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目的:研究钙结合蛋白D28k(calbindin-D28k,CaBP)体外表达及其对神经元细胞抗凋亡作用。方法:RT-PCR从小鼠脑组织中扩增出小鼠CaBP的cDNA片段;重组构建pTH-CB质(pcDNA3-TH-CB),脂质体包裹pTH-CB质粒转染MN-9D细胞(多巴胺能杂交瘤神经元细胞);RT-PCR、免疫沉淀法检测CaBP的cDNA在细胞内表达情况;Hoechst染色、Caspase-3活性检测、AnnexinV/PI法对CaBP过表达的抗凋亡作用进行检测。结果:小鼠脑组织RT-PCR扩增出785bp条带,测序结果与Genebank中报道完全一致。脂质体包裹重组构建pTH-CB质粒转染MN-9D细胞后,转染pTH-CB质粒的MN-9D细胞中,CaBP的mRNA表达量和CaBP水平显著高于转染pc-DAN3组和正常MN-9D细胞组(P<0.05),3种细胞凋亡检测方法结果显示,CaBP过表达的细胞抗凋亡能力显著提高。结论:体外克隆CaBP的cDNA能在细胞内表达,过表达的CaBP通过抑制细胞的caspase-3活性,起抗凋亡作用。
AIM: To investigate the expression of calbindin-D28k (CaBP) in vitro and its anti-apoptotic effects on neurons. Methods: cDNA fragment of mouse CaBP was amplified from mouse brain by RT-PCR. Recombinant plasmid pTH-CB (pcDNA3-TH-CB) was transfected into MN-9D cells by lipofectamine. The hybridoma neuron cells were obtained. The expression of CaBP cDNA was detected by RT-PCR and immunoprecipitation. The anti-apoptotic effect of CaBP overexpression was detected by Hoechst staining, Caspase-3 activity assay and AnnexinV / PI assay. Results: The 785bp band was amplified by RT-PCR from mouse brain tissue. The sequencing result was completely consistent with that reported in Genebank. Liposome-encapsulated recombinant plasmid pTH-CB transfected MN-9D cells, transfected pTH-CB plasmid MN-9D cells, CaBP mRNA expression levels and CaBP levels were significantly higher than the transfected pc-DAN3 group and normal MN-9D cells (P <0.05). The results of three apoptosis assays showed that the anti-apoptotic ability of CaBP overexpressed cells was significantly increased. CONCLUSION: cDNA of cloned CaBP in vitro can be expressed in cells. Overexpression of CaBP plays an anti-apoptotic role by inhibiting caspase-3 activity.