeded 95 ％.2.With regard to morphology and survival rates,there is no difference between astrocytes of normal and hypothermia groups after 8 hours exposure to hypoxia conditions.Reoxygenation could obviously rise astrocytes mortality with time went by ( H/R 37 ℃ group:12.87 ± 2.76 ( R4 ),31.55 ± 3.00 ( R12 ),46.40 ±8.50(R20) ;H/R 32℃ group:6.77 ± 1.53( R4),15.97 ±4.00(R12),28.33 ±5.69(R20) ;all P＜0.05).3.Immunofluorescence and western-blot revealed that reoxygenation increased Cyclin D1expression markedly,which was proportional to the duration of reoxygenation.Mild hypothermia could reduce Cyclin D1 expression of astrocytes severely under reoxygenation condition.ConclusionCyclin D1 expression can be regarded as a sensitive index of damage to astrocytes caused by hypoxia/reoxygenation conditions.
ABSTRACT
Objective; To express and purify HCA518 protein and prepare its monoclonal antibody ( McAb). Methods: The HCA518 protein was expressed with gene recombinant technique in prokaryotic system and purified with nickel chelate nitrilotriacetic acid(Ni-NTA) affinity chromatography column. Hybridoma cell lines that secreted anti-HCA518 McAb were established by cells fusion and screened by enzyme linked immunosorbent assay( ELISA). The specificity of anti-HCA518 McAb wa3 identified by Western blot assay. The HCA518 protein in tumor cells was stained by immunoflourescence assay. Results: Rcombinant HCA518 protein was expressed with a purity of 98%. Two hybridoma cell lines was selected and anti-HCA518 McAb was purified from mice ascites. The titers of anti HCA518 McAb in ascites were 1?10-4 and 5?10-4 respectively. The antibody belonged to IgG2b subtype and IgM. Anti-HCA518 McAb specifically reacted with recombinant HCA518 protein and tumor cells'nuclear protein (P100). The HCA518 protein was mainly located in cell nucleus. Conclusion: Stable hybridoma cell lines that secreted anti-HCA518 McAb have been established and anti HCA518 McAb was prepared with high specificity. It has important significance for detecting HCA518 protein in tumor tissues and determining malignant proliferation status of tumor cells and predicting its prognosis.