weaver基因对CAD细胞生物学功能的影响

Effect of weaver gene on biology function of CAD cells

目的 :评估weaver基因对神经细胞生长发育、蛋白质表达和生命力的影响。方法 :将一类酪氨酸脱氢酶阳性的神经细胞 (一种中枢神经源细胞 ) ,又称CAD (Cath .a -differentiated ,是Cath .a细胞的一种突变型 ,Cath .a是一种转基因大鼠的肿瘤细胞 )细胞 ,引入由DNA编码的野生型和突变型离子通道。采用DNA克隆、免疫组化、Westernblot等技术进行检测。用不同浓度 (0 2 5mg/L ,0 5mg/L ,1 0mg/L)的Girk2cDNA(对照组 )和wvGirk2cDNA(实验组 )质粒转染CAD细胞后 ,观察两组基因对转染CAD细胞的数目、蛋白质合成、神经突生长等指标的影响 ;并且观察MK80 1和Kir2 3对wvGirk2的抑制作用。结果 :发现高浓度wvGirk2转染的CAD细胞存活数目减少到约6 0 %的单纯Girk2转染细胞数目 ;低浓度wvGirk2没有引起细胞死亡但是减少转染基因的蛋白质产物 ,且神经轴突生长也受wvGirk2影响 ;MK80 1和Kir2 3对wvGirk2有抑制作用。结论 :wvGirk2基因在weaver动物中有一种特殊功能 ,可阻断wvGirk2通道防止细胞死亡。

AIM: To assess the impact of weaver gene on neuronal development,protein expression and vitality. METHODS: DNA encoding the wild-type and mutant ion channel was introduced into immortalized tyrosine hydroxylase-positive CNS-derived neurons named CAD(Cath.a-differentiated,a variant of Cath.a. Cath.a was established by targeted oncogenesis in transgenic mice) cells. DNA clone, immunostaining and Western blotting were used. Three different concentrations (0 25 mg/L, 0 5 mg/L, or 1 0 mg/L) of Girk2 and wv Girk2 expression plasmids were transfected into the CAD cells. The number of transfected cycling cells, protein synthesis and neurites growth were observed between two groups. RESULTS: The number of transfected cycling CAD cells with high concentration of wv Girk2 reduced to about 60%, compared to Girk2-transfected cells. Low concentration of wv Girk2 did not cause cell death but reduced the protein production of transfected genes. Neurite growth was also affected by wv Girk2. MK-801 and Kir2.3 altered the effect of wv Girk2. CONCLUSION: The results indicate that wv Girk2 functions as a blocker in weaver animals, which blockes the wv Girk2 channel to rescue the cells from death. Our data also suggest that the presence of channels and the level of wv Girk2 may have a significant impact on the fate of cells containing wv Girk2.