一氧化氮在8-Br-cAMP对视网膜母细胞瘤细胞株影响中的作用

The effect of nitric oxide induced by the 8-bromo-cyclic AMP in the human retinoblastoma HXO-Rb44 cells

目的研究８－Ｂｒ－ｃＡＭＰ对人视网膜母细胞瘤ＨＸＯ－Ｒｂ４４细胞作用后产生一氧化氮（ＮＯ）的效应，以探讨ＨＸＯ－Ｒｂ４４细胞分化和凋亡的机制。方法应用原位杂交和 ＲＮＡ斑点印迹技术检测 ＮＯＳ ｍＲＮＡ及 Ｂｃｌ－２ ｍＲＮＡ；应用硝酸还原酶法检测ＮＯ的含量；应用蛋白斑点印迹技术检测一氧化氮合成酶（ＮＯＳ）的酶活性；应用免疫细胞化学及蛋白质斑点印迹技术检测 ＮＳＥ的免疫反应性（ＩＲ）。结果 ＮＯＳ ｍＲＮＡ，ＮＯＳ酶活性，ＮＯ含量和 ＮＳＥ－ＩＲ均为实验组（ＥＧ）强于对照组（ＣＧ）（Ｐ＜０．０１），而Ｂｃｌ－２ ｍＲＮＡ为ＥＧ弱于ＣＧ（Ｐ＜０．０５），并在ＥＧ标本上可见ＨＸＯ－Ｒｂ４４细胞呈现神经样的突起。结论 ８－Ｂｒ－ｃＡＭＰ可使 ＨＸＯ－Ｒｂ４４细胞生成 ＮＯ增加，促进 ＮＯＳ的酶活性和 ＮＯＳ ｍＲＮＡ的表达，提高ＮＳＥ－ＩＲ，并降低 Ｂｃｌ－２ ｍＲＮＡ的表达。结果表明，８－Ｂｒ－ｃＡＭＰ具有促使 ＨＸＯ－Ｒｂ４４细胞向神经细胞分化并诱导该细胞凋亡的效应，提示ＮＯ可能参与此效应。

Objective To study the effect of nitric oxide(NO) induced by the 8-Br-cAMP in the human retinoblastoma HXO-Rb44 cells. The aim is to investigate the mechanism of cell differentiation and apoptosis induced by 8-Br-cAMP. Methods The NOS mRNA and Bcl-2 mRNA were detected with biotin-labeled NOS-cDNA probe by in situ hybridization and RNA dot blot. The NOS activity was detected by protein dot blot. NSE immunoreactivity(IR) was detected by immunocytochemistry and protein dot bolt. The NO was detected by nitrate reductase method. Results The NOS mRNA signal and NSE-IR were localized in the cytoplasm. The signals of NOS mRNA, NOS activity, NSE-IR and NO content in the EG were all higher than those in the CG(P<0.05-0. 01). The signals of Bcl-2 mRNA was lower in the EG than in the CG. Besides, the neurite-like processes in EG could be observed. Conclusions The results show that 8-Br-cAMP could increase NO product, NOS activity, NSE-IR (as the neuron specific marker) and the expression of NOS mRNA and decrease the expression of Bcl-2 mRNA. It indicates that 8-Br-cAMP could facilitate synthesis of NO in the HXO-Rb44 cells and have tendency toward neuron development and lead to cell apoptosis,suggesting that the increased NO may involve cell differentiation and apoptosis in the retinoblastoma HXO-Rb44 cells.