L-精氨酸和左旋-硝基精氨酸甲酯对纯化视网膜神经节细胞凋亡的影响

The influnce of L-Arg and L-NAME to purified retinal ganglion cells apoptosis of rats

目的 探讨 L-精氨酸 (L- arginine,L- Arg)和左旋 -硝基精氨酸甲酯 (L- nitro- arginine- m ethyl- ester,L- NAME)对纯化培养的视网膜神经节细胞 (retinal gangal cells,RGCs)凋亡的影响。 方法 同化培养液培养抗体纯化的 Sprague- Dawley(SD)系大鼠 RGCs;在纯化培养的 RGCs中分组加入体积百分浓度为 1× 10 - 6 、1× 10 - 5、 1× 10 - 4、1× 10 - 3、1× 10 - 2 、1× 10 - 1 mol/ L的 L - Arg及 L - NAME,培养 2 4 h后检测各组培养上清液中硝酸盐含量、细胞的活性 ;培养 4 8h后检测各组细胞的凋亡及细胞中 bcl- 2、bax及p5 3m RNA的表达。 结果 培养上清液中硝酸盐含量随着 L- Arg体积百分浓度的增高而增高 ,随着 L-NAME体积百分浓度的升高而降低。低体积百分浓度时 ,随着 L - Arg和 L - NAME体积百分浓度的增加bcl- 2 m RNA表达逐渐增高 ,bax及 p5 3m RNA的表达无明显变化。而高体积百分浓度时则相反。 结论 L - Arg低体积百分浓度时能促进 RGCs的生长 ,高体积百分浓度时则促进细胞凋亡 ;L - NAME对 L -

Objective To investigate the influnce of L arginine (L Arg) and L nitro arginine methyl ester(L NAME) to purified retinal ganglion cells(RGCs) apoptosis of rats cultured in different consistencies of L Arg and L NAME. Method RGCs from Sprague Dawley (SD) neonatal rats(postnatal 1~5 day) were cultured in assimilative culture solution in vitro and RGCs were purified by Thy1.1 with sheep anti rat FITC monoclonal antibody. RGCs were cultured in different consistencies of L Arg and L NAME: 1×10 -6 ,1×10 -5 ,1×10 -4 ,1×10 -3 ,1×10 -2 and 1×10 -1 mol/L for 24 hours and 48 hours, respectively. The changes of bcl 2, bax and p53 mRNA in RGCs in different consistencies of L Arg and L NAME were demonstrated qualitatively and quantitatively by in situ hybridization, and their apoptosis were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) method, respectively. Results After 24 hours in vitro, the purification rate of RGCs in the experiment arrived at 97 %. After 48 hours, there were a few apoptotic cells expression in the control group. Apoptotic cells expression in L Arg≥1×10 -3 mol/L and L NAME ≥1×10 -1 mol/L groups increased that had a significant difference with the control group ( P <0.05). In the group of L Arg≥1×10 -3 mol/L and L NAME ≥1×10 -1 mol/L, the expression of bcl 2 mRNA in RGCs became weaker and weaker as the consistencies were increased, but the expression of bax and p53 mRNA in RGCs became higher and higher and had a significant difference with control group ( P <0.05). Conclusion Lower concentration of L Arg can promote the growth of purified RGCs in vitro and higher concentration of L Arg can promote the apoptosis of RGCs.